sirolimus and Fibrosis

The calcineurin inhibitors (CNI) cyclosporine A (CsA) and tacrolimus represent potent immunosuppressive agents frequently used for solid organ transplantation and treatment of autoimmune disorders. Despite of their immense therapeutic benefits, residual fibrosis mainly in the kidney represents a common side effect of long-term therapy with CNI. Regardless of the immunosuppressive action, an increasing body of evidence implicates that a drug-induced increase in TGFβ and subsequent activation of TGFβ-initiated signaling pathways is closely associated with the development and progression of CNI-induced nephropathy. Mechanistically, an increase in reactive oxygen species (ROS) generation due to drug-induced changes in the intracellular redox homeostasis functions as an important trigger of the profibrotic signaling cascades activated under therapy with CNI. Although, inhibitors of the mechanistic target of rapamycin (mTOR) kinase have firmly been established as alternative compounds with a lower nephrotoxic potential, an activation of fibrogenic signaling cascades has been reported for these drugs as well. This review will comprehensively summarize recent advances in the understanding of profibrotic signaling events modulated by these widely used compounds with a specific focus put on mechanisms occurring independent of their respective immunosuppressive action. Herein, the impact of redox modulation, the activation of canonical TGFβ and non-Smad pathways and modulation of autophagy by both classes of immunosuppressive drugs will be highlighted and discussed in a broader perspective. The comprehensive knowledge of profibrotic signaling events specifically accompanying the immunomodulatory activity of these widely used drugs is needed for a reliable benefit-risk assessment under therapeutic regimens.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Cyclosporine; Fibrosis; Humans; Immunosuppressive Agents; Kidney; Kidney Diseases; Models, Animal; Reactive Oxygen Species; Signal Transduction; Sirolimus; Tacrolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Controlled trials of mTOR inhibitors in children following solid organ transplantation are scarce, although evidence from prospective single-arm studies is growing. Everolimus with reduced CNI therapy has been shown to be efficacious and safe in de novo pediatric kidney transplant patients in prospective trials. Prospective and retrospective data in children converted from CNI therapy to mTOR inhibition following kidney, liver, or heart transplantation suggest preservation of immunosuppressive efficacy. Good renal function has been maintained when mTOR inhibitors are used de novo in children following kidney transplantation or after conversion to mTOR inhibition with CNI minimization. mTOR inhibition with reduced CNI exposure is associated with a low risk for developing infection in children. Growth and development do not appear to be impaired during low-dose mTOR inhibition, but more studies are required. No firm conclusions can be drawn as to whether mTOR inhibitors should be discontinued in children requiring surgical intervention or whether mTOR inhibition delays progression of hepatic fibrosis after pediatric liver transplantation. In conclusion, current evidence suggests that use of mTOR inhibitors in children undergoing solid organ transplantation is efficacious and safe, but a number of issues remain unresolved and further studies are required.

Topics: Calcineurin Inhibitors; Child; Everolimus; Fibrosis; Heart Transplantation; Humans; Immunosuppressive Agents; Kidney Transplantation; Liver; Liver Transplantation; Lymphoproliferative Disorders; Postoperative Complications; Risk; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Wound Healing

Use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin in organ transplantation has evolved through different phases over the past two decades. After its discovery in the mid 1970s, antifungal and cytotoxic effects were the first of its properties to be explored, but the most significant advancement was found in its use as an immunosuppressive agent to reduce transplant rejection. This was viewed as an important step forward for immunosuppression, as early studies suggested that rapamycin was less nephrotoxic than calcineurin inhibitors (CNIs). Later, detrimental effects of rapamycin on kidney function were found in some patients. Nonetheless, a fascination with the mTOR pathway and its central role in multiple cellular processes has ensued. Among the potential positive clinically relevant effects is rapamycin's capacity to interfere with fibrotic processes that often accompany transplant rejection, and to influence the preferential development of immunological tolerance. A feature of increasing importance is that the mTOR pathway is central for vital aspects of tumor development, including angiogenesis and cell growth; rapamycin, therefore, has anticancer activities, which may prove critical in the fight against high cancer rates in transplant recipients. The final chapters defining the value of rapamycin have not been written yet, and indeed remain a work in progress. Only further research will reveal the full potential of rapamycin in organ transplantation.

Topics: Animals; Fibrosis; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Kidney; Neoplasms; Organ Transplantation; Sirolimus; TOR Serine-Threonine Kinases; Transplantation Tolerance

Kidney allograft fibrosis results from a reactive process mediated by humoral and cellular events and the activation of transforming growth factor beta1. It is a process that involves both parenchymal and graft infiltrating cells and can lead to organ failure if injury persists or if the response to injury is excessive. In this review, we will address the role of preventive and therapeutic strategies that target kidney allograft fibrogenesis. We conclude that in addition to preventive strategies, therapies based on bone morphogenetic protein 7, hepatocyte growth factor, connective tissue growth factor, and pirfenidone have shown promising results in preclinical studies. Clinical trials are needed to examine the effect of these therapies on long-term outcomes.

Topics: Chemokines; Fibrosis; Graft Rejection; Humans; Immunity, Humoral; Immunosuppressive Agents; Kidney Transplantation; Phenylurea Compounds; Piperidines; Receptors, Chemokine; Sirolimus; T-Lymphocytes; Transforming Growth Factor beta

The pathomechanisms underlying restenosis of the bioabsorbable sirolimus-eluting metallic scaffold (Magmaris) remain unknown. Using serial optical coherence tomography, we investigated causes of restenosis, including the contribution of late scaffold recoil versus neointimal hyperplasia.. Patients enrolled in BIOSOLVE-II undergoing serial angiography and optical coherence tomography (post-intervention and follow-up: 6 months and/or 1 year) were analyzed. Patients were divided into 2 groups according to angiographic in-scaffold late lumen loss (LLL) <0.5 or ≥0.5 mm. End points were late absolute scaffold recoil and neointimal hyperplasia area as assessed by optical coherence tomography.. In addition to neointimal hyperplasia, late scaffold recoil contributed significantly to LLL of sirolimus-eluting absorbable metal scaffolds. The extent of late scaffold recoil was dependent on the underlying plaque morphology and was the highest among fibrotic lesions. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01960504.

Topics: Absorbable Implants; Aged; Cardiovascular Agents; Coronary Restenosis; Coronary Vessels; Female; Fibrosis; Humans; Male; Metals; Middle Aged; Myocardial Ischemia; Neointima; Percutaneous Coronary Intervention; Predictive Value of Tests; Prospective Studies; Prosthesis Design; Sirolimus; Time Factors; Tomography, Optical Coherence; Treatment Outcome

In a 36-month, open-label, multicenter trial, 202 kidney transplant recipients were randomized at week 7 post-transplant to convert to everolimus or remain on cyclosporine: 182 were analyzed to month 36 (92 everolimus, 90 controls). Mean (SD) change in measured GFR (mGFR) from randomization to month 36 was 1.3 (14.0) ml/min with everolimus versus -1.7 (15.4) ml/min in controls (P = 0.210). In patients who remained on treatment, mean mGFR improved from randomization to month 36 by 7.9 (11.5) ml/min with everolimus (n = 37) but decreased by 1.4 (14.7) ml/min in controls (n = 62) (P = 0.001). During months 12-36, death-censored graft survival was 100%, patient survival was 98.9% and 96.7% in the everolimus and control groups, respectively, and 13.0% and 11.1% of everolimus and control patients, respectively, experienced mild biopsy-proven acute rejection (BPAR). Protocol biopsies in a limited number of on-treatment patients showed similar interstitial fibrosis progression. Donor-specific antibodies were present at month 36 in 6.3% (2/32) and 18.0% (9/50) of on-treatment everolimus and control patients with available data (P = 0.281). During months 12-36, adverse events were comparable, but discontinuation was more frequent with everolimus (33.7% vs. 10.0%). Conversion from cyclosporine to everolimus at 7 weeks post-transplant was associated with a significant benefit in renal function at 3 years when everolimus was continued.

Topics: Adult; Aged; Biopsy; Calcineurin Inhibitors; Cyclosporine; Everolimus; Female; Fibrosis; Glomerular Filtration Rate; Graft Rejection; Graft Survival; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Renal Insufficiency; Sirolimus; Treatment Outcome

The focus in renal transplantation is to increase long-term allograft survival. One of the limiting factors is calcineurin inhibitor (CNI)-induced fibrosis. This study attempted to examine the histological aspect of interstitial fibrosis and the modulation of the transforming growth factor-β (TGF-β) canonical signalling pathway following early withdrawal of CNI from sirolimus-based immunosuppressive therapy.. Forty-five kidney transplant recipients with low-medium immunologic risk were randomized and underwent protocol biopsies obtained at the time of transplantation and at 3 and 12 months thereafter. The recipients were taking tacrolimus, sirolimus and prednisone. After the 3rd month, patients were randomized into two groups: sirolimus (SRL) (removed CNI and increased sirolimus) and tacrolimus (TAC) (maintained CNI). Renal biopsies were analyzed according to Banff's 2007 criteria. The sum of Banff's ct and ci constituted the chronicity index. Fibrosis was evaluated by the histomorphometrical analysis of the total collagen and myofibroblast deposition. Immunohistochemical characterization and quantification of TGF-β, TGF-β receptor 1 (TGF-β-R1), receptor 2 (TGF-β-R2) and phospho-Smad2/3 (p-Smad2/3) were performed.. Maintenance of CNI was associated with the increase of the surface density of collagen and α-smooth muscle actin (α-SMA), (P = 0.001). Furthermore, increased TGF-β (P = 0.02), TGF-β-R1 (P = 0.02), p-Smad2/3 (P = 0.03) and stabilized TGF-β-R2. On the other hand, the removal of CNI with increase in the dose of sirolimus limited the enhancement of the chronicity index at 12 m (SRL, 2.18 vs TAC, 3.12, P = 0.0007), diminished the deposition of fibrosis and promoted the stabilization of TGF-β, TGF-β-R2, p-Smad2/3 and myofibroblasts as well as the reduction of TGF-β-R1 (P = 0.01).. The early withdrawal of CNI limited the fibrosis progression through the stabilization of chronicity index and of the canonical TGF-β signalling pathway.

Topics: Adult; Biopsy; Brazil; Calcineurin Inhibitors; Collagen; Drug Administration Schedule; Drug Therapy, Combination; Female; Fibrosis; Graft Rejection; Graft Survival; Humans; Immunohistochemistry; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Myofibroblasts; Prospective Studies; Signal Transduction; Sirolimus; Tacrolimus; Time Factors; Transforming Growth Factor beta; Treatment Outcome

There is no evidence on the incidence of subclinical inflammation and scaring lesions in patients receiving tacrolimus (TAC) minimization and elimination immunosuppressive regimens.. This study analyzed preimplantation, 3 and 24 months protocol biopsies and anti-HLA donor-specific antibodies (DSA) in 140 low immunological risk kidney transplant recipients receiving reduced TAC exposure, prednisone, and mycophenolate, randomized at 3 months to be converted or not to sirolimus (SRL).. Mean TAC concentrations were 6.0 ± 2.4 ng/mL and 5.8 ± 2.2 ng/mL at 3 and 24 months. The incidence of subclinical inflammation lesions at 3 months was 9.3%. The incidence of (interstitial fibrosis) IF/(tubular atrophy) TA at month 24 was 57.6%, higher in SRL compared to TAC group (68.8 vs 44.4%; P = 0.022). Patients converted to SRL showed higher incidence of acute rejection (7.3% vs 0%), proteinuria (59.6% vs 25%; P = 0.001), and DSA (17.8% vs 7.3%; P = 0.201), respectively. Biopsy-proven acute rejection (odds ratio [OR] 2.32, 95% confidence interval [95% CI], 0.979-5.518, P = 0.056), subclinical inflammation lesions at 3 months (OR, 11.75; 95% CI, 1.286-107.474; P = 0.029) and conversion to SRL (OR, 2.72; 95% CI, 1.155-6.383; P = 0.022) were associated with IF/TA at month 24. Black ethnicity (OR, 0.22; 95% CI, 0.058-0.873; P = 0.031), donor age (OR, 2.74; 95% CI, 1.329-5.649; P = 0.006), and conversion to SRL (OR, 2.34; 95% CI, 1.043-5.267; P = 0.039) were associated with inferior renal function at 24 months.. In kidney transplant recipients receiving reduced TAC exposure, subclinical inflammation lesions at 3 months were associated with IF/TA at 24 months. Conversion from TAC to SRL was associated with inferior renal function, higher incidence of IF/TA, and trends to higher incidence of DSA at 24 months.

Topics: Adult; Atrophy; Biomarkers; Biopsy; Calcineurin Inhibitors; Chi-Square Distribution; Drug Substitution; Female; Fibrosis; Graft Rejection; Graft Survival; Histocompatibility; HLA Antigens; Humans; Immunosuppressive Agents; Isoantibodies; Kidney; Kidney Transplantation; Logistic Models; Male; Middle Aged; Multivariate Analysis; Nephritis; Odds Ratio; Proteinuria; Risk Factors; Sirolimus; Tacrolimus; Time Factors; Treatment Outcome

In this ancillary study of the CONCEPT trial, we studied the role of CsA withdrawal at 3 months (3M) post-transplant on the intensity of epithelial phenotypic changes (EPC, an early marker for kidney fibrogenesis) on the 12 M surveillance biopsy. Although conversion from CsA to sirolimus (SRL) at 3M was reported to have improved mean graft function at 12 M, it did not reduce the score of EPC (1.73 ± 1.15 in the SRL group vs. 1.87 ± 1 in the CsA group, P = 0.61). Acute rejection, which had occurred twice more frequently in SRL-converted patients included here, was associated with 12 M EPC. Interestingly, we observed that the patients durably exposed to CsA and who developed 12 M EPC had a significant progression of blood pulse pressure (pp) from 1 to 6M post-transplantation (Δpp = +12.3 mmHg, P = 0.0035). Pulse pressure at 4, 6, and 9 M and pp progression from 1 to 6M were significantly associated with the development of EPC at 12 M in renal grafts. Logistic regression analysis revealed that a high 6M pp (≥ 60 mmHg) was an independent risk factor for 12 M EPC with an odds ratio of 2.25 per additional 10 mmHg pp (95%CI: 1.14-4.4, P = 0.02) after adjustment with recipient's and donor's age, acute rejection incidence and immunosuppressive regimen. A post hoc analysis of the data collected in the whole population CONCEPT study revealed that pp was significantly higher at 6 months in patients maintained on CsA and that at this time point pp correlated negatively with GFR at 1 year.

Topics: Adult; Blood Pressure; Calcineurin Inhibitors; Cyclosporine; Epithelium; Female; Fibrosis; Glomerular Filtration Rate; Graft Rejection; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Phenotype; Risk Factors; Sirolimus; Time Factors

Calcineurin-inhibitor therapy is a contributing factor to the origin of interstitial fibrosis and tubular atrophy (IFTA).. We conducted a prospective randomized trial of conversion of tacrolimus to sirolimus at 1-month posttransplant in kidney transplant recipients on rapid steroid withdrawal. We compared the chronic changes (IFTA and sum of Banff chronic scores--Total Score) on protocol biopsies at 1 month, 1 year, and 2 years in all randomized patients. We compared the outcomes between treatment groups and analyzed the impact of previous rejection on the chronic changes.. We randomized 122 patients, 62 to sirolimus and 60 to tacrolimus. The 1-year biopsy was performed in 54 patients (90%) of the tacrolimus group and 56 patients (90%) of the sirolimus group. The proportion of biopsies with IFTA more than or equal to 2 and the Total Score more than 2 increased over the 2 years but were not different between the study groups at any time point. On the 1-year biopsy, there was more IFTA, and the fraction with Total Score more than 2 was higher in the tacrolimus group with previous rejection. In the cohort without rejection, there was a significant progression of the IFTA and Total Score between 1 and 2 years in both the sirolimus and tacrolimus groups.. Conversion from tacrolimus to sirolimus at 1-month posttransplant in kidney transplant recipients on rapid steroid withdrawal does not decrease the progression of chronic changes on protocol biopsies during the first 2 years even in those patients without previous acute rejection.

Topics: Adult; Aged; Aged, 80 and over; Atrophy; Biopsy; Contraindications; Disease Progression; Female; Fibrosis; Graft Rejection; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Longitudinal Studies; Male; Middle Aged; Prospective Studies; Risk Factors; Sirolimus; Steroids; Tacrolimus; Transplantation, Homologous; Withholding Treatment

Serial intravascular ultrasound virtual histology (IVUS-VH) after implantation of metallic stents has been unable to show any changes in the composition of the scaffolded plaque overtime. The everolimus-eluting ABSORB scaffold potentially allows for the formation of new fibrotic tissue on the scaffolded coronary plaque during bioresorption. We examined the 12 month IVUS-VH changes in composition of the plaque behind the struts (PBS) following the implantation of the ABSORB scaffold. Using IVUS-VH and dedicated software, the composition of the PBS was analyzed in all patients from the ABSORB Cohort B2 trial, who were imaged with a commercially available IVUS-VH console (s5i system, Volcano Corporation, Rancho Cordova, CA, USA), immediately post-ABSORB implantation and at 12 month follow-up. Paired IVUS-VH data, recorded with s5i system, were available in 17 patients (18 lesions). The analysis demonstrated an increase in mean PBS area (2.39 ± 1.85 mm(2) vs. 2.76 ± 1.79 mm(2), P = 0.078) and a reduction in the mean lumen area (6.37 ± 0.90 mm(2) vs. 5.98 ± 0.97 mm(2), P = 0.006). Conversely, a significant decrease of 16 and 30% in necrotic core (NC) and dense calcium (DC) content, respectively, were evident (median % NC from 43.24 to 36.06%, P = 0.016; median % DC from 20.28 to 11.36%, P = 0.002). Serial IVUS-VH analyses of plaque located behind the ABSORB struts at 12-month demonstrated an increase in plaque area with a decrease in its NC and DC content. Larger studies are required to investigate the clinical impact of these findings.

Topics: Absorbable Implants; Aged; Australia; Cardiovascular Agents; Coronary Angiography; Coronary Artery Disease; Coronary Vessels; Europe; Everolimus; Female; Fibrosis; Humans; Male; Middle Aged; Necrosis; New Zealand; Percutaneous Coronary Intervention; Plaque, Atherosclerotic; Predictive Value of Tests; Prosthesis Design; Sirolimus; Time Factors; Tissue Scaffolds; Treatment Outcome; Ultrasonography, Interventional

To quantify the circumferential healing process at 6 and 12 months following scaffold implantation.. The healing process following stent implantation consists of tissue growing on the top of and in the space between each strut. With the ABSORB bioresorbable vascular scaffold (BVS), the outer circumference of the scaffold is detectable by optical coherence tomography (OCT), allowing a more accurate and complete evaluation of the intra-scaffold neointima.. A total of 58 patients (59 lesions), who received an ABSORB BVS 1.1 implantation and a subsequent OCT investigation at 6 (n=28 patients/lesions) or 12 (n=30 patients with 31 lesions) months follow-up were included in the analysis. The thickness of the neointima was calculated circumferentially in the area between the abluminal side of the scaffold and the lumen by means of an automated detection algorithm. The symmetry of the neointima thickness in each cross section was evaluated as the ratio between minimum and maximum thickness.. The neointima area was not different between 6 and 12 months follow-up (1.57±0.42 mm(2) vs. 1.64±0.77 mm(2); p=0.691). No difference was also found in the mean thickness of the neointima (median [IQR]) between the two follow-up time points (210 μm [180-260]) vs. 220 μm [150-260]; p=0.904). However, the symmetry of the neointima thickness was higher at 12 than at 6 months follow-up (0.23 [0.13-0.28] vs. 0.16 [0.08-0.21], p=0.019).. A circumferential evaluation of the healing process following ABSORB implantation is feasible, showing the formation of a neointima layer, that resembles a thick fibrous cap, known for its contribution to plaque stability.

Topics: Absorbable Implants; Algorithms; Cardiovascular Agents; Coronary Artery Disease; Coronary Vessels; Delayed-Action Preparations; Endovascular Procedures; Everolimus; Fibrosis; Humans; Image Processing, Computer-Assisted; Neointima; Polyesters; Predictive Value of Tests; Sirolimus; Time Factors; Tissue Scaffolds; Tomography, Optical Coherence; Treatment Outcome

This study sought to investigate in vivo the vascular response at the proximal and distal edges of the second-generation ABSORB everolimus-eluting bioresorbable vascular scaffold (BVS).. The edge vascular response after implantation of the BVS has not been previously investigated.. The ABSORB Cohort B trial enrolled 101 patients and was divided into B(1) (n = 45) and B(2) (n = 56) subgroups. The adjacent (5-mm) proximal and distal vessel segments to the implanted ABSORB BVS were investigated at either 6 months (B(1)) or 1 year (B(2)) with virtual histology intravascular ultrasound (VH-IVUS) imaging.. At the 5-mm proximal edge, the only significant change was modest constrictive remodeling at 6 months (Δ vessel cross-sectional area: -1.80% [-3.18; 1.30], p < 0.05), with a tendency to regress at 1 year (Δ vessel cross-sectional area: -1.53% [-7.74; 2.48], p = 0.06). The relative change of the fibrotic and fibrofatty (FF) tissue areas at this segment were not statistically significant at either time point. At the 5-mm distal edge, a significant increase in the FF tissue of 43.32% [-19.90; 244.28], (p < 0.05) 1-year post-implantation was evident. The changes in dense calcium need to be interpreted with caution since the polymeric struts are detected as "pseudo" dense calcium structures with the VH-IVUS imaging modality.. The vascular response up to 1 year after implantation of the ABSORB BVS demonstrated some degree of proximal edge constrictive remodeling and distal edge increase in FF tissue resulting in nonsignificant plaque progression with adaptive expansive remodeling. This morphological and tissue composition behavior appears to not significantly differ from the behavior of metallic drug-eluting stents at the same observational time points.

Topics: Absorbable Implants; Aged; Angioplasty, Balloon, Coronary; Cardiovascular Agents; Coronary Artery Disease; Coronary Vessels; Drug-Eluting Stents; Everolimus; Female; Fibrosis; Humans; Male; Middle Aged; Predictive Value of Tests; Prospective Studies; Prosthesis Design; Sirolimus; Time Factors; Tissue Scaffolds; Treatment Outcome; Ultrasonography, Interventional; Vascular Calcification

Implantation of a coronary stent results in a mechanical enlargement of the coronary lumen with stretching of the surrounding atherosclerotic plaque. Using intravascular ultrasound virtual-histology (IVUS-VH) we examined the temporal changes in composition of the plaque behind the struts (PBS) following the implantation of the everolimus eluting bioresorbable vascular scaffold (BVS). Using IVUS-VH and dedicated software, the composition of plaque was analyzed in all patients from the ABSORB B trial who were imaged with a commercially available IVUS-VH console (s5i system, Volcano Corporation, Rancho Cordova, CA, USA) post-treatment and at 6-month follow-up. This dedicated software enabled analysis of the PBS after subtraction of the VH signal generated by the struts. The presence of necrotic core (NC) in contact with the lumen was also evaluated at baseline and follow-up. IVUS-VH data, recorded with s5i system, were available at baseline and 6-month follow-up in 15 patients and demonstrated an increase in both the area of PBS (2.45 ± 1.93 mm(2) vs. 3.19 ± 2.48 mm(2), P = 0.005) and the external elastic membrane area (13.76 ± 4.07 mm(2) vs. 14.76 ± 4.56 mm(2), P = 0.006). Compared to baseline there was a significant progression in the NC (0.85 ± 0.70 mm(2) vs. 1.21 ± 0.92 mm(2), P = 0.010) and fibrous tissue area (0.88 ± 0.79 mm(2) vs. 1.15 ± 1.05 mm(2), P = 0.027) of the PBS. The NC in contact with the lumen in the treated segment did not increase with follow-up (7.33 vs. 6.36%, P = 0.2). Serial IVUS-VH analysis of BVS-treated lesions at 6-month demonstrated a progression in the NC and fibrous tissue content of PBS.

Topics: Absorbable Implants; Aged; Angioplasty, Balloon, Coronary; Australia; Cardiovascular Agents; Coronary Artery Disease; Coronary Vessels; Drug-Eluting Stents; Europe; Everolimus; Female; Fibrosis; Humans; Image Interpretation, Computer-Assisted; Male; Middle Aged; Necrosis; New Zealand; Predictive Value of Tests; Prosthesis Design; Sirolimus; Software; Time Factors; Treatment Outcome; Ultrasonography, Interventional

Chronic allograft nephropathy is the main cause of long-term renal transplant failure. Chronic use of calcineurin inhibitors contributes to its pathogenesis. Here, we report on a multicenter randomized trial to study the effects of withdrawal of cyclosporine A (CsA) from a triple immunosuppressive regimen containing CsA, prednisolone (P), and mycophenolate sodium (MPS) early after transplantation.. Patients continued on P/CsA, P/MPS, or P and everolimus (EVL). Before withdrawal, a transplant biopsy was performed ensuring no subclinical rejection was present. Drug levels were closely monitored. The primary outcome was interstitial graft fibrosis and hyalinosis. Secondary outcome was among others graft rejection.. According to trial regulations, an interim analysis was performed after enrollment of half of the intended number of patients (n=113). Mean follow-up was 14+/-5 months from transplantation and 8+/-5 months from conversion. After conversion, acute rejection percentages were 3% in the P/CsA group, 22% in the P/MPS group, and 0% in the P/EVL group (P<0.009).. We conclude that switching immunosuppressive therapy from P/CsA/MPS to therapy with P/CsA or P/EVL at 6 months after renal transplantation is effective in preventing rejection. Double therapy with P/MPS after withdrawal of P/CsA resulted in an increase in severe acute rejection episodes. These results were the immediate reason to halt the P/MPS arm. Serum creatinine values at the latest follow-up (8+/-5 months after conversion and 14+/-5 months after transplantation) in the P/EVL group were lower than in the P/CsA group.

Topics: Adult; Aged; Biomarkers; Biopsy; Creatinine; Cyclosporine; Drug Administration Schedule; Drug Monitoring; Drug Therapy, Combination; Everolimus; Female; Fibrosis; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Middle Aged; Mycophenolic Acid; Netherlands; Prednisolone; Prospective Studies; Sirolimus; Time Factors; Treatment Outcome

Conversion from cyclosporine (CsA) to sirolimus at week 12 after kidney transplantation is associated with a significant improvement in renal function. The aim of this analysis was to investigate the effect of this conversion on interstitial fibrosis (IF), a hallmark of chronic allograft injury, in patients taking part in the CONCEPT trial. This multicenter, prospective, trial included 193 renal recipients randomized at week 12 to switch from CsA to sirolimus or to continue CsA, with mycophenolate mofetil. Routine biopsy with automated, quantified assessment of IF by a program of color segmentation was performed at 1 year in 121 patients. At 1 year, renal function was significantly improved in the conversion group as assessed by estimated GFR (MDRD) and measured GFR. Biopsy results, however, showed no between-group difference in percentage of IF. Calculated GFR at 1 year was significantly associated with the percentage of IF (p = 0.004, R(2)= 0.07). By multivariate analysis diabetic patients had more fibrosis than non-diabetic patients. In conclusion, although kidney transplant patients converted from CsA to sirolimus showed significant improvement in renal function, we found no difference of IF on 1-year biopsies.

Topics: Adult; Biopsy; Chronic Disease; Cyclosporine; Female; Fibrosis; Glomerular Filtration Rate; Graft Rejection; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Sirolimus; Transplantation, Homologous; Treatment Outcome

Chronic allograft nephropathy (CAN) is characterized by deposition of extracellular matrix (ECM) in all renal compartments. PAI-1 seems to play a pivotal role in ECM turnover in CAN. Rapamycin has been shown to improve long-term graft survival in patients with CAN. The aim of the study was to evaluate the molecular mechanisms underlying the beneficial effects of rapamycin on CAN progression at glomerular and tubulointerstitial level.. After a biopsy-proven CAN diagnosis (T0), 18 patients on calcineurin inhibitors (CNI) were randomly assigned in a 2:1 ratio to continue CNI (6 patients) or to receive rapamycin (RAPA; 12 patients). After 2 years of treatment (T24), all patients underwent a second renal biopsy. Morphometric analysis was conducted at T0 and at T24. PAI-1 expression was evaluated at T0 and T24 by immunohistochemistry. We evaluated the effect of rapamycin on PAI-1 gene expression in cultured proximal tubular cells incubated with CD40L or thrombin, two potential CAN pathogenic mediators.. The RAPA group showed a significant regression of glomerulosclerotic lesions and only a 26% increase in interstitial fibrosis after 2 years compared to baseline, whereas the CNI group showed progression of glomerulosclerosis and 112% increase in fibrosis. Glomerular and tubulointerstitial PAI-1 expression was reduced compared to the baseline in the RAPA group, while they were unchanged in the CNI group. In vitro data showed that rapamycin significantly reduced PAI-1 gene expression induced by both CD40L and thrombin in proximal tubular epithelial cells.. These data suggest that rapamycin may modulate ECM deposition in CAN reducing PAI-1 expression.

Topics: Adult; Biopsy; CD40 Ligand; Cell Line; Cells, Cultured; Disease Progression; Fibrosis; Humans; Immunosuppressive Agents; Kidney Diseases; Kidney Glomerulus; Kidney Transplantation; Kidney Tubules; Middle Aged; Plasminogen Activator Inhibitor 1; Sclerosis; Sirolimus; Thrombin; Transplantation, Homologous

Analysis of 1-year surveillance biopsies was carried out for kidney transplant recipients participating in a randomized trial comparing tacrolimus- and sirolimus-based immunosuppression. The analysis was restricted to recipients remaining on assigned regimen throughout the first posttransplant year. Biopsies from 57 of 84 (68%) tacrolimus-randomized recipients were compared with 38 of 81 (47%) of sirolimus-randomized recipients, the discrepancy being explained by a higher rate of sirolimus discontinuation for non-graft-related complications. Included recipients from the two groups did not differ for baseline characteristics or 1-year iothalamate clearance. Histologic analysis indicated no differences between the groups for glomerular, arterial/arteriolar, or acute interstitial abnormalities. There were, however, significantly higher mean scores in the tacrolimus group for interstitial fibrosis and tubular atrophy with a trend toward higher estimated percent interstitial fibrosis. The results indicate that sirolimus may be associated with reduced early graft fibrosis compared with tacrolimus. This potential benefit is offset by lower success rate in maintaining the regimen and was not accompanied by superior glomerular filtration rate at 1 year.

Topics: Adult; Aged; Female; Fibrosis; Glomerular Filtration Rate; Humans; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Middle Aged; Sirolimus; Tacrolimus

Topics: Amiodarone; Animals; Fibrosis; Lung; Mammals; Nicorandil; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Rats; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Topics: Cell Proliferation; Epidural Space; Fibroblasts; Fibrosis; Humans; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

CD4+ cytotoxic T lymphocytes (CTLs) were recently implicated in immune-mediated inflammation and fibrosis progression of Graves' orbitopathy (GO). However, little is known about therapeutic targeting of CD4+ CTLs. Herein, we studied the effect of rapamycin, an approved mTOR complex 1 (mTORC1) inhibitor, in a GO mouse model, in vitro, and in patients with refractory GO. In the adenovirus-induced model, rapamycin significantly decreased the incidence of GO. This was accompanied by the reduction of both CD4+ CTLs and the reduction of orbital inflammation, adipogenesis, and fibrosis. CD4+ CTLs from patients with active GO showed upregulation of the mTOR pathway, while rapamycin decreased their proportions and cytotoxic function. Low-dose rapamycin treatment substantially improved diplopia and the clinical activity score in steroid-refractory patients with GO. Single-cell RNA-Seq revealed that eye motility improvement was closely related to suppression of inflammation and chemotaxis in CD4+ CTLs. In conclusion, rapamycin is a promising treatment for CD4+ CTL-mediated inflammation and fibrosis in GO.

Topics: Animals; CD4-Positive T-Lymphocytes; Fibrosis; Graves Ophthalmopathy; Inflammation; Mice; Sirolimus; T-Lymphocytes, Cytotoxic; TOR Serine-Threonine Kinases

The incidence of heart failure mainly resulting from cardiac hypertrophy and fibrosis increases sharply in post-menopausal women compared with men at the same age, which indicates a cardioprotective role of estrogen. Previous studies in our group have shown that the novel estrogen receptor G Protein Coupled Receptor 30 (GPR30) could attenuate myocardial fibrosis caused by ischemic heart disease. However, the role of GPR30 in myocardial hypertrophy in ovariectomized mice has not been investigated yet. In this study, female mice with bilateral ovariectomy or sham surgery underwent transverse aortic constriction (TAC) surgery. After 8 weeks, mice in the OVX + TAC group exhibited more severe myocardial hypertrophy and fibrosis than mice in the TAC group. G1, the specific agonist of GPR30, could attenuate myocardial hypertrophy and fibrosis of mice in the OVX + TAC group. Furthermore, the expression of LC3II was significantly higher in the OVX + TAC group than in the OVX + TAC + G1 group, which indicates that autophagy might play an important role in this process. An in vitro study showed that G1 alleviated AngiotensionII (AngII)-induced hypertrophy and reduced the autophagy level of H9c2 cells, as revealed by LC3II expression and tandem mRFP-GFP-LC3 fluorescence analysis. Additionally, Western blot results showed that the AKT/mTOR pathway was inhibited in the AngII group, whereas it was restored in the AngII + G1 group. To further verify the mechanism, PI3K inhibitor LY294002 or autophagy activator rapamycin was added in the AngII + G1 group, and the antihypertrophy effect of G1 on H9c2 cells was blocked by LY294002 or rapamycin. In summary, our results demonstrate that G1 can attenuate cardiac hypertrophy and fibrosis and improve the cardiac function of mice in the OVX + TAC group through AKT/mTOR mediated inhibition of autophagy. Thus, this study demonstrates a potential option for the drug treatment of pressure overload-induced cardiac hypertrophy in postmenopausal women.

Topics: Animals; Aortic Valve Stenosis; Autophagy; Cardiomegaly; Female; Fibrosis; Mice; Mice, Inbred C57BL; Myocardium; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Receptors, G-Protein-Coupled; Sirolimus; TOR Serine-Threonine Kinases

Retroperitoneal fibrosis (RPF) is a rare autoimmune disease with fibrous tissue growth and inflammation in retroperitoneum. Its current treatments involve long-term uptake of glucocorticoids (e.g., prednisone) for controlling inflammation; however, side effects are common. We strived for an improved therapy for fibrosis remission while reducing side effects.. We surveyed gene-disease-drug databases and discovered that mammalian target of rapamycin (mTOR) was a key signalling protein in RPF and the mTOR inhibitor compound sirolimus affected many RPF pathways. We designed a therapy combining a gradual reduction of prednisone with a long-term, stable dosage of sirolimus. We then implemented a single-arm clinical trial and assessed the effects in eight RPF patients at 0, 12 and 48 weeks of treatment by measuring fibrous tissue mass by CT, markers of inflammation and kidney functions by lab tests, immune cell profiles by flow cytometry and plasma inflammatory proteins by Olink proteomics.. With the combined therapy, fibrous tissue shrunk about by half, markers of acute inflammation reduced by 70% and most patients with abnormal kidney functions had them restored to normal range. Molecularly, fibrosis-related T cell subsets, including T. Our combined therapy resulted in significant fibrosis remission and an overall regression of the immune system towards healthy states, while achieving good tolerance. We concluded that this new therapy had the potential to replace the steroid monotherapy for treating RPF.

Topics: Fibrosis; Humans; Inflammation; Prednisone; Retroperitoneal Fibrosis; Sirolimus; TOR Serine-Threonine Kinases

Atractylenolide III (ATL-III) is a major active constituent of the natural plant Atractylodes rhizome. Our previous study has shown that ATL-III may alleviate alveolar macrophage apoptosis via the inhibition of the mammalian target of rapamycin (mTOR)-mediated autophagy of human silicosis. Therefore, we aimed to further explore the function of ATL-III in autophagy, apoptosis, and pulmonary fibrosis by establishing the ATL-III-intervened silicosis mouse model in this study. Meanwhile, we sought and then verified potential autophagy-related signaling pathways by matching differentially expressed genes (attained by RNA sequencing) and the autophagy database. In this study, RNA-sequencing results implied that the epidermal growth factor receptor, the crucial upstream activator of mTOR, was seen as a potential autophagy-regulatory molecule in the ATL-III-intervened silicosis mouse model. The finding of this study was that ATL-III might improve the disorder of autophagic degradation via the activation of epidermal growth factor receptor-mTOR signals in the pulmonary tissue of the silicosis mouse model. ATL-III also alleviated cell apoptosis and silicotic fibrosis. Overall, we supposed that ATL-III might be a potential protective medicine, which had a regulatory effect on autophagy, for the intervention of silicotic fibrosis. In the future, the therapeutic drugs for silicosis should be further focused on the development and application of such natural autophagy agents.

Topics: Animals; Autophagy; ErbB Receptors; Fibrosis; Humans; Mice; Silicosis; Sirolimus; TOR Serine-Threonine Kinases

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Topics: Animals; Constriction, Pathologic; Drug-Eluting Stents; Fibrosis; Humans; Laryngostenosis; Mice; Sirolimus; Th17 Cells; TOR Serine-Threonine Kinases; Tracheal Stenosis

Robust activation of mTOR (mammalian target of rapamycin) signaling in diabetes exacerbates myocardial injury following lethal ischemia due to accelerated cardiomyocyte death with cardiac remodeling and inflammatory responses. We examined the effect of rapamycin (RAPA, mTOR inhibitor) on cardiac remodeling and inflammation following myocardial ischemia/reperfusion (I/R) injury in diabetic rabbits. Diabetic rabbits (DM) were subjected to 45 min of ischemia and 10 days of reperfusion by inflating/deflating a previously implanted hydraulic balloon occluder. RAPA (0.25 mg/kg, i.v.) or DMSO (vehicle) was infused 5 min before the onset of reperfusion. Post-I/R left ventricular (LV) function was assessed by echocardiography and fibrosis was evaluated by picrosirius red staining. Treatment with RAPA preserved LV ejection fraction and reduced fibrosis. Immunoblot and real-time PCR revealed that RAPA treatment inhibited several fibrosis markers (TGF-β, Galectin-3, MYH, p-SMAD). Furthermore, immunofluorescence staining revealed the attenuation of post-I/R NLRP3-inflammasome formation with RAPA treatment as shown by reduced aggregation of apoptosis speck-like protein with a caspase recruitment domain and active-form of caspase-1 in cardiomyocytes. In conclusion, our study suggests that acute reperfusion therapy with RAPA may be a viable strategy to preserve cardiac function with the alleviation of adverse post-infarct myocardial remodeling and inflammation in diabetic patients.

Topics: Animals; Diabetes Mellitus; Fibrosis; Inflammation; Ischemia; Mammals; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rabbits; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Remodeling

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the

Topics: Animals; Death, Sudden, Cardiac; Disease Models, Animal; Everolimus; Fibrosis; Humans; Mammals; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Myocarditis; Sarcoidosis; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Vocal fold scar formation due to vocal fold injury (VFI) is a common cause of surgery or trauma-induced voice disorders. Severe scar formation can lead to reduced voice quality or even be life-threatening. Here, we investigated the role of autophagy in VFI, focusing on fibrosis as a consequence of autophagy in inducing VFI. A VFI model was constructed in rats by dissecting the lamina propria tissue from the thyroarytenoid muscle. Real-time PCR and Western blot were used to analyze expressions of autophagy markers, including Beclin1 and Atg7, in VFI. Tgfb1 and Col1a1 were assessed to determine the correlation of fibrosis with VFI progression and autophagy levels. Rat vocal fold fibroblasts were also treated with TGF-β1 or rapamycin, which activates and suppresses autophagy respectively, to explore how autophagy regulates fibrosis in VFI. Initially, we observed that autophagy was downregulated in vocal fold mucosa after VFI in rats. This was particularly evident by the time-dependent downregulation of Beclin1 and Atg7 following VFI. Concurrently, levels of Tgfb1 and Col1a1 also surged, hinting at elevated fibrosis levels. Furthermore, our experiments with TGF-β1 stimulation revealed that it inhibited autophagy in rat vocal fold fibroblasts. Interestingly, when we introduced rapamycin, this effect was reversed. Our data suggest that autophagy is a suppressor of VFI by alleviating fibrosis, making targeting autophagy a potential therapeutic route in VFI.

Topics: Animals; Beclin-1; Cicatrix; Fibrosis; Rats; Sirolimus; Transforming Growth Factor beta1; Vocal Cords

Topics: Actins; Adenosine; AMP-Activated Protein Kinases; Animals; Autophagy; Cadherins; Cathepsin D; Chloroquine; Endometrium; Epithelial-Mesenchymal Transition; Female; Fibronectins; Fibrosis; Iodide Peroxidase; Lipopolysaccharides; Lysosomal-Associated Membrane Protein 1; Mice; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Phosphates; Proto-Oncogene Proteins c-akt; Sequestosome-1 Protein; Serine; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Triiodothyronine

Interstitial fibrosis and tubular atrophy, a major cause of kidney allograft dysfunction, has been linked to premature cellular senescence. The mTOR inhibitor Rapamycin protects from senescence in experimental models, but its antiproliferative properties have raised concern early after transplantation particularly at higher doses. Its effect on senescence has not been studied in kidney transplantation, yet. Rapamycin was applied to a rat kidney transplantation model (3 mg/kg bodyweight loading dose, 1.5 mg/kg bodyweight daily dose) for 7 days. Low Rapamycin trough levels (2.1-6.8 ng/mL) prevented the accumulation of p16INK4a positive cells in tubules, interstitium, and glomerula. Expression of the cytokines MCP-1, IL-1β, and TNF-α, defining the proinflammatory senescence-associated secretory phenotype, was abrogated. Infiltration with monocytes/macrophages and CD8+ T-lymphocytes was reduced and tubular function was preserved by Rapamycin. Inhibition of mTOR was not associated with impaired structural recovery, higher glucose levels, or weight loss. mTOR inhibition with low-dose Rapamycin in the immediate posttransplant period protected from premature cellular senescence without negative effects on structural and functional recovery from preservation/reperfusion damage, glucose homeostasis, and growth in a rat kidney transplantation model. Reduced senescence might maintain the renal regenerative capacity rendering resilience to future injuries resulting in protection from interstitial fibrosis and tubular atrophy.

Topics: Animals; Atrophy; Cellular Senescence; Female; Fibrosis; Glucose; Humans; Kidney; Kidney Diseases; Kidney Transplantation; Male; Rats; Sirolimus; TOR Serine-Threonine Kinases

As a central organ of energy metabolism, the liver is closely related to selenium for its normal function and disease development. However, the underlying roles of mitochondrial energy metabolism and mitophagy in liver fibrosis associated with selenium remain unclear.. 28 rats were randomly divided into normal, low-selenium, nano-selenium supplement-1, and supplement-2 groups for a 12-week intervention. We observed pathological and ultrastructural changes in the liver and analyzed the effects of selenium deficiency and nano-selenium supplementation on liver metabolic activities and crucial proteins expression of mammalian target of the rapamycin (mTOR) signaling pathway.. Selenium deficiency caused liver pathological damage and fibrosis with the occurrence of mitophagy by disrupting normal metabolic activities; meanwhile, the mTOR signaling pathway was up-regulated to enhance mitophagy to clear damaged mitochondria. Furthermore, nano-selenium supplements could reduce the severity of pathological damage and fibrosis in livers and maintain normal energy metabolic activity. With the increased concentrations of nano-selenium supplement, swelling mitochondria and mitophagy gradually decreased, accompanied by the higher expression of mTOR and phosphorylation-modified mTOR proteins and lower expression of unc-51 like autophagy activating kinase 1 (ULK1) and phosphorylation-modified ULK1 proteins.. Mitophagy regulated by the mTOR signaling pathway plays a dual protective role on low-selenium inducing liver fibrosis and nano-selenium supplements preventing liver fibrosis. Mitochondrial energy metabolism plays an important role in these processes as well.

Topics: Animals; Autophagy; Fibrosis; Liver Cirrhosis; Mammals; Mitophagy; Rats; Selenium; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model.. Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg. Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (β-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis.. SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.

Topics: Acetyl-CoA C-Acetyltransferase; Acetyl-CoA Carboxylase; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cardiomegaly; Coenzyme A-Transferases; Endoplasmic Reticulum Stress; Fatty Acids; Fibrosis; Glucose; Hydroxybutyrate Dehydrogenase; Insulins; Keto Acids; Ketones; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Sirolimus; Sodium; Sodium-Glucose Transporter 2; Sodium-Glucose Transporter 2 Inhibitors; TOR Serine-Threonine Kinases

The global burden of chronic kidney disease is increasing, and the majority of these diseases are progressive. Special site-targeted drugs are emerging as alternatives to traditional drugs. Oligonucleotides (ODNs) have been proposed as effective therapeutic tools in specific molecular target therapies for several diseases. We designed ring-type non-coding RNAs (ncRNAs), also called mTOR ODNs to suppress mammalian target rapamycin (mTOR) translation. mTOR signaling is associated with excessive cell proliferation and fibrogenesis. In this study, we examined the effects of mTOR suppression on chronic renal injury. To explore the regulation of fibrosis and inflammation in unilateral ureteral obstruction (UUO)-induced injury, we injected synthesized ODNs via the tail vein of mice. The expression of inflammatory-related markers (interleukin-1β, tumor necrosis factor-α), and that of fibrosis (α-smooth muscle actin, fibronectin), was decreased by synthetic ODNs. Additionally, ODN administration inhibited the expression of autophagy-related markers, microtubule-associated protein light chain 3, Beclin1, and autophagy-related gene 5-12. We confirmed that ring-type ODNs inhibited fibrosis, inflammation, and autophagy in a UUO mouse model. These results suggest that mTOR may be involved in the regulation of autophagy and fibrosis and that regulating mTOR signaling may be a therapeutic strategy against chronic renal injury.

Topics: Actins; Animals; Autophagy; Beclin-1; Disease Models, Animal; Fibronectins; Fibrosis; Inflammation; Interleukin-1beta; Kidney; Mammals; Mice; Microtubule-Associated Proteins; Oligonucleotides; Renal Insufficiency, Chronic; RNA, Untranslated; Sirolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Ureteral Obstruction

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-β1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-β1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-β1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-β1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-β1 induction, TGF-β1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.

Topics: Actins; Animals; Blotting, Western; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Conjunctiva; Fibroblasts; Fibronectins; Fibrosis; Humans; Immunohistochemistry; Male; Naphthyridines; Rabbits; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Trabeculectomy; Transforming Growth Factor beta1

Acrylamide (ACRL) was demonstrated to induce hepatotoxicity and programmed cell death (PCD). Rapamycin (RAPA)-induced autophagy had been reported to limit the progression of hepatocellular injury in experimental models. This research was designed to study two death pathways involved in ACRL-induced hepatotoxicity and the modulating effect of RAPA on the resulting hepatic injury. Thirty-six adult male rats were divided into three groups: control group, ACRL-treated group (20 mg kg/day), and the last group co-treated with ACRL plus RAPA (0.5 mg kg/day). Drugs were administered for 21 days via oral gavage. Blood samples were collected to assess alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Livers were dissected; parts were used for detection of superoxide dismutase (SOD) and malondialdehyde (MDA) tissue levels. Other parts were processed for hematoxylin and eosin, Masson's trichrome staining, immunostaining for microtubule-associated proteins 1A/1B light chain 3B (LC3), ubiquitin-binding protein (p62), caspase-3, and receptor-interacting protein kinase 1 (RIPK1). ACRL induced a significant elevation in ALT, AST, MDA levels, and reduction in the SOD level. ACRL also induced hepatocellular injury, fibrosis, and defective autophagy indicated by elevation of LC3 and p62 and increased p62/LC3 ratio. Moreover, it increased the apoptotic (caspase-3) and necroptotic (RIPK1) markers expression. RAPA significantly reduced liver enzymes, oxidative stress, fibrosis, and improved liver histology. Moreover, RAPA decreased p62/LC3 ratio indicated enhanced autophagy, and significantly reduced caspase-3 and RIPK1 expression. In conclusion, RAPA maintained autophagic activity which may save the hepatocytes from PCD and enhance cell viability.

Topics: Acrylamide; Animals; Apoptosis; Autophagy; Caspase 3; Chemical and Drug Induced Liver Injury; Fibrosis; Male; Necroptosis; Rats; Sirolimus; Superoxide Dismutase

To investigate the effect and significance of mammalian target of rapamycin (mTOR) inhibitors on the expression of α-SMA in lung injury induced by high volume fraction of inspired oxygen (hyperoxygen) in SD rat pups.. Seventy-two Sprague-Dawley rat pups (age: 3 weeks) were randomly divided into air + saline, hyperoxia + saline, hyperoxia + OSI-027, and hyperoxia + rapamycin groups. Animal models were constructed (n = 18). Hyperoxia was induced by continuous administration of 90% oxygen. Normal saline, OSI-027, and rapamycin are administered by intraperitoneal injection on 1d, 3d, 6d, 8d, 10d, 13d of the observation period, respectively. Following assessments were made on the 3rd, 7th, and 14th day of modeling: pathological changes in lung tissues, lung injury score, Western Blot to assess the distribution and expressions of mTOR, pS6K1, and α-SMA protein in lung tissues.. In terms of time factors, the protein expressions of mTOR, pS6K1, and α-SMA increased with time. Except for the air group, the lung injury scores of the other groups increased with time, In terms of grouping factors, lung injury score in the air group was significantly lower than that in the other groups. In the hyperoxia group, the protein expressions of mTOR, PS6K1, and α-SMA were significantly higher than those in the other groups. The lung injury score in the hyperoxia group was significantly higher than that in the other groups. The lung injury score in the hyperoxia OSI group was significantly lower than that in the hyperoxia rapamycin group.. In hyperoxia lung injury, inhibiting the activation of mTOR signaling pathway can effectively reduce the expression of α-SMA; however, only mTORC1/2 dual inhibitor OSI-027 exhibited an anti-proliferative effect, and alleviated hyperoxia-induced lung injury and fibrosis in SD rat pups.

Topics: Actins; Animals; Female; Fibrosis; Hyperoxia; Imidazoles; Lung; Lung Injury; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Triazines

In this study, we investigated the effect of mTOR inhibitor (mTORi) drug-eluting biodegradable stent (DE stent), a putative restenosis-inhibiting device for coronary artery, on thermal-injury-related ureteral stricture in rabbits. In vitro evaluation confirmed the dose-dependent effect of mTORi, i.e., rapamycin, on fibrotic markers in ureteral component cell lines. Upper ureteral fibrosis was induced by ureteral thermal injury in open surgery, which was followed by insertion of biodegradable stents, with or without rapamycin drug-eluting. Immunohistochemistry and Western blotting were performed 4 weeks after the operation to determine gross anatomy changes, collagen deposition, expression of epithelial-mesenchymal transition markers, including Smad, α-SMA, and SNAI 1. Ureteral thermal injury resulted in severe ipsilateral hydronephrosis. The levels of type III collagen, Smad, α-SMA, and SNAI 1 were increased 28 days after ureteral thermal injury. Treatment with mTORi-eluting biodegradable stents significantly attenuated thermal injury-induced urinary tract obstruction and reduced the level of fibrosis proteins, i.e., type III collagen. TGF-β and EMT signaling pathway markers, Smad and SNAI 1, were significantly modified in DE stent-treated thermal-injury-related ureteral stricture rabbits. These results suggested that intra-ureteral administration of rapamycin by DE stent provides modification of fibrosis signaling pathway, and inhibiting mTOR may result in fibrotic process change.

Topics: Absorbable Implants; Animals; Drug-Eluting Stents; Fibrosis; Rabbits; Sirolimus; TOR Serine-Threonine Kinases; Ureteral Obstruction

We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

Topics: Actins; Blood Proteins; Cell Movement; Cell Proliferation; Collagen Type I; Fibronectins; Fibrosis; Humans; Middle Aged; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; rho-Associated Kinases; Sirolimus; TOR Serine-Threonine Kinases; Trabecular Meshwork; Transforming Growth Factor beta2; Up-Regulation

Wound healing is a multicellular process that involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, and endothelial cells. This process is also regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, proliferation, and differentiation. A recent study showed that mTORC1 activation in epithelial cells dramatically enhanced epithelial cell proliferation, migration, and cutaneous wound healing; however, the roles of mTORC1 in fibroblasts during wound healing remain unknown. Here, we generated genetically mutated mice with activated mTORC1 in fibroblasts by conditionally deleting the mTORC1 inhibitor, TSC1. Activation of mTORC1 in fibroblasts significantly increased fibroblastic cell proliferation and contractile α-smooth muscle actin expression, thus promoting wound closure. Elevated mTORC1 activity also adversely induced excessive collagen production, leading to excessive scaring and fibrosis. Importantly, both accelerated wound healing and fibrotic phenotypes were largely reversed by the mTORC1 inhibitor, rapamycin. These observations were also replicated in primary human dermal fibroblasts. These results collectively demonstrated that mTORC1 activity in skin fibroblasts was a critical orchestrator in cutaneous wound healing and scarring.

Topics: Actins; Animals; Apoptosis; Cell Proliferation; Cicatrix; Collagen; Fibroblasts; Fibrosis; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Primary Cell Culture; Protein Kinase Inhibitors; Sirolimus; Tuberous Sclerosis Complex 1 Protein; Wound Healing

Autophagy plays a crucial role in the pathological process of cardiovascular diseases. However, little is known about the pathological mechanism underlying autophagy regulation in dilated cardiomyopathy (DCM).. We explored whether up-regulating autophagy could improve cardiac function in mice with experimental DCM through the mTOR-4EBP1 pathway. Animal model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Both up- or down-regulation of autophagy were studied by administration of rapamycin or 3-MA in parallel. Morphology, Western blotting, and echocardiography were applied to confirm the pathological mechanisms.. Autophagy was activated and autophagosomes were significantly increased in the rapamycin group. The collagen volume fraction (CVF) was decreased in the rapamycin group compared with the DCM group (9.21 ± 0.82% vs 14.38 ± 1.24%, P < 0.01). The expression of p-mTOR and p-4EBP1 were significantly decreased in rapamycin-induced autophagy activation, while the levels were increased by down-regulating autophagy with 3-MA. In the rapamycin group, the LVEF and FS were significantly increased compared with the DCM group (54.12 ± 6.48% vs 45.29 ± 6.68%, P < 0.01; 26.89 ± 4.04% vs 22.17 ± 2.82%, P < 0.05). As the inhibitor of autophagy, 3-MA aggravated the progress of maladaptive cardiac remodeling and declined cardiac function in DCM mice.. The study indicated a possible mechanism for improving cardiac function in mice with experimental DCM by up-regulating autophagy via the mTOR-4EBP1 pathway, which could be a promising therapeutic strategy for DCM.

Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagosomes; Autophagy; Cardiomyopathy, Dilated; Cell Cycle Proteins; Disease Models, Animal; Fibrosis; Male; Mice, Inbred BALB C; Myocytes, Cardiac; Recovery of Function; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ventricular Function, Left; Ventricular Remodeling

Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown.. Here, we developed the first mouse model of severe MR. Valvular damage was achieved by severing the mitral valve leaflets and chords with iridectomy scissors, and MR was confirmed by echocardiography. Serial echocardiography was performed to follow up LV morphology and systolic function. Analysis of cardiac tissues was subsequently performed to evaluate valve deformation, cardiomyocyte morphology, LV fibrosis, and cell death. Finally, dysregulated pathways were assessed by RNA-sequencing analysis and immunofluorescence.. In the ensuing 15 weeks after the induction of MR, gradual LV dilatation and dysfunction occurred, resulting in severe systolic dysfunction. Further analysis revealed that severe MR resulted in a marked increase in cardiac mass and increased cardiomyocyte length but not width, with electron microscopic evidence of sarcomere disarray and the development of sarcomere disruption. From a mechanistic standpoint, severe MR resulted in activation of multiple components of both the mammalian target of rapamycin and calcineurin pathways. Inhibition of mammalian target of rapamycin signaling preserved sarcomeric structure and prevented LV remodeling and systolic dysfunction. Immunohistochemical analysis uncovered a differential pattern of expression of the cell polarity regulator Crb2 (crumbs homolog 2) along the longitudinal axis of cardiomyocytes and close to the intercalated disks in the MR hearts. Electron microscopy images demonstrated a significant increase in polysome localization in close proximity to the intercalated disks and some areas along the longitudinal axis in the MR hearts.. These results indicate that LV dysfunction in response to severe MR is a form of maladaptive eccentric cardiomyocyte hypertrophy and outline the link between cell polarity regulation and spatial localization protein synthesis as a pathway for directional cardiomyocyte growth.

Topics: Animals; Cell Adhesion Molecules; Cell Shape; Cell Size; Disease Models, Animal; Echocardiography; Fibrosis; Gene Expression Profiling; Hypertrophy; Infusion Pumps, Implantable; Magnetic Resonance Imaging; Male; Mice; Mitral Valve; Mitral Valve Insufficiency; Myocytes, Cardiac; Polyribosomes; RNA, Messenger; Sirolimus; Systole; TOR Serine-Threonine Kinases; Ventricular Dysfunction, Left

Conjunctival fibrosis is a process of extracellular matrix accumulation and the appearance of myofibroblasts in subconjunctival fibroblasts induced by injury or inflammation, which can significantly reduce the filtration efficiency of glaucoma filtration surgery. In this study, autophagy was confirmed to be involved in regulating the fibrosis of human subconjunctival fibroblasts (hSCFs) induced by TGF-β1. Following the addition of rapamycin, we detected that autophagy activation could reduce the increased expression level of αSMA and the accumulation of extracellular matrix component proteins namely fibronectin and type I collagen induced by TGF-β1 via the inhibition of SMAD2 phosphorylation. Following the addition of HCQ, the inhibition of autophagy aggravated TGF-β1-induced fibrosis of hSCFs. We further verified that trehalose, a safe clinical drug, could alleviate TGF-β1-induced fibrosis of hSCFs by activating autophagy and that these effects could be blocked by autophagy inhibition. In summary, autophagy was shown to be involved in the regulation of TGF-β1-induced fibrosis of hSCFs, which provided a novel perspective for exploring the progression of this lesion. More importantly, the protective effects of trehalose on TGF-β1-induced fibrosis of hSCFs were mediated by the activation of autophagy and could provide possible new approaches for the clinical treatment of conjunctival fibrosis.

Topics: Autophagy; Cells, Cultured; Collagen Type I; Conjunctiva; Disease Progression; Extracellular Matrix; Fibroblasts; Fibronectins; Fibrosis; Humans; Inflammation; Myofibroblasts; Phosphorylation; Sirolimus; Smad2 Protein; Transforming Growth Factor beta1; Trehalose

Endoplasmic reticulum stress (ERS) is involved in a variety of diseases. Recently, it was found that ERS induces not only apoptosis but also autophagy. Previous studies showed that inhibition of autophagy alleviates cell injury. The purpose of our study was to investigate the involvement of the R-like ER kinase (PERK) in ERS-induced autophagy in H9c2 cardiomyoblasts. To address this aim, therefore, H9c2 cells were treated with PERK agonist and inhibitor after establishment of rapamycin-induced ERS models in H9c2 cardiomyoblasts. Transmission electron microscopy and immunofluorescence staining were used to detect degrees of ERS-induced autophagy, apoptosis and myocardial fibrosis. Western blotting was employed to detect the levels of total and phosphorylated PERK, light chain 3 (LC3), P62, Caspase3, Bcl2 and Bax. Immunofluorescence staining was used to assess α-SMA density. TGF-β induced H9c2 cardiomyoblasts time-dependently upregulated col I, col III, FN, and LC3 expressions, PERK phosphorylation and α-SMA density, and downregulated P62 level compared with control cells. Treatment with PERK agonist and inhibitor respectively increased and decreased LC3 expression, conversely in P62 level, which is consistent with effect of ERS agonists and inhibitors. And a PERK inhibitor upregulated the expressions of Caspase3 and Bax, and downregulated Bcl2 level, which developed H9c2 cardiomyoblasts. Moreover, siRNA-mediated knockdown of PERK reduced ERS mediated autophagy activity and increased cells apoptosis. On the other hand, elevated autophagy activity could downregulated PERK level. Our finding showed that PERK activity mediates upregulation of ERS-induced autophagy and regulation of cardiomyocyte apoptosis in H9c2 cardiomyoblasts.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Butadienes; Cell Line; eIF-2 Kinase; Endoplasmic Reticulum Stress; Fibrosis; Humans; Myocytes, Cardiac; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Rats; Signal Transduction; Sirolimus

Topics: Aging; Animals; Antioxidants; Autophagy; Beclin-1; beta-Galactosidase; Cell Line; Cyclin-Dependent Kinase Inhibitor p16; Fibrosis; Glutathione; Heart; Indoles; Lipid Peroxidation; Mice; Myoblasts, Cardiac; Myocardium; Oxidative Stress; Oximes; Proto-Oncogene Proteins c-myc; Rats; Reactive Oxygen Species; Sirolimus; Superoxide Dismutase; Tumor Suppressor Protein p53

We recently observed reduced autophagy in Crohn's disease patients and an anti-inflammatory effect of autophagy stimulation in murine colitis, but both anti- and pro-fibrotic effects are associated with autophagy stimulation in different tissues, and fibrosis is a frequent complication of Crohn's disease. Thus, we analyzed the effects of pharmacological modulation of autophagy in a murine model of intestinal fibrosis and detected that autophagy inhibition aggravates, while autophagy stimulation prevents, fibrosis. These effects are associated with changes in inflammation and in collagen degradation in primary fibroblasts. Thus, pharmacological stimulation of autophagy may be useful against intestinal fibrosis.

Topics: Animals; Autophagy; Collagen; Crohn Disease; Disease Models, Animal; Fibroblasts; Fibrosis; Immunosuppressive Agents; Inflammation; Intestines; Mice; Mice, Inbred C57BL; Sirolimus

Topics: Administration, Ophthalmic; Animals; Burns, Chemical; Cell Proliferation; Cells, Cultured; Cornea; Corneal Injuries; Corneal Neovascularization; Corneal Opacity; Disease Models, Animal; Drug Carriers; Drug Compounding; Eye Burns; Fibroblasts; Fibrosis; Humans; Lipids; Male; Mice, Inbred BALB C; Nanomedicine; Nanoparticles; Sirolimus; Sodium Hydroxide; Wound Healing

Huangkui capsule (HKC), an anti-inflammatory Chinese modern patent medicine, has been now widely applied to the clinical therapy of diabetic nephropathy (DN). However, the overall therapeutic mechanisms in vivo are still unclear. Renal tubular epithelial-to-mesenchymal transition (EMT) is one of the major pathogenesis of renal interstitial fibrosis in DN. Recently, the physiological roles of NLRP3 inflammasome activation and toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling are closely linked to EMT. But, it remains elusive whether HKC regulates renal tubular EMT in vivo through targeting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys.. This study thereby aimed to clarify the therapeutic effects of HKC on renal tubular EMT in DN and its underlying mechanisms in vivo, compared to rapamycin (RAP).. Thirty-two rats were randomly divided into 4 groups: the Sham group, the Vehicle group, the HKC group and the RAP group. The early DN rat models were induced by unilateral nephrectomy combined with intraperitoneal injection of streptozotocin, and administered with HKC suspension or RAP suspension or vehicle after modeling for 4 weeks. Changes in the incipient renal lesions-related parameters in urine and blood were analyzed, respectively. Renal interstitial tissues were isolated for histomorphometry, immunohistochemistry and Western blotting at sacrifice.. For the early DN rat models, HKC at the suitable dose of 2 g/kg/day ameliorated the general condition and biochemical parameters partially including kidney weight (KW), urinary albumin (UAlb), serum creatinine (Scr) and serum albumin (Alb), attenuated renal tubular EMT significantly and inhibited the activation of NLRP3 inflammasome in the kidneys obviously, which was superior to RAP generally. In addition to these, HKC also suppressed TLR4/NF-κB signaling in the kidneys of the DN model rats accurately, which was different from RAP specifically.. The results of this study further indicated that HKC, different from RAP, can alleviate renal tubular EMT in the DN model rats, likely by inhibiting NLRP3 inflammasome activation and TLR4/NF-κB signaling in the kidneys. Our findings thus provide the more accurate information in vivo about a clinical value of HKC, a traditional anti-inflammatory phytomedicine, in the treatment of the early DN patients.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Diabetic Nephropathies; Disease Models, Animal; Drugs, Chinese Herbal; Epithelial-Mesenchymal Transition; Fibrosis; Inflammasomes; Kidney; Male; Nephrectomy; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Toll-Like Receptor 4

Fibrosis and dysphagic stricture of the esophagus is a major unaddressed problem often accompanying endoscopic removal of esophageal cancers and precancerous lesions. While weekly injections of antiproliferative agents show potential for improved healing, repeated injections are unlikely clinically and may alternatively be replaced by creating an esophageal drug delivery system. Affinity-based polymers have previously shown success for continuous delivery of small molecules for weeks to months. Herein, we explored the potential of an affinity-based microparticle to provide long-term release of an antiproliferative drug, sirolimus. In molecular docking simulations and surface plasmon resonance experiments, sirolimus was found to have suitable affinity for beta-cyclodextrin, while dextran, as a low affinity control, was validated. Polymerized beta-cyclodextrin microparticles exhibited 30 consecutive days of delivery of sirolimus during in vitro release studies. In total, the polymerized beta-cyclodextrin microparticles released 36.9 mg of sirolimus per milligram of polymer after one month of incubation in vitro. Taking daily drug release aliquots and applying them to PT-K75 porcine mucosal fibroblasts, we observed that cyclodextrin microparticle delivery preserved bioactivity of sirolimus inhibiting proliferation by 27-67% and migration of fibroblasts by 28-100% of buffer treated controls in vitro. Testing for esophageal injection site losses, no significant loss was incurred under simulated saliva flow for 10 min, and 16.7% of fluorescently labeled polymerized cyclodextrin microparticle signal was retained at 28 days after submucosal injection in esophageal tissue ex vivo versus only 4% of the initial amount remaining for free dye molecules injected alone. By combining affinity-based drug delivery for continuous long-term release with a microparticle platform that is injectable yet remains localized in tissue interstitium, this combination platform demonstrates promise for preventing esophageal fibrosis and stricture.

Topics: Animals; Cell Movement; Cell Proliferation; Cellulose; Cyclodextrins; Drug Delivery Systems; Esophageal Diseases; Fibroblasts; Fibrosis; Immunosuppressive Agents; Sirolimus; Swine; Wound Healing

Cardiac pressure overload (for example due to aortic stenosis) induces irreversible myocardial dysfunction, cardiomyocyte hypertrophy and interstitial fibrosis in patients. In contrast to adult, neonatal mice can efficiently regenerate the heart after injury in the first week after birth. To decipher whether insufficient cardiac regeneration contributes to the progression of pressure overload dependent disease, we established a transverse aortic constriction protocol in neonatal mice (nTAC). nTAC in the non-regenerative stage (at postnatal day P7) induced cardiac dysfunction, myocardial fibrosis and cardiomyocyte hypertrophy. In contrast, nTAC in the regenerative stage (at P1) largely prevented these maladaptive responses and was in particular associated with enhanced myocardial angiogenesis and increased cardiomyocyte proliferation, which both supported adaptation during nTAC. A comparative transcriptomic analysis between hearts after regenerative versus non-regenerative nTAC suggested the transcription factor GATA4 as master regulator of the regenerative gene-program. Indeed, cardiomyocyte specific deletion of GATA4 converted the regenerative nTAC into a non-regenerative, maladaptive response. Our new nTAC model can be used to identify mediators of adaptation during pressure overload and to discover novel potential therapeutic strategies.

Topics: Angiogenesis Inducing Agents; Animals; Cell Proliferation; Cytokinesis; Disease Models, Animal; Female; Fibrosis; GATA4 Transcription Factor; Gene Expression; Heart; Heart Failure; Male; Mice; Mice, Inbred ICR; Mice, Knockout; Myocytes, Cardiac; Pressure; Rats; Sirolimus; Transcriptome

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser

Topics: Animals; Chemokine CCL2; Drug Therapy, Combination; Female; Fibrosis; Humans; Kidney; Lupus Nephritis; Mice; Mycophenolic Acid; Phosphorylation; Rabbits; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Cirrhosis is often associated with portal hypertension and portal-systemic collateral vessels formation attributed to angiogenesis, which leads to severe complications as hepatic encephalopathy. Sirolimus has anti-fibrosis and anti-angiogenesis effects, but whether it influences the severity of portal-systemic collaterals and hepatic encephalopathy is unknown. This study was thus designed to address this issue in rats with common bile duct ligation-induced liver cirrhosis. Sham-operated rats were surgical controls. Rats were intraperitoneally administered with 0.5 and 2 mg/kg/day sirolimus or vehicle for 2 weeks. Four weeks post operations, motor activities, body weight, biochemistry and hemodynamic data were measured. The liver was dissected for histopathology, immunohistochemical stains and protein analysis. On the parallel cirrhotic groups, the portal-systemic shunting was determined. The results showed that the body weight gain was significantly lower in sirolimus-treated rats. Sirolimus reduced portal pressure and plasma levels of alanine aminotransferase, aspartate aminotransferase and ammonia, and attenuated hepatic inflammation and fibrosis in cirrhotic rats. In addition, the hepatic phosphorylated mammalian target of rapamycin (mTOR) and P70S6K protein expressions were significantly downregulated and endothelial nitric oxide synthase (eNOS) expression upregulated by sirolimus. Sirolimus did not influence portal-systemic shunting and motor activities of cirrhotic rats. In conclusion, sirolimus significantly improved hepatic inflammation and fibrosis accompanied by portal pressure reduction in cirrhotic rats, in which down-regulated mTOR/P70S6K and up-regulated eNOS expressions might play a role. However, sirolimus did not significantly change the severity of portal-systemic collaterals and motor activities, suggesting that the multifactorial pathogenesis of hepatic encephalopathy could not be fully overcome by sirolimus.

Topics: Animals; Body Weight; Common Bile Duct; Fibrosis; Hemodynamics; Hepatic Encephalopathy; Ligation; Liver Cirrhosis; Male; Motor Activity; Rats; Rats, Sprague-Dawley; Sirolimus

Renal ischemia/reperfusion (IR) can induce acute kidney injury (AKI), which often progresses to chronic kidney disease (CKD). Dexmedetomidine (Dex), a highly selective α2 adrenergic receptor (α2-AR) agonist, protects against acute renal IR-induced injury. However, the effects of Dex on the transition of AKI to CKD remain unclear. Therefore, we investigated the mechanisms of Dex on renal fibrosis.. Adult male C57BL/6 mice were pretreated with Dex, a specific α2A-adrenergic receptor (AR) blocker (BRL-44408), or a cell senescence inhibitor (rapamycin) in a surgical bilateral renal IR model. The diagnoses of AKI and chronic renal fibrosis were performed by histopathological staining and western blotting. Histopathological changes, cell senescence, tubular fibrotic markers, and the expression of inflammatory factors were studied.. Pretreatment with Dex alleviated renal IR-induced AKI and chronic tubulointerstitial fibrosis in later stages. Similar to the effects of rapamycin, pretreatment with Dex also decreased the number of senescent tubular cells and weakened the protein expression of senescence-associated markers such as p53, p21, and p16. Furthermore, the expression of inflammatory markers was also decreased in Dex-treated IR mice; and these protective effects of Dex could be abolished by treatment with the specific α2A-AR blocker, BRL-44408.. The administration of a single dose of Dex protects against AKI and CKD. Dex inhibits tubular cell senescence and inflammation as well as improves renal fibrosis to moderate the AKI-to-CKD transition. The renal protective potential of Dex may provide a novel treatment strategy for high-risk renal injury patients.

Topics: Acute Kidney Injury; Adrenergic alpha-2 Receptor Agonists; Animals; Cellular Senescence; Dexmedetomidine; Fibrosis; Imidazoles; Inflammation; Isoindoles; Kidney; Male; Mice; Mice, Inbred C57BL; Receptors, Adrenergic, alpha-2; Reperfusion Injury; Signal Transduction; Sirolimus

Regulation of microRNA gene expression by DNA methylation may represent a key mechanism to drive cardiac fibrosis progression. Cardiac fibroblast autophagy is the primary source of cardiac fibrosis, but the mechanisms underlying this process are incompletely understood. Here we found that DNMT3A suppression of the microRNA-200b (miR-200b) through pathway leads to cardiac fibroblast autophagy in cardiac fibrosis.. To understand the impact of DNMT3A on miR-200b at cardiac fibrosis, the rat cardiac fibrosis model was established via the abdominal aortic coarctation. Cardiac fibroblasts (CFs) were harvested from SD neonate rats and cultured. The expression of DNMT3A, miR-200b, collagen I was measured by western blotting, immunohistochemistry and qRT-PCR. Gain- or loss-of-function approaches were used to manipulate DNMT3A and miR-200b.. DNMT3A level was upregulated and negatively correlated with miR-200b expression in fibrosis tissues and cardiac fibroblast. We found that autophagy was activated by miR-200b inhibitor and inactivated by miR-200b mimic in the rat cardiac fibroblast. Knockdown of DNMT3A notably increased the expression of miR-200b.. Taken together, these findings indicate that DNMT3A regulation of miR-200b controls cardiac fibroblast autophagy during cardiac fibrosis and provide a basis for the development of therapies for cardiac fibrosis.

Topics: Animals; Animals, Newborn; Autophagy; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; Fibroblasts; Fibrosis; Male; MicroRNAs; Microtubule-Associated Proteins; Myocardium; Rats, Sprague-Dawley; Sirolimus

The aim of this study was to investigate whether the underlying plaque type affects the neointimal coverage after drug-eluting stent implantation.. A total of 1793 struts in 22 zotarolimus-eluting stents were assessed using optical coherence tomography imaging within 3 months of implantation. Neointimal coverage was evaluated within 5 mm from each stent edge on cross-sectional optical coherence tomography images at every 1-mm interval. The percentage of struts covered by neointima was compared among the normal segment group, the fibrous plaque group, and the lipid plaque group on the basis of the underlying plaque type.. The percentage of covered strut was significantly lower in the normal segment group than in the fibrous plaque group (35.9±30.2 vs. 57.1±31.0%, P<0.05) and the lipid plaque group (vs. 64.7±23.5%, P<0.01). The neointima was significantly thinner in the normal segment group than in the lipid plaque group (19.0±22.3 vs. 32.0±18.8 μm, P<0.01). The percentage of struts on the normal segment was significantly higher in cross-sections with a ratio of uncovered to total struts per section more than 0.3 than in cross-sections with a ratio up to 0.3 (32.4±31.7 vs. 19.5±33.8%, P<0.01).. Struts on the normal segment were less covered and had thinner neointima than struts on the lipid plaque at the stent edge within 3 months after zotarolimus-eluting stent implantation. Caution should be exercised when implanting longer drug-eluting stents to achieve uniform strut coverage in the early phase.

Topics: Aged; Cardiovascular Agents; Coronary Angiography; Coronary Artery Disease; Coronary Vessels; Drug-Eluting Stents; Female; Fibrosis; Humans; Male; Middle Aged; Neointima; Percutaneous Coronary Intervention; Plaque, Atherosclerotic; Prosthesis Design; Retrospective Studies; Sirolimus; Time Factors; Tomography, Optical Coherence; Treatment Outcome

TGFβ1 and mTOR are considered to play important roles in fibrotic diseases. Rapamycin has been reported to inhibit urethral stricture formation in a rabbit model of urethral fibrosis.. To evaluate if dual mTOR inhibitor has a superior efficacy compared with rapamycin on inhibiting cell proliferation and collagen expression in human urethral scar fibroblasts (HUSFs).. We established HUSF cultures from fresh surgical specimen. The HUSFs were identified with typical fibroblast markers using immunofluorescence. Then we examined the effect of TGFβ1 on HUSFs using Cell Counting Kit-8 and Western blot. The inhibiting effects of OSI-027 (a dual mTOR inhibitor) on cell proliferation and collagen expression in TGFβ1-induced HUSFs were compared with rapamycin using Cell Counting Kit-8, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).. HUSFs were stained positive for vimentin, collagen I, and collagen III. TGFβ1 had no effect on cell proliferation but increased collagen I and collagen III expressions in HUSFs. OSI-027 was more effective inhibiting cell proliferation and collagen expression compared with rapamycin in TGFβ1-induced HUSFs. OSI-027 played a more important role in inhibiting TGFβ1-induced mTOR pathway and phosphorylation of Smad2 compared with rapamycin in HUSFs.. OSI-027 can inhibit the pro-fibrotic effects of TGFβ1 significantly compared with rapamycin in HUSFs. These findings may provide a new therapy in the adjunctive treatment of urethral stricture disease.

Topics: Cell Proliferation; Cells, Cultured; Cicatrix; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; Dose-Response Relationship, Drug; Fibroblasts; Fibrosis; Humans; Imidazoles; Phosphorylation; Primary Cell Culture; Protein Kinase Inhibitors; Signal Transduction; Sirolimus; Smad2 Protein; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Triazines; Urethra; Vimentin

Attributing to their antiproliferative effect, both rapamycin and peroxisome proliferator-activated receptor-γ (PPARγ) can halt the progression of autosomal dominant polycystic kidney disease (ADPKD). Whether combined use could enhance this effect is unknown. The present study used rapamycin and the PPARγ agonist rosiglitazone concomitantly to observe their combined effects on the proliferation of ADPKD cyst-lining epithelial cells and the progression of ADPKD in Han:SPRD rats. Concomitant use of the two drugs inhibited the proliferation of WT9-12 cells significantly through a superimposition effect. Rosiglitazone inhibited the phosphorylation of mammalian target of rapamycin p70S6K. Concomitant use of rosiglitazone and rapamycin further downregulated the p-p70S6K level. Rosiglitazone also inhibited the phosphorylation of Akt and antagonized the activation of Akt induced by rapamycin. Concomitant use of rosiglitazone and rapamycin significantly retarded the deterioration of renal function, decreased cyst cell proliferation and interstitial fibrosis in Han:SPRD rats. Rapamycin significantly increased cholesterol levels in the blood, whereas rosiglitazone mitigated rapamycin-induced hyperlipidemia. These results indicate that the effects of concomitant use of rosiglitazone and rapamycin in inhibiting the proliferation of WT9-12 cells and delaying the progression of ADPKD in Han:SPRD rats are stronger than those of either drug alone. The present study may provide a new strategy for the long-term treatment of ADPKD.

Topics: Animals; Cell Line; Cell Proliferation; Disease Models, Animal; Disease Progression; Drug Therapy, Combination; Epithelial Cells; Fibrosis; G1 Phase Cell Cycle Checkpoints; Humans; Kidney; Lipids; Male; Phosphorylation; Polycystic Kidney, Autosomal Dominant; PPAR gamma; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Renal Agents; Ribosomal Protein S6 Kinases, 70-kDa; Rosiglitazone; Signal Transduction; Sirolimus

Islet microencapsulation is an attractive strategy for the minimization or avoidance of life-long immunosuppression after transplantation. However, the clinical implementation of this technique is currently limited by incomplete biocompatibility. Thus, the aim of the present study was to demonstrate the improved biocompatibility of rapamycin-containing polyethylene glycol (Rapa-PEG)-coating on alginate microcapsules containing xenogeneic islets. The Rapa-PEG-coating on the alginate layer was observed using scanning electron microscopy (SEM) and the molecular cut-off weight of the microcapsules was approximately 70 kDa. The viabilities of the alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets were lower than the viability of the naked islets just after encapsulation, but these the differences diminished over time in culture dishes. Rapa-PEG-coating on the alginate capsules effectively decreased the proliferation of macrophage cells compared to the non-coating and alginate coating of xenogeneic pancreas tissues. Glucose-stimulated insulin secretion did not significantly differ among the groups prior to transplantation. The random blood glucose levels of diabetic mice significantly improved following the transplantation of alginate-encapsulated and Rapa-PEG-coated alginate-encapsulated islets, but there were no significant differences between these two groups. However, there was a significant decrease in the number of microcapsules with fibrotic cell infiltration in the Rapa-PEG-coated alginate microcapsule group compared to the alginate microcapsule group. In conclusion, Rapa-PEG-coating might be an effective technique with which to improve the biocompatibility of microcapsules containing xenogeneic islets. Copyright © 2015 John Wiley & Sons, Ltd.

Topics: Alginates; Animals; Biocompatible Materials; Blood Glucose; Capsules; Cell Proliferation; Cell Survival; Coated Materials, Biocompatible; Diabetes Mellitus, Experimental; Fibrosis; Glucose; Glucuronic Acid; Hexuronic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Mice; Mice, Inbred BALB C; Polyethylene Glycols; RAW 264.7 Cells; Sirolimus; Sus scrofa; Transplantation, Heterologous

Topics: Animals; Carrier Proteins; Dilatation; Fibrosis; GATA4 Transcription Factor; Heart Ventricles; Intracellular Signaling Peptides and Proteins; Lithium Compounds; Male; MAP Kinase Signaling System; Myocardial Contraction; Myocardial Infarction; NFATC Transcription Factors; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Rotenone; Sirolimus; Stimulation, Chemical; Time Factors; TOR Serine-Threonine Kinases; Ventricular Remodeling

To investigate the influence of the expression profile of mammalian target of rapamycin-related proteins on the development of interstitial fibrosis after kidney transplantation.. Immunohistochemical staining was carried out to evaluate the expression of five mammalian target of rapamycin-related proteins (phosphorylated-Akt, Ras homolog enriched in brain, phosphorylated-mammalian target of rapamycin, phosphorylated-p70 ribosomal S6 kinase and phosphorylated-4E binding protein 1) in graft biopsy specimens obtained from 77 patients at 3 months after kidney transplantation. The change of the estimated glomerular filtration rate and the change of the fibrosis index (defined as the change in the percent area of fibrosis on Masson's trichrome-stained sections of biopsy specimens) from 3 months to 3 years after kidney transplantation were determined.. There was a significant correlation between change of the estimated glomerular filtration and change of the fibrosis index in the 77 patients. Univariate analysis identified expression of phosphorylated-Akt, phosphorylated-mammalian target of rapamycin and phosphorylated-p70 ribosomal S6 kinase, as well as donor type and pre-transplant dialysis duration, as significant predictors of a change of the fibrosis index >10%. However, only phosphorylated-mammalian target of rapamycin expression, phosphorylated-p70 ribosomal S6 kinase expression and donor type were independently associated with a change of the fibrosis index >10% according to multivariate analysis.. These findings suggest that mammalian target of rapamycin-related proteins are involved in the development of interstitial fibrosis after kidney transplantation.

Topics: Adult; Biopsy; Female; Fibrosis; Glomerular Filtration Rate; Graft Rejection; Humans; Immunohistochemistry; Immunosuppressive Agents; Kidney; Kidney Transplantation; Logistic Models; Male; Middle Aged; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptome; Transplantation, Homologous; Young Adult

Autophagy enables cells to digest endogenous/exogenous waste products, thus potentially prolonging the cellular lifespan. Early endothelial progenitor cells (eEPCs) protect mice from ischemic acute kidney injury (AKI). The mid-term prognosis in AKI critically depends on vascular rarefication and interstitial fibrosis with the latter partly being induced by mesenchymal transdifferentiation of endothelial cells (EndoMT). This study aimed to determine the impact of eEPC preconditioning with different autophagy inducing agents [suberoylanilide hydroxamic acid (SAHA)/temsirolimus] in ischemic AKI.. Male C57/Bl6 N mice were subjected to bilateral renal ischemia (40 min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48 h and 4 weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-β ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS-senescence-associated β-galactosidase, SA-β-Gal), and were evaluated 96 h later.. Cultured eEPCs showed reduced perinuclear density of LC3-II + vesicles and elevated levels of SA-β-Gal after treatment with TGF-β alone, indicating impaired autophagy and aggravated SIPS. These effects were completely abrogated by SAHA. Systemic administration of either SAHA or tems pretreated eEPCs resulted in elevated intrarenal endothelial p62 at 48 h and 4 weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4 weeks endothelial expression of mesenchymal alpha-smooth muscle actin (αSMA) was reduced in animals receiving untreated and SAHA-pretreated cells. In addition, SAHA-treated cells reduced fibrosis at week 4. Tems in contrast aggravated EndoMT. Postischemic renal function declined after renal ischemia and remained unaffected in all experimental cell treatment groups.. In ischemic AKI, intrarenal endothelial autophagy may be stabilized by systemic administration of pharmacologically preconditioned eEPCs. Early EPCs can reduce postischemic EndoMT and fibrosis in the mid-term. Autophagy induction in eEPCs either increases or decreases the mesenchymal properties of intrarenal endothelial cells, depending on the substance being used. Thus, endothelial autophagy induction in ischemic AKI, mediated by eEPCs is not a renoprotective event per se.

Topics: Actins; Acute Kidney Injury; Animals; Autophagy; beta-Galactosidase; Cells, Cultured; Disease Models, Animal; Endothelial Progenitor Cells; Epithelial-Mesenchymal Transition; Fibrosis; Hydroxamic Acids; Ischemia; Kidney; Male; Mice, Inbred C57BL; Microtubule-Associated Proteins; Phenotype; Sirolimus; Time Factors; Transforming Growth Factor beta; Vorinostat

Spleen enlargement is often detected in patients with liver cirrhosis, but the precise pathogenetic mechanisms behind the phenomenon have not been clearly elucidated. We investigated the pathogenetic mechanisms of splenomegaly in both portal hypertensive patients and rats, and tried to identify the possible therapy for this disease.. Spleen samples were collected from portal hypertensive patients after splenectomy. Rat models of portal hypertension were induced by common bile duct ligation and partial portal vein ligation. Spleen samples from patients and rats were used to study the characteristics of splenomegaly by histological, immunohistochemical, and western blot analyses. Rapamycin or vehicle was administered to rats to determine the contribution of mTOR signaling pathway in the development of splenomegaly.. We found that not only spleen congestion, but also increasing angiogenesis, fibrogenesis, inflammation and proliferation of splenic lymphoid tissue contributed to the development of splenomegaly in portal hypertensive patients and rats. Intriguingly, splenomegaly developed time-dependently in portal hypertensive rat that accompanied with progressive activation of mTOR signaling pathway. mTOR blockade by rapamycin profoundly ameliorated splenomegaly by limiting lymphocytes proliferation, angiogenesis, fibrogenesis and inflammation as well as decreasing portal pressure.. This study provides compelling evidence indicating that mTOR signaling activation pathway plays a key role in the pathogenesis of splenomegaly in both portal hypertensive patients and rats. Therapeutic intervention targeting mTOR could be a promising strategy for patients with portal hypertension and splenomegaly.

Topics: Animals; Anti-Infective Agents; Female; Fibrosis; Humans; Hypertension, Portal; Male; Middle Aged; Neovascularization, Pathologic; Portal Pressure; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Spleen; Splenomegaly; TOR Serine-Threonine Kinases

The fusing of the epicardium and sternum due to adhesion is a common problem during repeated cardiac surgery and carries with it an increased risk of bleeding. The use of barriers and patches has been tested to prevent the formation of adhesions, but the very presence of a patch can provoke adhesion formation. The objective of this study was, therefore, to investigate both biodegradable and bioresorbable polylactone patches [(polycaprolactone-poly(ethylene oxide)-polycaprolactone tri-block copolymer (PCE)]. The patches were also tested with a controlled release of rapamycin, which prevents cell migration and extracellular matrix deposition. The clinical effectiveness of rapamycin in pericardial patches has not previously been examined.. Three groups of 6 female Danish Landrace pigs underwent sternotomy and abrasion of the epicardium, before being randomized to either group 1--the control group (with no patch), group 2--PCE patch implanted between the sternum and epicardium, or group 3--PCE patch and slow-release 1.6-mg rapamycin. After a median time period of 26 days, the pigs were euthanized and their hearts removed en bloc with the sternum, for macroscopic, histological and pathological examination.. Upon macroscopic examination, a significantly lower degree of adhesion in group 2, as compared to group 1 (p < 0.05), was found. Histological analysis of the tissues showed significantly more fibrosis, inflammation and foreign body granulomas (p < 0.05) in both group 2 and group 3, when compared to group 1.. A PCE patch following sternotomy in animal subjects reduces postoperative macroscopic adhesions without reducing microscopic fibrosis or inflammation. Loading the patch with rapamycin was found not to increase the antifibrotic effect.

Topics: Absorbable Implants; Animals; Cardiac Surgical Procedures; Ethylene Oxide; Female; Fibrosis; Lactones; Models, Anatomic; Myocardium; Sirolimus; Swine; Tissue Adhesions

To investigate the effect of rapamycin on TGFβ1 and MMP1 expression in a rabbit model of urethral stricture.. Twenty-four adult New Zealand male rabbits underwent an electrocoagulation of the bulbar urethra with a 13Fr pediatric resectoscope. Then rabbits were randomly divided into three groups: (1) normal control group: normal saline (NS), (2) the vehicle control group: dimethyl sulfoxide (DMSO), and (3) the treatment group: effective-dose rapamycin in DMSO (Ra), with 12, 6, and 6 rabbits in each group, respectively. Drugs were given by urethral irrigation daily for 4 weeks. Urethral tissue was harvested for histological and molecular analyses. TGFβ1 and MMP1 expression levels were evaluated by real-time quantitative PCR and immunohistochemistry.. Ten, six, and six rabbits were evaluated finally in Ra, DMSO, and NS group, respectively. Histological examination revealed the distribution of fibrosis and the degree of collagen deposition in the Ra group were smaller and slighter than the two control groups. Collagen content was significantly less in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). qRT-PCR analysis showed a higher expression of MMP1 mRNA in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). Immunohistochemistry showed the protein levels of MMP1 in the Ra group were significantly increased when compared with the DMSO group (P < 0.01) and the NS group (P < 0.01). On the other hand, no statistical difference could be found between every two groups in both mRNA and protein levels of TGFβ1.. Rapamycin enhances the expression of MMP1 in a rabbit model of urethral stricture, but has no direct effect on the expression of TGFβ1.

Topics: Animals; Antibiotics, Antineoplastic; Collagen; Disease Models, Animal; Fibrosis; Gene Expression; Male; Matrix Metalloproteinase 1; Protein Biosynthesis; Rabbits; RNA, Messenger; Sirolimus; Transforming Growth Factor beta1; Urethral Stricture

Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5mg/kg) and/or human recombinant ACE2 (rhACE2; 2mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF-β1 and collagen I and elevated renal Ang-(1-7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100nmolL(-1)) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-β1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1mgmL(-1)) or mTOR inhibitor rapamycin (10μmolL(-1)). These protective effects of rhACE2 were eradicated by the Ang-(1-7)/Mas receptor antagonist A779 (1μmolL(-1)). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1-7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders.

Topics: Angiotensin I; Angiotensin-Converting Enzyme 2; Animals; Apolipoproteins E; Atherosclerosis; Fibrosis; Kidney; Kidney Diseases; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Knockout; Peptide Fragments; Peptidyl-Dipeptidase A; Sirolimus; TOR Serine-Threonine Kinases

Epidural fibrosis is a common complication after laminectomy. It is associated with intractable lower back pain and additional complications. To date, no study has evaluated whether the local application of rapamycin (RAPA) can inhibit fibroblast proliferation and reduce epidural scar adhesion after laminectomy. The results of the present study showed that the local application of RAPA reduces epidural fibrosis after laminectomy in rats.. In this study, 32 male Sprague-Dawley rats were randomly divided into four groups (0.2 mg/ml RAPA-treated group, 0.1 mg/ml RAPA-treated group, 0.05 mg/ml RAPA-treated group and physiological saline group). Laminectomy was performed at the level of lumbar segment 1 to 2, and different concentrations of RAPA or saline were applied to the laminectomy sites for 10 min. Four weeks after laminectomy, the rats were sacrificed, and the degrees of epidural adhesion in each group were evaluated. Macroscopic assessment, analysis of hydroxyproline content, and histological analysis were used to determine the therapeutic effect of the local application of RAPA on the inhibition of fibroblast proliferation and the reduction of epidural fibrosis after laminectomy. Next, we cultured fibroblasts from epidural scar tissues of rats that had undergone laminectomy. Fibroblasts were exposed to the indicated concentrations of RAPA, and western blotting and TUNEL assays were used to assess the effects of RAPA on inhibiting fibroblasts proliferation and promoting fibroblast apoptosis.. The results of macroscopic assessments, analysis of hydroxyproline content, and histological analyses indicated that RAPA significantly inhibited fibroblast proliferation and reduced epidural fibrosis in the treated groups in the rat model. The western blotting results indicated that the expression levels of the pro-apoptotic proteins cleaved-PARP and Bax were up-regulated, whereas those of Bcl-2 were reduced. TUNEL assay indicated that the apoptosis rates of fibroblasts were significantly increased after exposure to the indicated concentrations of RAPA.. The local application of RAPA reduced epidural fibrosis after laminectomy by inhibiting the proliferation of fibroblasts, stimulating their apoptosis, and decreasing collagen synthesis. This protocol may be used in new clinical treatment strategies to reduce epidural fibrosis after laminectomy.

Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Cicatrix; Epidural Space; Fibroblasts; Fibrosis; Hydroxyproline; Immunosuppressive Agents; In Situ Nick-End Labeling; Laminectomy; Male; Random Allocation; Rats, Sprague-Dawley; Sirolimus

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

Topics: Angiotensin II; Animals; Autophagy; Autophagy-Related Protein 5; Cell Cycle; Cell Cycle Checkpoints; Cell Division; Collagen Type I; Epithelial Cells; Fibrosis; G2 Phase; Gene Deletion; Humans; Kidney; Kidney Diseases; Kidney Tubules; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Signal Transduction; Sirolimus

TGF-β plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-β in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-β. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is shown to impair TGF-β-driven fibrogenic processes, including cell proliferation and production of profibrotic mediators. These findings provide new perspectives on the interrelation of metabolism and profibrotic TGF-β signaling and present opportunities for potential therapeutic intervention.-Andrianifahanana, M., Hernandez, D. M., Yin, X., Kang, J.-H., Jung, M.-Y., Wang, Y., Yi, E. S., Roden, A. C., Limper, A. H., Leof, E. B. Profibrotic up-regulation of glucose transporter 1 by TGF-β involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

Topics: Animals; Cell Proliferation; Connective Tissue Growth Factor; Fibroblasts; Fibrosis; Glucose Transporter Type 1; Lung; MAP Kinase Signaling System; Mice; Phosphatidylinositol 3-Kinases; Signal Transduction; Sirolimus; Transforming Growth Factor beta; Up-Regulation

Experimental uremic cardiomyopathy causes cardiac fibrosis and is causally related to the increased circulating levels of the cardiotonic steroid, marinobufagenin (MBG), which signals through Na/K-ATPase. Rapamycin is an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR) implicated in the progression of many different forms of renal disease. Given that Na/K-ATPase signaling is known to stimulate the mTOR system, we speculated that the ameliorative effects of rapamycin might influence this pathway.. Biosynthesis of MBG by cultured human JEG-3 cells is initiated by CYP27A1, which is also a target for rapamycin. It was demonstrated that 1 μmol/L of rapamycin inhibited production of MBG in human JEG-2 cells. Male Sprague-Dawley rats were subjected to either partial nephrectomy (PNx), infusion of MBG, and/or infusion of rapamycin through osmotic minipumps. PNx animals showed marked increase in plasma MBG levels (1025±60 vs 377±53 pmol/L; P<0.01), systolic blood pressure (169±1 vs 111±1 mm Hg; P<0.01), and cardiac fibrosis compared to controls. Plasma MBG levels were significantly decreased in PNx-rapamycin animals compared to PNx (373±46 vs 1025±60 pmol/L; P<0.01), and cardiac fibrosis was substantially attenuated by rapamycin treatment.. Rapamycin treatment in combination with MBG infusion significantly attenuated cardiac fibrosis. Our results suggest that rapamycin may have a dual effect on cardiac fibrosis through (1) mTOR inhibition and (2) inhibiting MBG-mediated profibrotic signaling and provide support for beneficial effect of a novel therapy for uremic cardiomyopathy.

Topics: Animals; Blood Pressure; Bufanolides; Cardiomyopathies; Cells, Cultured; Enzyme Inhibitors; Fibroblasts; Fibrosis; Heart; Humans; Immunosuppressive Agents; Male; Myocardium; Nephrectomy; Rats; Rats, Sprague-Dawley; Sirolimus; Sodium-Potassium-Exchanging ATPase; Uremia

Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.

Topics: Animals; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Mice; Mice, Transgenic; Muscle, Skeletal; Myositis Ossificans; Ossification, Heterotopic; Osteogenesis; Regeneration; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Topics: Animals; Anti-Inflammatory Agents; Capsules; Female; Fibrosis; Gene Expression Regulation; Kidney; Microscopy, Electron, Scanning; Microspheres; Myofibroblasts; Rats, Inbred F344; Sirolimus; T-Lymphocytes; Tissue Distribution; TOR Serine-Threonine Kinases; Treatment Outcome; Ureteral Obstruction

Aldosterone (Aldo), a mediator of kidney fibrosis, is implicated in the pathogenesis of chronic kidney diseases (CKD). The aim of this study was to evaluate the regulatory role of rapamycin (Rap) in Aldo-induced tubulointerstitial inflammation and fibrosis.. Uninephrectomized, Sprague-Dawley rats were given 1% NaCl (salt) to drink and were randomized to receive treatment for 28 days as follows: vehicle infusion (control), 0.75 μg/h Aldo subcutaneous infusion, or Aldo infusion plus 1 mg/kg/day of Rap by intraperitoneal injection. The effect of Rap on Aldo-induced fibrosis and renal inflammation was investigated using Masson's technique, immunohistochemistry, and western blotting. The effects of Rap on the Aldo-induced epithelial-mesenchymal transition (EMT) process and on TNF-α mRNA expression and secretion in cultured HK-2 cells were investigated by immunofluorescent staining, western blot, qRT-PCR and ELISA.. An in vivo study indicated that signaling by the mammalian target of Rap (mTOR) was activated in rats in the Aldo group compared to controls, as indicated by up-regulated expression of p-mTOR and p-S6K. In addition, the inflammatory response increased, as evidenced by increases in inflammatory markers (MCP-1, ICAM-1, F4/80), and the accumulation of extracellular matrix (ECM), as indicated by increased collagen I and fibronectin expression and pro-fibrogenic gene (PAI-1 and TGF-β1) expression. These changes were attenuated by Rap treatment. An in vitro study showed that Rap significantly suppressed the Aldo-induced EMT process and TNF-α mRNA expression and secretion in cultured HK-2 cells.. Rap can ameliorate tubulointerstitial inflammation and fibrosis by blocking mTOR signaling. Tubular cells may be a major cell type involved in this physiologic process.

Topics: Aldosterone; Animals; Antibiotics, Antineoplastic; Cell Line; Epithelial-Mesenchymal Transition; Extracellular Matrix; Fibrosis; Humans; Male; Nephritis, Interstitial; Phosphorylation; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Up-Regulation

Peridural fibrosis (PF) is a normal complication after lumbar surgery. It is a challenge for both surgeons and patients. Rapamycin (RPM), a novel antibiotic with anti-proliferative and immunosuppressive properties, has been shown to be effective in preventing uncontrolled scar proliferation diseases. The object of the present research was to investigate the effects of RPM on inhibiting PF in vitro and in vivo.. In vitro, the fibroblasts collected and isolated from the rat tail skin were cultured with/without RPM and cell counting was performed. In vivo, the double-blinded study was conducted in 60 healthy Wistar rats divided randomly into 3 groups: 1) RPM treatment group; 2) Vehicle treatment group; 3) Control group. Rats underwent a L1-L2 level laminectomy with a satisfactory anesthetization. Four weeks post-operatively, the Rydell score, histological analysis, hydroxyproline content, vimentin expressional level, and inflammatory cytokines expressional levels were assessed.. In vitro, RPM showed ability to prevent fibroblast proliferation. In vivo, the laminectomy was well tolerated by all rats, which were killed 4 weeks post-operatively. The Rydell score, histological evaluation, hydroxyproline content, vimentin expression level, and inflammatory activity showed the positive effect of RPM in preventing peridural adhesion, inhibiting fibrotic formation and collagen synthesis, and down-regulating inflammation.. In the present primary study, RPM showed good efficacy in preventing the proliferation of fibroblasts. RPM can prevent rat peridural adhesion through inhibiting collagen synthesis, fibroblasts proliferation, and inflammatory activity.

Topics: Analysis of Variance; Animals; Cell Proliferation; Cells, Cultured; Cytokines; DNA Primers; Drug Evaluation, Preclinical; Fibroblasts; Fibrosis; Hydroxyproline; Laminectomy; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Spinal Cord; Vimentin

Topics: Biomarkers; Biopsy, Needle; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Imidazoles; Immunohistochemistry; Molecular Targeted Therapy; Phosphorylation; Polymerase Chain Reaction; Scleroderma, Diffuse; Sensitivity and Specificity; Sirolimus; Smad2 Protein; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Triazines

Topics: Fibrosis; Humans; Sirolimus

Septic kidney injury is one of the most common complications in critically ill patients with a high risk of developing chronic kidney disease (CKD). Emerging data indicate that mammalian target of rapamyci (mTOR) signaling plays a major role in septic inflammation by regulating the immune response of macrophage. This study was designed to evaluate the role of mTOR signaling in kidney macrophages during endotoxemia-induced chronic kidney injury and subsequent fibrogenesis.. Male C57BL/6 mice were used for all animal studies (n=9 for each group). Lipopolysaccharide (LPS) was injected intraperitoneally (1 mg/kg) every 2 days to induce persistent endotoxemia. Rapamycin (1 mg/kg·day) was administered to a subgroup of mice 1 day prior to LPS treatment and continued to termination of the experiment. In ex-vivo experiment, RAW264.7 cells were cultured and treated with LPS (2 µg/ml) for 48 h while a subgroup of cells were incubated in the presence of rapamycin (50 nmol) for 2 h.. Continuous administration of LPS resulted in progressive macrophage infiltration, tubular injury and collagen deposition in mice kidneys. Rapamycin markedly ameliorated LPS-induced kidney pathological changes. Expression of pS6K was rarely observed in normal kidney macrophages, but significantly increased with time by LPS treatment. In ex-vivo study, LPS induced prominent production of IL-1β and MCP-1 in cultured RAW264.7 cells, which was significantly suppressed by rapamycin.. Taken together, our findings show that endotoxemia results in activation of mTOR signaling in macrophages, leading to progressive kidney inflammatory injuries and subsequent fibrosis. Our study may reveal a mechanism involved in the development of sepsis-associated CKD and kidney fibrosis.

Topics: Animals; Blotting, Western; Cell Line; Chemokine CCL2; Collagen; Endotoxemia; Fibrosis; Fluorescent Antibody Technique; Immunohistochemistry; Immunosuppressive Agents; Injections, Intraperitoneal; Interleukin-1beta; Kidney; Kidney Tubules; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Renal Insufficiency, Chronic; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension.. Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1β) were assessed by western blot.. The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin.. The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.

Topics: Animals; Extracellular Matrix; Fibrosis; Hemodynamics; Inflammation; Liver; Liver Cirrhosis; Liver Function Tests; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Multiprotein Complexes; Portal Pressure; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; Splenomegaly; TOR Serine-Threonine Kinases

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.

Topics: Adrenal Cortex Hormones; Animals; Autophagy; Cell Death; Creatine Kinase; Drug Delivery Systems; Fibrosis; Immunosuppressive Agents; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle Strength; Muscular Dystrophy, Duchenne; Myocardial Contraction; Myocardium; Nanoparticles; Regeneration; Sirolimus; Tissue Distribution

Conversion from calcineurin inhibitor (CNI) to mTOR inhibitors may reduce and even halt the progression of chronic allograft dysfunction (CAD) which is the most important cause of renal allograft loss. We aimed to investigate the effects of conversion from CNI to everolimus on parameters of fibrosis, inflammation, glomerulotubular damage and vascular functions in renal transplant recipients.. Fifteen stable renal transplant recipients who were under CNI treatment (male/female 13/2, mean age 41 ± 10 years) were enrolled and switched to everolimus. Serum and urinary transforming growth factor-β (TGF-β), urinary neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemoattractant protein-1 (MCP-1) were measured as markers of fibrosis, tubular damage and inflammation. As parameters of vascular functions, pulse wave velocity (PWV), augmentation index (AIx), serum asymmetric dimethyl-arginine and fibroblast growth factor-23 (FGF-23) were measured. All these measurements were repeated at the 3rd month of conversion.. Estimated GFR (52 ± 7-57 ± 11 ml/min/l.73 m(2), p = 0.02) (was increased after conversion to everolimus. However, serum uric acid levels were significantly decreased (6.21 ± 1.21-5.50 ± 1.39 mg/dL, p = 0.01). Serum TGF-β levels (8727 ± 2897-1943 ± 365 pg/mL, p = 0.03) and urinary NGAL levels (26 ± 10-12 ± 2 ng/mg creatinine, p = 0.05) were significantly decreased. However, urinary MCP-1, FGF-23, PWV and AIx did not change. Urinary TGF-β was associated with urinary NGAL (r = 0.62, p = 0.01), urinary MCP-1 (r = 0.68, p = 0.005) and proteinuria (r = 0.50, p = 0.05).. Conversion from CNI to everolimus resulted in significant decreases of serum TGF-β and urinary NGAL which may represent less fibrosis and tubular damage. Association of urinary TGF-β with NGAL and MCP-1 suggests that tubular damage, fibrosis and inflammation may act together for progression of CAD.

Topics: Acute-Phase Proteins; Adult; Calcineurin Inhibitors; Chemokine CCL2; Everolimus; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibrosis; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Transplantation; Kidney Tubules; Lipocalin-2; Lipocalins; Male; Middle Aged; Nephritis; Proto-Oncogene Proteins; Pulse Wave Analysis; Renal Artery; Risk Factors; Sirolimus; Transforming Growth Factor beta; Transplant Recipients

IgA nephropathy (IgAN) is the most frequent glomerulonephritis worldwide. Different therapeutic approaches have been tested against IgAN. The present study was designed to explore the renoprotective potential of low-dose mammalian target of rapamycin (mTOR) inhibitor rapamycin in an IgAN rat model and the possible mechanism of action.. After establishing an IgAN model, the rats were randomly divided into four groups: control, control with rapamycin treatment, IgAN model, and IgAN model with rapamycin treatment. Coomassie Brilliant Blue was utilized to measure 24-hour urinary protein levels. Hepatic and renal function was determined with an autoanalyzer. Proliferation was assayed via 5-bromo-2'-deoxyuridine incorporation. Real-time PCR and immunohistochemistry were utilized to detect the expression of α-SMA, collagen I, collagen III, TGF-β1 and platelet-derived growth factor. Western blotting and immunohistochemistry were performed to determine p-S6 protein levels.. Low-dose mTOR inhibitor rapamycin prevented an additional increase in proteinuria and protected kidney function in a model of IgAN. Rapamycin directly or indirectly interfered with multiple key pathways in the progression of IgAN to end-stage renal disease: (1) reduced the deposition of IgA and inhibited cell proliferation; (2) decreased the expression of fibrosis markers α-SMA and type III collagen, and (3) downregulated the expression of the profibrotic growth factors platelet-derived growth factor and TGF-β1. The expression of p-S6 was significantly elevated in IgAN rats.. The mTOR pathway was activated in IgAN rats and the early application of low-dose mTOR inhibitor rapamycin may slow the renal injury of IgAN in rats.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Disease Progression; Fibrosis; Glomerulonephritis, IGA; Immunosuppressive Agents; Kidney; Kidney Glomerulus; Liver; Male; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Topics: Angioscopy; Autopsy; Biopsy; Cardiovascular Agents; Coronary Artery Disease; Coronary Restenosis; Coronary Vessels; Drug-Eluting Stents; Fatal Outcome; Fibrosis; Humans; Male; Middle Aged; Neointima; Percutaneous Coronary Intervention; Prosthesis Design; Sirolimus; Tomography, Optical Coherence

The purpose of this study was to explore effects of rapamycin on renal hypoxia, interstitial inflammation and fibrosis, and the expression of transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), Flk-1 and Flt-1 in a rat model of unilateral ureteral obstruction (UUO).. Male Sprague-Dawley rats (n=36) were randomly divided into three groups (n=12 per group): sham surgery, UUO and UUO plus rapamycin (0.2 mg/kg/d). Serum creatinine (Scr), blood urea nitrogen, uric acid, triglycerides, cholesterol and 24-h urine protein levels were measured. The extent of interstitial fibrosis was determined by Masson's trichrome staining. ED-1 positive macrophages, type III collagen, hypoxia, TGF-1, VEGF, Flk-1, and Flt-1 mRNA and protein expressions were detected using immunohistochemical staining, real-time PCR and Western blot.. UUO induced an elevation in Scr, renal hypoxia, inflammation, interstitial fibrosis, TGF-β1, VEGF, Flk-1, and Flt-1 mRNA and protein expression levels (P < 0.05). Rapamycin alleviated the UUO-induced renal hypoxia, infiltration of inflammatory cells and tubulointerstitial fibrosis (at days 3 and 7). Rapamycin also down-regulated the UUO-induced elevated expression levels of TGF-β1 and Flt-1 mRNA and protein (P < 0.05). Rapamycin decreased VEGF mRNA and protein expression at day 3, and increased Flk-1 mRNA and protein expression at day 7, compared with the UUO group (P < 0.05).. Rapamycin shows beneficial effects by reducing UUO-induced renal hypoxia, inflammation and tubulointerstitial fibrosis.

Topics: Animals; Blotting, Western; Fibrosis; Hypoxia; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Sirolimus; Transforming Growth Factor beta1; Ureteral Obstruction; Vascular Endothelial Growth Factor Receptor-1

Uterine leiomyomata are the most common tumors found in the female reproductive tract. Despite the high prevalence and associated morbidities of these benign tumors, little is known about the molecular basis of uterine leiomyoma development and progression. Loss of the Tuberous Sclerosis 2 (TSC2) tumor suppressor has been proposed as a mechanism important for the etiology of uterine leiomyomata based on the Eker rat model. However, conflicting evidence showing increased TSC2 expression has been reported in human uterine leiomyomata, suggesting that TSC2 might not be involved in the pathogenesis of this disorder. We have produced mice with conditional deletion of the Tsc2 gene in the myometria to determine whether loss of TSC2 leads to leiomyoma development in murine uteri. Myometrial hyperplasia and increased collagen deposition was observed in Tsc2(cKO) mice compared with control mice, but no leiomyomata were detected by post-natal week 24. Increased signaling activity of mammalian target of rapamycin complex 1, which is normally repressed by TSC2, was also detected in the myometria of Tsc2(cKO) mice. Treatment of the mutant mice with rapamycin significantly inhibited myometrial expansion, but treatment with the progesterone receptor modulator, mifepristone, did not. The ovaries of the Tsc2(cKO) mice appeared normal, but half the mice were infertile and most of the other half became infertile after a single litter, which was likely due to oviductal blockage. Our study shows that although TSC2 loss alone does not lead to leiomyoma development, it does lead to myometrial hyperplasia and fibrosis.

Topics: Animals; Female; Fertility; Fibrosis; Gene Deletion; Hyperplasia; Leiomyoma; Mechanistic Target of Rapamycin Complex 1; Mesoderm; Mice; Mullerian Ducts; Multiprotein Complexes; Myometrium; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Topics: Fibrosis; Humans; Immunosuppressive Agents; Models, Biological; Sirolimus

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates cell growth, differentiation, and survival by upregulating mammalian target of rapamycin complex 1 (mTORC1) signaling. The role of Rheb/mTORC1 signaling in the activation of kidney fibroblasts and the development of kidney fibrosis remains largely unknown. In this study, we found that Rheb/mTORC1 signaling was activated in interstitial myofibroblasts from fibrotic kidneys. Treatment of rat kidney interstitial fibroblasts (NRK-49F cell line) with TGFβ1 also activated Rheb/mTORC1 signaling. Blocking Rheb/mTORC1 signaling with rapamycin or Rheb small interfering RNA abolished TGFβ1-induced fibroblast activation. In a transgenic mouse, ectopic expression of Rheb activated kidney fibroblasts. These Rheb transgenic mice exhibited increased activation of mTORC1 signaling in both kidney tubular and interstitial cells as well as progressive interstitial renal fibrosis; rapamycin inhibited these effects. Similarly, mice with fibroblast-specific deletion of Tsc1, a negative regulator of Rheb, exhibited activated mTORC1 signaling in kidney interstitial fibroblasts and increased renal fibrosis, both of which rapamycin abolished. Taken together, these results suggest that Rheb/mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis, possibly providing a therapeutic target for progressive renal disease.

Topics: Animals; Cell Line; Disease Models, Animal; Fibroblasts; Fibrosis; Immunohistochemistry; Kidney; Mechanistic Target of Rapamycin Complex 1; Mice; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Neuropeptides; Proteins; Ras Homolog Enriched in Brain Protein; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Deficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known.. In the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients.. AML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA.. These data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients.

Topics: Actins; Cell Line; Enzyme Activation; Fibrosis; Gene Expression Regulation; Humans; Intracellular Space; Kidney Neoplasms; Models, Biological; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Transport; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; YY1 Transcription Factor

Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the past decade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian target of rapamycin complex (mTORC)1. We recently reported that the hypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagen expression regardless of ambient oxygen tension. A modulator of HIF expression other than oxygen tension is mTORC1. We therefore sought to evaluate a possible role for mTORC1 activity in TGF-β-induced fibrogenesis. mTORC1 activity was increased in human mesangial cells treated with TGF-β in a TGF-β receptor-dependent manner. Short hairpin (sh)RNA to Smad3 decreased, while overexpression of Smad3 increased, the mTORC1 activity, suggesting that TGF-β stimulation of mTORC1 also requires Smad3. Pretreatment with rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity and collagen I protein expression. mTORC1 inhibition also prevented the TGF-β-stimulated increase in both hypoxia-responsive element (HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-induced HRE activity. shRNA to Smad3 reduced HRE activity, while overexpression of Smad3 increased HIF-1α protein expression and activity in an mTORC1-dependent manner. Lastly, overexpression of HIF-1α bypassed the inhibitory effect of mTORC1 blockade on collagen expression. These results suggest that Smad3/mTORC1 interaction to promote HIF-1 expression is a key step in normoxic kidney fibrogenesis.

Topics: Blotting, Western; Cell Culture Techniques; Collagen; Fibrosis; Glomerular Mesangium; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoprecipitation; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; RNA, Small Interfering; Signal Transduction; Sirolimus; Smad3 Protein; TOR Serine-Threonine Kinases; Transfection; Transforming Growth Factor beta

Endovascular interventions on peripheral arteries are limited by high rates of restenosis. Our hypothesis was that adventitial injection of rapamycin nanoparticles would be safe and reduce luminal stenosis in a porcine femoral artery balloon angioplasty model.. Eighteen juvenile male crossbred swine were included. Single-injury (40%-60% femoral artery balloon overstretch injury; n=2) and double-injury models (endothelial denudation injury 2 weeks before a 20%-30% overstretch injury; n=2) were compared. The double-injury model produced significantly more luminal stenosis at 28 days, P=0.002, and no difference in medial fibrosis or inflammation. Four pigs were randomized to the double-injury model and adventitial injection of saline (n=2) or 500 μg of nanoparticle albumin-bound rapamycin (nab-rapamycin; n=2) with an endovascular microinfusion catheter. There was 100% procedural success and no difference in endothelial regeneration. At 28 days, nab-rapamycin led to significant reductions in luminal stenosis, 17% (interquartile range, 12%-35%) versus 10% (interquartile range, 8.3%-14%), P=0.001, medial cell proliferation, P<0.001, and fibrosis, P<0.001. There were significantly fewer adventitial leukocytes at 3 days, P<0.001, but no difference at 28 days. Pharmacokinetic analysis (single-injury model) found rapamycin concentrations 1500× higher in perivascular tissues than in blood at 1 hour. Perivascular rapamycin persisted ≥8 days and was not detectable at 28 days.. Adventitial nab-rapamycin injection was safe and significantly reduced luminal stenosis in a porcine femoral artery balloon angioplasty model. Observed reductions in early adventitial leukocyte infiltration and late medial cell proliferation and fibrosis suggest an immunosuppressive and antiproliferative mechanism. An intraluminal microinfusion catheter for adventitial injection represents an alternative to stent- or balloon-based local drug delivery.

Topics: Adventitia; Angioplasty, Balloon; Animals; Arteritis; Cell Movement; Cell Proliferation; Constriction, Pathologic; Disease Models, Animal; Dose-Response Relationship, Drug; Femoral Artery; Fibrosis; Injections, Intra-Arterial; Leukocytes; Male; Nanoparticles; Sirolimus; Swine; Tunica Media

Cyclosporin A (CsA) is associated with significant chronic nephrotoxicity, which typically manifests as renal fibrosis. In contrast, rapamycin (RAPA) has been shown to inhibit fibrosis. This study sought to determine the effect of CsA and RAPA on the expression of connective tissue growth factor (CTGF) and E-cadherin in a rat kidney model of chronic allograft nephropathy.. Left renal grafts from male Fisher (F344, RT1(1v1)) rats were orthotopically transplanted into Lewis (LEW, RT1(1)) rats. After transplantation, all recipients were given CsA 10 mg/kg(-1) d(-1) for 10 d and divided into three groups (n = 9/group): (1) vehicle, administered orally; (2) CsA, 6 mg/kg(-1) d(-1); (3) RAPA, 0.8 mg/kg(-1) d(-1). At 4, 8, and 12 wk posttransplantation, the kidney allografts were harvested and serum creatinine levels were measured. Connective tissue growth factor expression was determined using real-time polymerase chain reaction and Western blot. Kidney allografts sections also underwent hematoxylin-eosin and Masson trichrome staining, in addition to CTGF and E-cadherin immunostaining.. The serum creatinine levels were increased at 8 and 12 wk posttransplantation and were significantly lower in the RAPA group (P < 0.05). The Banff score also showed a significant decrease at 4, 8, and 12 wk (P < 0.05). CTGF messenger ribonucleic acid and protein levels were significantly lower in the RAPA group (P < 0.05), whereas E-cadherin expression was higher in the RAPA group at 4, 8, and 12 wk (P < 0.05). Masson's trichrome staining showed a significant decrease in collagen deposition at 8 and 12 wk after RAPA treatment.. RAPA can ameliorate fibrogenesis in kidney allografts by inhibiting epithelial-mesenchymal transition process, whereas CsA did not have this effect.

Topics: Animals; Cadherins; Connective Tissue Growth Factor; Creatinine; Cyclosporine; Epithelial-Mesenchymal Transition; Fibrosis; Graft Rejection; Immunosuppressive Agents; Kidney; Kidney Transplantation; Male; Models, Animal; Rats; Rats, Inbred F344; Rats, Inbred Lew; Renal Insufficiency, Chronic; Sirolimus; Transplantation, Homologous

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.

Topics: Age Factors; Aging; Animals; Animals, Newborn; Cardiomyopathy, Hypertrophic; Cell Size; Cells, Cultured; Disease Models, Animal; Fibrosis; Mice; Mice, Transgenic; Mutagenesis, Site-Directed; Mutation; Myocardial Contraction; Myocytes, Cardiac; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Ventricular Function, Left

Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-β(1). Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts.

Topics: Animals; Aristolochic Acids; Cell Line; Cell Proliferation; Extracellular Matrix; Fibroblasts; Fibrosis; Immunosuppressive Agents; Intestines; Kidney Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mutagens; Myofibroblasts; NIH 3T3 Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

Chronic renal transplant dysfunction is histopathologically characterized by interstitial fibrosis and tubular atrophy. This study investigated the relative contribution of baseline donor, recipient, and transplant characteristics to interstitial fibrosis and tubular atrophy score at month 12 after renal transplantation.. This retrospective study includes all 109 consecutive recipients with adequate implantation and month 12 biopsies transplanted between April of 2003 and February of 2007. Immunosuppression regimen was tacrolimus and steroids (10 days) plus either sirolimus or mycophenolate mofetil.. Average interstitial fibrosis and tubular atrophy score increased from 0.70 to 1.65 (P<0.001). In an adjusted multiple linear regression analysis, interstitial fibrosis and tubular atrophy score at month 12 was significantly related to donor type (donors after cardiac death versus living donor had interstitial fibrosis and tubular atrophy score+0.41, 95% confidence interval=0.05-0.76, P=0.02), baseline interstitial fibrosis and tubular atrophy, and immunosuppression regimen. Because of interaction between the latter two variables (P=0.002), results are given separately: recipients with a baseline interstitial fibrosis and tubular atrophy score of zero had a 0.60 higher score at month 12 (95% confidence interval=0.09-1.10, P=0.02) when mycophenolate mofetil-treated, whereas recipients with a baseline interstitial fibrosis and tubular atrophy score more than zero had a 0.38 higher score at month 12 (95% confidence interval=0.01-0.74, P=0.04) when sirolimus-treated. A higher score at month 12 correlated with a lower estimated GFR (ρ=-0.45, P<0.001).. These findings suggest that histologic assessment of a preimplantation biopsy may guide choice of immunosuppresion to maximize transplant survival and its interaction with type of immunosuppression.

Topics: Adult; Aged; Atrophy; Biopsy; Drug Therapy, Combination; Female; Fibrosis; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Kidney Transplantation; Kidney Tubules; Linear Models; Male; Middle Aged; Multivariate Analysis; Mycophenolic Acid; Netherlands; Proteinuria; Retrospective Studies; Risk Assessment; Risk Factors; Sirolimus; Steroids; Tacrolimus; Time Factors; Treatment Outcome

The present study aimed to investigate differences in plaque morphology and components in between the target coronary artery lesion with and without late-acquired incomplete stent apposition (LISA) using radiofrequency analysis (virtual histology) of intravascular ultrasound data.. Incomplete stent apposition is frequently observed in patients with very late stent thrombosis after sirolimus-eluting stent implantation.. The study group consisted of 70 coronary artery lesions in 43 patients who underwent elective coronary stenting for stable angina pectoris. Virtual histology intravascular ultrasound was performed at the implantation of stent and 12-month follow-up. LISA was defined as a separation of stent struts from the intimal surface of the arterial wall that had not been present at the time of stent implantation. The plaque eccentricity index (EI) was calculated as (lumen radius+maximal plaque thickness)/(lumen radius+minimal plaque thickness).. At 12-month follow-up, LISA occurred in 15 plaques (LISA group). Compared with the non-LISA group, the LISA group had significantly longer stents, a higher EI, smaller amount of fibro-fatty component (7.7±4.2 vs. 12.5±7.0%, P=0.01) and larger amount of necrotic core component (16.6±9.8 vs. 11.1±6.4%, P=0.06). Multivariate logistic regression analysis revealed that amount of necrotic core and plaque EI were independent positive predictors for LISA (odds ratio=1.4, 95% confidence interval=1.1-1.6, P=0.04 and 11.2, 1.9-64.9, P<0.01, respectively).. Plaques with increased amounts of necrotic core and higher eccentricity are associated with subsequent LISA after sirolimus-eluting stent implantation.

Topics: Aged; Cardiovascular Agents; Chi-Square Distribution; Coronary Artery Disease; Coronary Thrombosis; Coronary Vessels; Drug-Eluting Stents; Female; Fibrosis; Humans; Logistic Models; Male; Middle Aged; Multivariate Analysis; Necrosis; Odds Ratio; Percutaneous Coronary Intervention; Plaque, Atherosclerotic; Predictive Value of Tests; Prosthesis Design; Risk Assessment; Risk Factors; Sirolimus; Time Factors; Treatment Outcome; Ultrasonography, Interventional

Rapamycin (RPM) has antiangiogenic and antiproliferative effects on cells. The aim of this study was to evaluate the mechanism of RPM as a novel antifibrotic agent by assessing its effect on interstitial fibrosis (IF). Among 60 renal transplant recipients, group 1 patients (n=20) were treated with RPM and group 2 (n=40), with cyclosporine. The proportions of infiltrating macrophages and lymphocytes in the interstitium were evaluated in 1-year biopsies. The microvessels were highlightened with CD34. After an initial biopsy, the development of diffuse IF over 18 months was evaluated by follow-up biopsies. The mean microvessel density (MVD) was significantly lower among group 1 (69.3±16) versus group 2 (96.5±30; P<.001). The proportions of macrophages and lymphocytes were lower in group 1 compared to group 2 biopsies (P<.001 for both). Fourteen (35%) group 2 and only 2 (10%) group 1 cases developed IF over 18 months (P<.05). The mean MVD in the initial biopsy was 75.6±18 in cases that did not versus 120±28 among those who did develop IF (P<.001). The amount of interstitial inflammation was greater among patients who did compared with cases who did not develop IF (P<.01). The overall 1-, 3-, and 5-year graft survival rates for group 1 were 95%, 95%, and 89% versus 95%, 65%, and 45% for group 2 patients, respectively (P<.001). RPM-treated patients showed a lower incidence of diffuse IF, which can be explained by antiproliferative and antiangiogenic effects of RPM. In conclusion, RPM therapy displayed an independently positive impact on long-term graft survival.

Topics: Adult; Antigens, CD34; Biopsy; Cell Proliferation; Cyclosporine; Female; Fibrosis; Graft Survival; Humans; Immunosuppressive Agents; Inflammation; Kidney Transplantation; Lymphocytes; Macrophages; Male; Neovascularization, Pathologic; Sirolimus; Transplantation, Homologous

Endomyocardial remodeling is characterized by progressive fibrosis and scars and may develop after heart transplantation. The role of everolimus in preventing this process has not been evaluated as yet.. We prospectively studied 132 patients at baseline pretransplant and at 4 weeks, 1 year, and 3 years after heart transplantation. Fibrosis, scars (Zeiss Vision, in Sirius), and acute cellular rejection (hematoxylin-eosin) were studied in biopsy. Transplant vasculopathy was assessed by coronary angiography (focal stenoses, peripheral obliterations, negative vascular remodeling defined by peripheral obliterations, and diffusely narrowed proximal and mid vessel segments).. Patients on everolimus versus patients on mycophenolate mofetil presented with significantly less fibrosis at 4 weeks (3.8%±0.3% vs. 5.5%±0.3%, P=0.007), 1 year (4.1%±0.3% vs. 4.8%±0.3%, P=0.015), and 3 years (4.2%±0.3% vs. 5.5%±0.7%, P=0.049) posttransplant and showed less scarring at 3 years posttransplant (19.9±1.9% vs. 31.9±4.6% vs. baseline biopsy 26.0±2.8%; P=0.017). Angiographic peripheral obliterations correlated with higher amounts of endomyocardial fibrosis. The negative correlation of everolimus and the positive correlation of peripheral obliterations with fibrosis were confirmed by regression analysis. Angiographic stenoses or acute cellular rejection had no effect on the development of fibrosis. Negative vascular remodeling in 1-year follow-up tended to be less frequent in everolimus-treated patients (24% vs. 76%; P=0.053).. Everolimus prevents endomyocardial remodeling after heart transplantation and might have beneficial effects on vascular remodeling of epicardial coronary arteries too. Angiographic peripheral obliterations correlate with increased amounts of endomyocardial fibrosis, suggesting a relevant effect on microvascular perfusion.

Topics: Coronary Angiography; Cytomegalovirus Infections; Everolimus; Female; Fibrosis; Graft Rejection; Heart Transplantation; Humans; Immunosuppressive Agents; Linear Models; Male; Middle Aged; Myocardium; Sirolimus

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

Topics: Animals; Benzamides; Disease Models, Animal; Dose-Response Relationship, Drug; Fibroblasts; Fibrosis; Imatinib Mesylate; Kidney; Kidney Diseases; Male; Piperazines; Proto-Oncogene Proteins c-abl; Pyrimidines; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Smad2 Protein; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; Ureteral Obstruction

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Cell Division; Disease Models, Animal; Fibrosis; Gene Expression; Humans; Immunosuppressive Agents; Intermediate Filament Proteins; Intracellular Signaling Peptides and Proteins; Kidney Tubules, Collecting; Kidney Tubules, Distal; Mice; Mosaicism; Nerve Tissue Proteins; Nestin; Phenotype; Polycystic Kidney Diseases; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; TRPP Cation Channels

Splenomegaly is a frequent hallmark of portal hypertension that, in some cases, can be very prominent and cause symptoms like abdominal pain, splenic infarction, and cytopenia. This study characterizes the pathogenetic mechanisms leading to spleen enlargement in portal hypertensive rats and focuses on mTOR pathway as a potential modulator of splenomegaly in portal hypertension.. Characterization of splenomegaly was performed by histological, hematological, immunohistochemical and Western blot analyses in rats with portal hypertension induced by portal vein ligation, and compared with sham-operated animals. The contribution of the mTOR signaling pathway to splenomegaly was determined in rats with fully developed portal hypertension and control rats by treatment with rapamycin or vehicle.. Our results illustrate that splenomegaly in portal hypertensive rats arises as a consequence of the interplay of several factors, including not only spleen congestion, as traditionally thought, but also enlargement and hyperactivation of the splenic lymphoid tissue, as well as increased angiogenesis and fibrogenesis. Since mTOR signaling plays a central role in immunological processes, angiogenesis and fibrogenesis, we next determined the involvement of mTOR in splenomegaly. Interestingly, mTOR signaling was overactivated in the spleen of portal hypertensive rats, and mTOR blockade by rapamycin profoundly ameliorated splenomegaly, causing a 44% decrease in spleen size. This effect was most likely accounted for the inhibitory action of rapamycin on lymphocyte proliferation, neovascularization and fibrosis.. These findings shed light on the pathogenesis of splenomegaly in portal hypertension, and identify mTOR signaling as a potential target for therapeutic intervention in this disease.

Topics: Animals; Apoptosis; Cell Division; Fibrosis; Hematocrit; Hypertension, Portal; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Lymphocytes; Lymphoid Tissue; Male; Neovascularization, Pathologic; Organ Size; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Spleen; Splenomegaly; TOR Serine-Threonine Kinases

Rapamycin, a novel macrolide immunosuppressive drug, is increasingly used as an agent for posttransplant immunosuppression and treatment of autoimmune disease. The molecular mechanism related to rapamycin-mediated immunosuppression is that rapamycin binds to FK-506 binding protein 12, and the formed complex inhibits the function of the mammalian target of rapamycin (mTOR), which in turn reduces protein phosphorylation, cell cycle progression, and cytokine production. The aim of this study was to examine the effect of rapamycin against the development of fibrosis and autoimmunity in 2 different types of systemic sclerosis (SSc) model mice.. Tight skin (TSK/+) mice and bleomycin- induced SSc model mice were used to evaluate the effect of rapamycin on fibrosis and immunologic abnormalities. Furthermore, the antifibrotic effect of rapamycin was assessed using TSK/+ mouse fibroblasts.. Treatment with rapamycin reduced skin fibrosis of TSK/+ mice and skin and lung fibrosis of bleomycin-induced SSc model mice. The production of fibrogenic cytokines, such as interleukin-4 (IL-4), IL-6, IL-17, and transforming growth factor beta1, was attenuated by rapamycin. Hypergammaglobulinemia and anti-topoisomerase I antibody production were also reduced by rapamycin treatment in TSK/+ mice. In addition, mTOR expression levels were increased in TSK/+ mouse fibroblasts compared with those in wild-type mouse fibroblasts. Rapamycin treatment inhibited proliferation and collagen production of TSK/+ mouse fibroblasts in a dose-dependent manner.. This study is the first to show that rapamycin has a significant inhibitory effect on fibrosis in both TSK/+ and bleomycin-induced SSc model mice. These results suggest that rapamycin might be an attractive candidate for clinical trials in SSc patients.

Topics: Animals; Cell Proliferation; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Immunohistochemistry; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Lung; Mice; Mice, Transgenic; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Sirolimus; Skin; Statistics, Nonparametric; TOR Serine-Threonine Kinases

Rapamycin is an immunosuppressive drug with potent antifibrotic activity. We evaluated the effect of rapamycin on murine adriamycin nephropathy, a model of progressive glomerulosclerosis and tubulointerstitial fibrosis.. Adriamycin nephropathy was induced in Balb/c mice by a single intravenous injection of adriamycin. The mice were treated orally with either saline or rapamycin, beginning at the time of adriamycin injection or rapamycin starting 1 week after adriamycin injection. The mice were sacrificed 6 weeks after adriamycin injection.. Saline-treated mice developed massive proteinuria and impaired renal function. Kidney sections from saline-treated mice showed marked focal segmental glomerulosclerosis, tubular dilation with protein cast deposition, interstitial fibrosis, and numerous infiltrating macrophages and T lymphocytes. The intrarenal expression of Collagen I and RANTES was also increased. In contrast, both groups of rapamycin-treated mice had markedly reduced proteinuria and preserved renal function, with only mild histological abnormalities. The intrarenal expression of Collagen I and RANTES was reduced, concomitant with a significant reduction in interstitial inflammatory cell infiltration.. Rapamycin is effective in attenuating the glomerular and tubulointerstitial abnormalities in adriamycin nephropathy. The beneficial effects of rapamycin are mediated, at least in part, through reduced RANTES expression and inflammatory cell infiltration.

Topics: Albuminuria; Animals; Antibiotics, Antineoplastic; Body Weight; Chemokine CCL5; Collagen Type I; Disease Models, Animal; Doxorubicin; Fibrosis; Gene Expression; Glomerulosclerosis, Focal Segmental; Immunosuppressive Agents; Kidney; Male; Mice; Mice, Inbred BALB C; Severity of Illness Index; Sirolimus; Survival Rate

Because the size of renal cysts in the native kidneys of patients with ADPKD who have been transplanted was found to be reduced when rapamycin was the immunosuppressant, we tested the involvement of the mTOR pathway in cyst enlargement. Here, male pcy mice, with mutation in one of the nephronophthisis genes, were treated with rapamycin at an early (6 to 12 weeks of age) or a later (20 to 30 weeks of age) disease stage by means of slow-release pellets containing placebo or rapamycin. Effectiveness of the rapamycin dose and delivery was shown by the inhibition of insulin-stimulated phosphorylation of p70S6K, a marker of mTOR activity, in skeletal muscle. Early treatment did not affect initial cyst development but when started late, there was a significant reduction in the rate of cyst enlargement, kidney fibrosis, and the progressive loss of renal function as measured by blood urea nitrogen. Kidneys of the mice treated through 30 weeks of age tended to be smaller and have less fibrosis compared with those of untreated or placebo-treated pcy/pcy mice at 20 weeks when treatment was initiated. Our study shows that rapamycin can prevent the late- but not the early-stage progression of renal pathology and deterioration of renal functional in this model of nephronophthisis, presumably by inhibiting mTOR activity.

Topics: Animals; Carrier Proteins; Cysts; Disease Models, Animal; Disease Progression; Fibrosis; Kidney Diseases, Cystic; Kidney Function Tests; Male; Mice; Mice, Inbred Strains; Phosphotransferases (Alcohol Group Acceptor); Sirolimus; TOR Serine-Threonine Kinases

Rapamycin represents a recognized drug-based therapeutic approach to treat cardiovascular disease. However, at least in the female heart, rapamycin may suppress the recruitment of putative signalling events conferring cardioprotection. The present study tested the hypothesis that rapamycin-sensitive signalling events contributed to the cardioprotective phenotype of the female rat heart after an ischemic insult. Rapamycin (1.5 mg/kg) was administered to adult female Sprague-Dawley rats 24 h after complete coronary artery ligation and continued for 6 days. Rapamycin abrogated p70S6K phosphorylation in the left ventricle of sham rats and the noninfarcted left ventricle (NILV) of 1-week postmyocardial-infarcted (MI) rats. Scar weight (MI 0.028 +/- 0.006, MI+rapamycin 0.064 +/- 0.004 g) and surface area (MI 0.37 +/- 0.08, MI+rapamycin 0.74 +/- 0.03 cm2) were significantly larger in rapamycin-treated post-MI rats. In the NILV of post-MI female rats, rapamycin inhibited the upregulation of eNOS. Furthermore, the increased expression of collagen and TGF-beta3 mRNAs in the NILV were attenuated in rapamycin-treated post-MI rats, whereas scar healing was unaffected. The present study has demonstrated that rapamycin-sensitive signalling events were implicated in scar formation and reactive fibrosis. Rapamycin-mediated suppression of eNOS and TGF-beta3 mRNA in post-MI female rats may have directly contributed to the larger infarct and attenuation of the reactive fibrotic response, respectively.

Topics: Animals; Blotting, Western; Cicatrix; Collagen; Disease Models, Animal; Female; Fibrosis; Heart Ventricles; Myocardial Infarction; Myocardium; Nitric Oxide Synthase Type III; Phosphorylation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Transforming Growth Factor beta3; Ventricular Remodeling

Although both sirolimus (CYPHER) and paclitaxel (TAXUS) drug-eluting stents have demonstrated efficacy and safety in clinical trials, human autopsy data have raised concerns about long-term healing and the potential for local inflammatory reactions.. Overlapping stents (CYPHER drug-eluting stents, Bx SONIC bare metal stents, TAXUS drug-eluting stents, and Liberté bare metal stents) were implanted in noninjured coronary arteries of 58 domestic swine. Histopathological evaluation of proximal, overlapped, and distal stented segments was determined with emphasis on inflammation at 30, 90, and 180 days. Circumferential granulomatous inflammation in all stented segments was defined as inflammation consisting of macrophages, multinucleated giant cells, lymphocytes, and granulocytes, including many eosinophils, adjacent to almost all struts. Circumferential granulomatous inflammation was more prevalent in CYPHER (9 of 23, 39%) compared with TAXUS (1 of 21, 5%; P=0.01) and control bare metal stents (0 of 44) in the combined 90- and 180-day cohorts. Only CYPHER specimens showed marked adventitial inflammation (P=0.0025) and fibrosis (P=0.0055) accompanied by extensive remodeling. Fibrin deposition within neointima and medial smooth muscle cell death were greater (both P<0.001) in TAXUS than CYPHER at 30 days, with more fibrin in TAXUS than CYPHER through 90 days (P<0.05).. Although these data cannot be directly extrapolated to humans, the high prevalence in this porcine model of diffuse granulomatous inflammation seen with CYPHER stents, persisting at 180 days and associated with extensive remodeling of the artery, and persistent para-strut fibrin deposition with TAXUS stents emphasize the need for further investigation of biocompatibility with these and other novel combination drug/polymer drug-eluting stents.

Topics: Animals; Arteritis; Coronary Vessels; Disease Models, Animal; Drug-Eluting Stents; Eosinophils; Female; Fibrin; Fibrosis; Granuloma, Foreign-Body; Paclitaxel; Sirolimus; Swine; Tunica Intima

Hepatic stellate cell transdifferentiation, epithelial-mesenchymal cell transition, and the ductular reaction each contribute to the development of hepatic fibrosis in cholestatic liver diseases. Inhibitors of mammalian target of rapamycin have antifibrotic properties. We evaluated the hypothesis that the antifibrotic action of rapamycin is due to attenuated myofibroblast proliferation in addition to an inhibitory effect on epithelial-mesenchymal transition and the ductular reaction. Hepatic fibrosis was induced by bile duct ligation, and rodents received 1.5 mg/kg/day rapamycin by subcutaneous infusion for 21 days. The expression of various markers of hepatic fibrosis, stellate cell transactivation, epithelial-mesenchymal transition, and the ductular reaction was compared between treated and untreated animals. Hepatic fibrosis, hepatic procollagen type 1 messenger RNA, and alpha-smooth muscle actin expression were significantly reduced in treated animals. Hepatic stellate cell procollagen expression and proliferation were also reduced by rapamycin. The following markers of epithelial-mesenchymal transition--vimentin protein expression, S100 calcium binding protein A4 and transforming growth factor beta 1 messenger RNA, and the mothers against decapentaplegic homolog signaling pathway--were all reduced after rapamycin treatment. The intensity of the ductular reaction was reduced by rapamycin as assessed by histopathological scoring and by reduced cytokeratin 19 expression. Rapamycin caused a reduction in hepatic progenitor cell proliferation. Together, these data show that multiple profibrogenic pathways are activated in an animal model of cholestasis and that rapamycin attenuates epithelial-mesenchymal transition and the ductular reaction as well as hepatic stellate cell activation.

Topics: Animals; Cell Differentiation; Cell Proliferation; Epithelium; Fibrinogen; Fibrosis; Immunosuppressive Agents; Liver; Liver Cirrhosis; Male; Mesoderm; Models, Biological; Rats; Rats, Wistar; Sirolimus; Stem Cells

Fibrocytes are collagen-type-I-producing cells that arise at low frequency from hematopoietic cells. We have analyzed in mice which leukocyte subsets are required for generation of fibrocytes and show that murine fibrocytes develop from the subpopulation of CD11b(+) CD115(+) Gr1(+) monocytes under the control of CD4(+) T cells. In the absence of CD4(+) T cells, differentiation of fibrocytes was markedly reduced in vitro and in vivo. In the presence of CD4(+) T cells, the characteristics of T-cell activation critically determined development of fibrocytes. Polyclonal activation of CD4(+) T cells induced the release of soluble factors that completely prevented the outgrowth of fibrocytes and could be identified as IL-2, TNF, IFN-gamma, and IL-4. Application of IL-2 and TNF significantly reduced the appearance of fibrocytes and the severity of fibrosis in the model of unilateral ureteral obstruction. In contrast, activation of CD4(+) T cells in the presence of calcineurin inhibitors, but not mTOR inhibitors, markedly enhanced the outgrowth of fibrocytes and renal deposition of collagen I. Taken together, we show that differentiation of fibrocytes is critically dependent on CD4(+) T cells and that the context of T-cell activation determines whether development of fibrocytes is supported or blocked. Our data may have implications for prevention of organ fibrosis in autoimmune diseases and transplantation.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Differentiation; Cells, Cultured; Collagen Type I; Cyclosporine; Cytokines; Female; Fibrosis; Immunosuppressive Agents; Kidney; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Monocytes; Rats; Rats, Inbred Lew; Receptors, Chemokine; Sirolimus

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using alpha-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.

Topics: AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Atrial Fibrillation; Carrier Proteins; Collagen Type I; Collagen Type II; Fibrosis; Gene Expression Regulation; Heart Atria; Hypertrophy, Left Ventricular; Mice; Mice, Knockout; Myocardium; Organ Specificity; Peptide Elongation Factor 2; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Ischemia reperfusion injury (IRI) has long-term sequelae on kidney allograft function. Early initiation of rapamycin can retard surgical wound healing and recovery from IRI. In contrast, rapamycin may paradoxically retard long-term fibrotic effects of kidney IRI. We, therefore, hypothesized that delayed initiation of rapamycin after kidney ischemia, started after the initial week of wound healing, would decrease the long-term inflammation and fibrosis caused by IRI. C57BL/6 male mice were subjected to either 45 or 60 minutes of unilateral kidney ischemia or a sham operation. Mice were given rapamycin (subcutaneous, 1.5 mg/kg/d) or vehicle starting at 1 week after IRI surgery for 3 weeks. Urine albumin excretion, kidney histology, and kidney cytokine proteins were examined at 4 weeks after surgery. The 3-week treatment course of rapamycin significantly reduced body weight gain in all 3 groups and reduced postischemic kidney weight in both the 45- and 60-minute ischemia groups, but unexpectedly increased urine albumin excretion in all rapamycin-treated sham or IRI mice compared with vehicle-treated mice. Rapamycin treatment showed minimal effects on postischemic kidney fibrosis with variable effects on various cytokine/chemokine protein expressions, namely, decreasing interleukin (IL)-1alpha, IL-6, tumor necrosis factor (TNF)-alpha, and regulated on activation normal T cell expressed and secreted (RANTES) while increasing IL-4, keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP-1alpha), and IL-10 in the ischemic kidney. These data demonstrated that rapamycin reduced mouse body weight and ischemic kidney weight, while increasing urinary albumin excretion. Delayed initiation of rapamycin after IRI had a minimal effect on renal fibrosis and mixed effects on proinflammatory mediator production. These data do not support delayed initiation of rapamycin after IRI to attenuate IRI-induced progressive fibrosis and inflammation, and They raise further caution regarding rapamycin and albuminuria.

Topics: Albuminuria; Animals; Body Weight; Cytokines; Fibrosis; Immunosuppressive Agents; Injections, Subcutaneous; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Organ Size; Reperfusion Injury; Sirolimus; Weight Gain

To investigate the anti-fibrotic effect of sirolimus (rapamycin) at the cell level.. The primary cultured rat renal cortical myofibroblasts were divided into two groups, control group and sirolimus 40 mg/L group at each time point. The protein levels of alpha-SMA, Col-I, fibronectin(FN) were analyzed by Western blot in both the whole cell lysates and supernatant culture media 12 h , 24 h and 48 h after incubation, respectively. Real-time quantitative PCR was carried out to measure the levels of procollagen-I mRNA 1 h, 2 h, 4 h, and 6 h after cell incubation. The activities of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell media were assayed by gelatin zymography.. (1) Sirolimus had no effect on the expression of alpha-SMA of myofibroblasts at different time points. (2) The expression of Col-I in the whole cell lysates both reduced at the end of 24 h and 48 h in sirolimus group significantly [(0.58+/-0.05) and (0.63+/-0.18), P < 0.05] compared with control group at each time point, respectively. (3) The levels of procollagen-I mRNA reduced significantly at the end of 1 h and 2 h compared with control group at each time point [(0.38+/-0.05) and (0.55+/-0.16), P < 0.05], but increased to basic level at the end of 4 h. (4) The myofibroblasts had basic expression of Col-I early at the end of 12 h, its expression in supernatant culture medium reduced obviously both at 24 h and 48 h in sirolimus group compared with control group of each time point [(0.59+/-0.25) and (0.52+/-0.21), P < 0.05]. (5) The expression of FN in the whole cell lysates had the same trend as that in supernatant culture medium, which reduced obviously at the end of 24 h in sirolimus group compared with control group at each time point [(0.44+/-0.09) and (0.40+/-0.15), P < 0.05], but the inhibitive effect of sirolimus on FN disappeared at the end of 48 h. (6) The activities of MMP-2 and MMP-9 in the supernatant culture media were not significantly changed along with the experimental time points.. Sirolimus may exert its anti-fibrotic effect through the inhibition of the expression of Col-I and/or FN in cultured renal cortical myofibroblasts.

Topics: Actins; Animals; Cells, Cultured; Collagen Type I; Fibroblasts; Fibronectins; Fibrosis; Kidney Cortex; Rats; RNA, Messenger; Sirolimus

Scleroderma is a common autoimmune disorder with no effective therapy. Current concepts of scleroderma include the hypothesis that scleroderma results from excess conversion of endothelial cells to fibroblast like cells, called endothelial mesenchymal transformation. This process is thought to be mediated by cytokines including transforming growth factor beta (TGFb), which causes increased collagen synthesis, resulting in fibrosis, the hallmark of the disease. In vitro studies have hypothesized that rapamycin may be of benefit in scleroderma due to antagonism of collagen synthesis. Given that rapamycin has antiangiogenic activities, inhibits wound healing, and prevents the synthesis of collagen in vivo, we tried rapamycin in a patient with scleroderma. We observed rapid improvement in skin stiffness and mobility. Our results provide the rationale for larger clinical trials of rapamycin in scleroderma and other fibrotic disorders.

Topics: Adult; Angiogenesis Inhibitors; Collagen; Female; Fibrosis; Humans; Immunosuppressive Agents; Mesoderm; Pregnancy; Pregnancy Complications; Scleroderma, Systemic; Sirolimus; Transforming Growth Factor beta; Treatment Outcome

Treatment options in human mesangioproliferative glomerulonephritis/sclerosis, mostly IgA nephropathy, are limited. Progressive mesangioproliferative nephropathy represents a major cause of end-stage kidney disease. The present study explores the efficacy of low-dose mTOR inhibition by rapamycin in a chronic-progressive model of mesangioproliferative glomerulosclerosis (cGS). cGS was induced by high-dose anti-thy1 antibody injection into uninephrectomized rats. Rapamycin administration (2.5 mg.kg(-1).body wt(-1)) was started 10 days after antibody injection and continued until week 20. cGS was characterized by advancing proteinuria, increased blood pressure, marked tubulointerstitial and glomerular fibrosis, cell proliferation and round cell infiltration, and impaired renal function. Kruskal-Wallis and Mann-Whitney U-tests were used for statistical analysis. The course of chronic anti-thy1-induced glomerulosclerosis was significantly attenuated by low-dose rapamycin treatment. In week 20, this was demonstrated by improvements in proteinuria (-38%), systolic blood pressure (-16 mmHg), tubulointerstitial and glomerular histological matrix accumulation (-61 and -24%), transforming growth factor-beta1 overexpression (-41 and -47%), collagen I deposition (-53 and -65%), cell proliferation (-90 and -76%), and leukocyte number (macrophages -52 and -53%; lymphocytes -58 and 51%), respectively. Rapamycin improved renal function as well (blood creatinine -0.68 mg/dl, urea -66.7 mg/day, and creatinine clearance +0.13 ml.min(-1).100 g body wt(-1)). In conclusion, low-dose mTOR inhibition by rapamycin limits the progressive course of anti-thy1-induced renal disease toward chronic glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency. Renoprotection by rapamycin involved significant beneficial effects on multiple key pathways in the progression of chronic renal disease, i.e., proteinuria, extracellular matrix accumulation, renal cell proliferation, and inflammatory cell infiltration.

Topics: Animals; Blood Pressure; Body Weight; Cell Movement; Cell Proliferation; Collagen Type I; Creatinine; Disease Models, Animal; Extracellular Matrix Proteins; Fibrosis; Glomerular Filtration Rate; Glomerulonephritis, Membranoproliferative; Isoantibodies; Kidney; Kidney Function Tests; Leukocytes, Mononuclear; Lipids; Male; Protein Kinases; Rats; Rats, Wistar; Sirolimus; Tissue Inhibitor of Metalloproteinase-1; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1; Urea

The association of calcineurin inhibitors (CNIs) with mTOR inhibitors (mTORi) is still a problem in clinical practice and there is substantial interest in better understanding the impact of these associations on kidney toxicity. We aimed to analyse the functional and histological profiles of damage and to define the contribution of inflammatory and pro-fibrotic mediators in the association of cyclosporine (CsA) and/or tacrolimus (Tac) with sirolimus (SRL).. A well-defined model of nephrotoxicity in salt-depleted male rats was used. Monotherapy groups were distributed as a non-treated control group with saline solution (n = 12), the Tac group (n = 16) (tacrolimus 6 mg/kg/day) and the CsA group (n = 13) (CsA 15 mg/kg/day). The groups with different associations were scattered as the Tac + SRL group (n = 14) (tacrolimus 6 mg/kg/day and rapamycin 3 mg/kg/day) and the CsA + SRL group (n = 7) (CsA 15 mg/kg/day and rapamycin 3 mg/kg/day). Groups were divided into 30 and 70 days of follow-up, but the CsA + SRL group was only studied for 30 days because animals became sick.. Rats with the CsA + SRL association were the only ones which showed a significant reduction in body weight, impairment of renal function and severe and diffuse tubular vacuolization and tubular atrophy following a striped distribution, and scarce areas of the kidney were still preserved. The Tac + SRL association did not produce renal function impairment, and mild histological damage including enhanced periglomerular tubular atrophy was observed. This local damage affected the distal convoluted tubule involving macula densa and juxtaglomerular apparatus. Pro-inflammatory mediators paralleled functional and structural data. ED-1 and TNF-alpha were noticeably higher in the CsA + SRL than in the Tac + SRL association. Only in the CsA + SRL association an important increase in alpha-SMA+ cells was seen, mainly found in the areas with tubular atrophy. TGF-beta1 was also markedly enhanced in the CsA + SRL association whilst monotherapy or Tac + SRL groups at 30 days TGF-beta1 did not show any changes. However, at 70 days of treatment TGF-beta1 was significantly increased in the Tac + SRL group. Animals receiving SRL showed a decrease in renal vascular endothelial growth factor (VEGF) expression. This growth factor was significantly down-regulated in both CNI associations than in SRL monotherapy. P-glycoprotein (Pgp) was overexpressed in CsA and CsA + SRL therapy whilst Tac and TAC + SRL showed a middle increase Pgp expression but higher than the control and SRL group.. We conclude that the association of SRL with high doses of CsA or Tac produces a different functional, histological, inflammatory and pro-fibrogenic pattern. Thus, the addition of SRL to high doses of CsA leads to severe renal injury. Combination with high doses of Tac is clearly less deleterious in the short term. However, there is a low grade of pro-fibrotic inflammatory expression when this association is prolonged.

Topics: Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcineurin Inhibitors; Cyclosporine; Drug Interactions; Fibrosis; Immunosuppressive Agents; Interferon-gamma; Kidney; Male; Protein Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; Tacrolimus; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Decorin, a small leucine-rich proteoglycan, affects the synthesis of the elastic fiber component fibrillin-1 in the kidney via hitherto unknown mechanisms. Here, we show that decorin binds to and induces phosphorylation of insulin-like growth factor-I (IGF-I) receptor in renal fibroblasts. Inhibition of the IGF-I receptor tyrosine kinase and its downstream target phosphoinositide-3 kinase prevented decorin-mediated synthesis of fibrillin-1. Furthermore, decorin induced phosphorylation of phosphoinositide-dependent kinase 1, protein kinase B/Akt, mammalian target of rapamycin (mTOR), and p70 S6 kinase. Accordingly, the enhanced synthesis of fibrillin-1 was blocked by rapamycin, an inhibitor of mTOR. Notably, IGF-I, which signals through the same pathway, also stimulated fibrillin-1 synthesis. Systemic administration of rapamycin to mice subjected to unilateral ureteral obstruction, a model of renal fibrosis and increased fibrillin-1 synthesis, markedly reduced the number of interstitial fibroblasts and fibrillin-1 deposition. In streptozotocin-induced diabetes, IGF-I receptor was up-regulated in the kidneys from decorin-null mice. However, this could not compensate for the decorin deficiency, resulting ultimately in decreased fibrillin-1 content. This study provides evidence for the involvement of decorin and the IGF-I receptor/mTOR/p70 S6 kinase signaling pathway in the translational regulation of fibrillin-1.

Topics: Animals; Cells, Cultured; Decorin; Diabetes Mellitus, Type 1; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Fibroblasts; Fibrosis; Humans; Immunohistochemistry; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Protein Binding; Protein Kinases; Proteoglycans; Rats; Receptor, IGF Type 1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Renal fibrosis is a hallmark of end-stage renal diseases and of chronic allograft nephropathy (CAN). Rapamycin, besides its action through blockade of lymphocyte proliferation, also has antiproliferative, antiviral, and antitumor actions. Its use in clinical in patients with CAN has recently been advocated.. Our goal was to evaluate the effect of rapamycin in an established model of renal fibrosis, unilateral ureteral obstruction.. C57BL/6 mice were divided into two groups, treated or not with daily doses of rapamycin (0.2 mg/kg) beginning on day-1. The obstruction was performed as day 0. Blood and kidney tissues were collected at 1, 4, 7, and 14 days after the surgery to quantify bone morphogenic protein (BMP)-7 and transforming growth factor (TGF)-beta mRNA by real time PCR.. Daily treatment with rapamycin caused a significant reduction in serum creatinine at day 1 (0.57 +/- 0.03 vs 0.95 +/- 0.15 mg/dL, P = .002) and at day 14 (0.56 +/- 0.04 vs 0.73 +/- 0.07 mg/dL, P = .040). This profile was corroborated by histological morphometric analyses showing less fibrosis at day 14. However, rapamycin surprisingly induced an upregulation of TGF-beta at day 4 (3.05 +/- 0.46 vs 1.85 +/- 0.41, P = .006) and at day 7 (6.33 +/- 0.55 vs 4.97 +/- 0.38, P = .024) with a reduced expression by day 14 (4.03 +/- 1.07 vs 7.89 +/- 0.83, P < .001). Surprisingly, rapamycin also promoted an increment in BMP-7, completely reversing the ratio of TGF-beta to BMP-7, allowing a more protective phenotype.. Rapamycin slightly ameliorated the renal dysfunction and, at later time points, induced less fibrosis and less decrease in the TGF-beta to BMP-7 ratio.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Creatinine; Disease Models, Animal; Disease Progression; Fibrosis; Immunosuppressive Agents; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Male; Mice; Mice, Inbred C57BL; RNA, Messenger; Sirolimus; Transforming Growth Factor beta

Is a common feature of progressive renal diseases regardless of the initiating insult To clarify the role of connective tissue growth factor (CTGF) in after (UUO) in renal interstitial fibrosis and effects of rapamycin (RAP) thereupon.. Eighteen Sprague-Dawley rats were randomly divided into 3 equal groups: unilateral ureteral obstruction (UUO) model group, undergoing ligation of the left ureter; RAP treatment group, undergoing ligation of the left ureter and intraperitoneal injection of RAP 0.04 mg.kg(-1).d(-1); and sham operation group. The right kidneys were taken out 7 and 14 days after the operation respectively to undergo renal pathological examination by Masson staining. Semi-quantitative RT-PCR was used to detect the mRNA expression of CTGF. Western blotting was performed to examine the protein expression of CTGF and fibronectin (FN).. In comparison with the sham operation group, renal interstitial fibrosis was significant more expression in the 2 UUO groups, especially the UUO model group (P < 0.01). Seven and 14 days after the operation the levels of CTGF mRNA expression of the UUO model and RAP treatment groups (both P < 0.01), and the level of CTGF mRNA expression of the RAP treatment group was significantly lower than that of the UUO model group (P < 0.01), however, there was no significant difference in the level of CTGF mRNA expression between the 2 UUO groups 14 days after the operation. Seven and 14 days after the operation the levels of CTGF protein expression of the UUO model and RAP treatment groups were both significantly higher than that of the sham operation group (both P < 0.01), and the levels of CTGF protein expression of the RAP treatment group were significantly lower than that of the UUO model group (P < 0.05 and P < 0.01). The levels of FN expression 7 and 14 days after the operation of the 2 UUO groups were both significantly higher than that of the sham operation group (both P < 0.01). and the level of FN expression 7 days after of the RAP treatment group was significantly lower than that of the UUO model group (P < 0.01), however, there was no significant difference in the level of FN expression between the 2 UUO groups 14 days after the operation.. The expression of CTGF mRNA and that of CTGF protein increase after UUO. Rapamycin play a protective role in the kidney by downregulating the CTGF expression and alleviating the renal interstitial fibrosis following UUO.

Topics: Animals; Blotting, Western; Connective Tissue Growth Factor; Disease Models, Animal; Fibrosis; Gene Expression; Immediate-Early Proteins; Immunosuppressive Agents; Injections, Intraperitoneal; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a well-characterized hydronephrosis model exhibiting interstitial inflammatory-cell infiltration and tubular dilatation followed by tubulointerstitial fibrosis of the obstructed kidney. Our recent report indicates that rapamycin is effective for 50% of transplant recipients with chronic allograft nephropathy. In this study, we investigate the effect of rapamycin on UUO-induced renal fibrosis. UUO or sham-operated rats were randomly assigned to rapamycin or vehicle and were killed on days 7 and 14 after UUO or sham operation. Rapamycin decreased cross-sectional and gross-morphology changes in the obstructed kidney significantly. Rapamycin markedly blunted the increase in weight of the obstructed kidney, obstructed kidney length, and the obstructed/non-obstructed kidney weight ratio (by 74.6, 42.8, and 61.6% on day 14, respectively, all P<0.01). The scores for tubular dilatation, interstitial volume, interstitial collagen deposition, and alpha-smooth muscle actin (alpha-SMA) after UUO were significantly reduced by rapamycin. Rapamycin also decreased the number of infiltrative anti-ED1-positive cells and the gene expression of transforming growth factor (TGF)-beta1 (84.8 and 80.2% on day 7) after UUO (both P<0.01). By double immunostaining and Western analysis, rapamycin blocked the TGF-beta1-induced loss of E-cadherin expression and de novo increase of the expression of alpha-SMA in a dose-dependent manner. In conclusion, rapamycin significantly attenuated tubulointerstitial damage in a UUO-induced rat model of renal fibrosis, suggesting that rapamycin may have the potential to delay the progression of tubulointerstitial renal fibrosis.

Topics: Animals; Fibrosis; Immunosuppressive Agents; Kidney; Male; Rats; Rats, Sprague-Dawley; Sirolimus; Ureteral Obstruction

Inhibition of established left ventricular hypertrophy (LVH) and fibrosis may bring clinical benefits by reducing cardiac morbidity and mortality. The mammalian target of rapamycin, mTOR, is known to play a critical role in determining cell and organ size. We investigated whether mTOR inhibition can inhibit the chronic pressure-overload-induced LVH and fibrosis.. Male FVB/N mice underwent transverse aortic constriction (TAC) for 5 weeks to allow for establishment of LVH, followed by treatment with the mTOR inhibitor, Rapamune (2 mg/kg per day, gavage), for 4 weeks. Echocardiography was used to monitor changes in LVH and function. Haemodynamic, morphometric, histological and molecular analyses were conducted.. Inhibition of mTOR by Rapamune was confirmed by a suppression of activated phosphorylation of ribosomal S6 protein and eukaryotic translation initiation factor-4E due to pressure overload. Despite a comparable degree of pressure overload between the vehicle- or Rapamune-treated TAC groups, Rapamune treatment for 4 weeks attenuated TAC-induced LVH by 46%, estimated by LV weight or myocyte size, and LV fractional shortening was also preserved versus vehicle-treated control (39 +/- 1 versus 32 +/- 2%, P < 0.05). Inhibition of established LVH by Rapamune was associated with a 38% reduction in collagen content. Moreover, altered gene expression due to pressure overload was largely restored.. Despite sustained pressure overload, inhibition of mTOR by a 4-week period of Rapamune treatment attenuates chronically established LVH and cardiac fibrosis with preserved contractile function.

Topics: Analysis of Variance; Animals; Atrial Natriuretic Factor; Blood Pressure; Chronic Disease; Disease Models, Animal; Down-Regulation; Eukaryotic Initiation Factor-4E; Fibrosis; Heart Rate; Hypertrophy, Left Ventricular; Immunosuppressive Agents; Male; Mice; Mitogen-Activated Protein Kinase 3; Myosin Heavy Chains; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Ribosomal Protein S6; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Sirolimus; STAT3 Transcription Factor; Stroke Volume; TOR Serine-Threonine Kinases

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-beta, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect alpha1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle alpha-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis approximately 2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.

Topics: Animals; Cell Cycle; Cell Proliferation; Cells, Cultured; Chromones; Collagen Type I; Cyclins; DNA; Enzyme Inhibitors; Fibrosis; Gene Expression Regulation; Hepatocytes; Humans; Liver; Male; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Transforming Growth Factor beta

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-beta(1), and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.

Topics: Animals; Cytokines; Disease Models, Animal; Fibrosis; Glomerulonephritis, Membranous; Immunosuppressive Agents; Kidney; Male; Proteinuria; Rats; Rats, Sprague-Dawley; Sirolimus

We have previously shown the long-term influence of renal ischemia/reperfusion (I/R) injury and immunosuppression on fibrotic genes and apoptosis in a rat model. For the first time, we have now investigated the effects of I/R and immunosuppression on inflammation and caspase activation.. I/R injury was induced in the right kidney and the left was removed. Cyclosporin (CsA) (10 mg/kg), tacrolimus (0.2 mg/kg), rapamycin (1 mg/kg), or mycophenolate mofetil (MMF) (10 mg/kg) was then administered for 16 weeks. The effects of I/R and immunosuppressants on interstitial inflammation, interleukin (IL)-1beta expression, caspase-1 and caspase-3 activation, tubulointerstitial damage, and fibrosis were evaluated.. ED-1+ (a specific rat monocyte/macrophage marker) cells were mainly localized in the tubulointerstitium and periglomerular areas and increased in I/R group compared to controls (P < 0.01). This was further increased by CsA, but decreased by tacrolimus, rapamycin, or MMF (P < 0.05). The 17 kD active IL-1beta remained unchanged, but 35 kD IL-1beta precursor was decreased by rapamycin in comparison with I/R group (P < 0.05). The 45 kD or 20 kD caspase-1 was increased by I/R or CsA, respectively, and decreased by rapamycin (P < 0.05). The 24 kD caspase-3, which proved to be an active caspase-3 subunit, was increased in I/R and CsA groups and deceased by tacrolimus, rapamycin, or MMF (P < 0.05), but not 32 kD precursor or 17 kD active caspase-3. The activity data of caspase-1 and caspase-3 exhibited the same trend as Western blotting data. The staining of active caspase-3 was scattered in kidneys, mainly in tubular and interstitial areas, which was consistent with that of ED-1+ cells. There was a strong positive correlation between interstitial inflammation and 24 kD caspase-3 expression or caspase-3 activity (r = 0.814 or 0.484), all of which were also closely related with urinary protein (r = 0.537, 0.529, or 0.517), serum creatinine (r = 0.463, 0.573, or 0.539), tubulointerstitial damage (r = 0.794, 0.618, or 0.712) and fibrosis (r = 0.651, 0.567, or 0.469), all P < 0.01.. This study shows that the mechanisms of long-term I/R injury and immunosuppressants treatment include interstitial inflammation and caspase activation, most clearly demonstrated by the 24 kD active caspase-3.

Topics: Animals; Apoptosis; Caspase 1; Caspase 3; Caspases; Chronic Disease; Cyclosporine; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Fibrosis; Glomerulonephritis; Immunosuppressive Agents; Interleukin-1; Male; Mycophenolic Acid; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; Tacrolimus

Modern immunosuppressive agents such as tacrolimus and rapamycin are claimed to be associated with a reduction in vascular narrowing, a central feature of chronic rejection. This study assesses the effect of cyclosporine, tacrolimus and rapamycin on the development of intimal thickening, fibrosis-associated genes and deposition of extracellular matrix (ECM) proteins in a model of intimal hyperplasia. Male Sprague-Dawley rats received either no treatment or 5 mg/kg cyclosporine, 0.1 mg/kg tacrolimus or 0.05 mg/kg rapamycin. Animals underwent left common carotid balloon angioplasty, and intima medial ratios, pro-fibrotic gene expression and ECM accumulation were calculated at 14 and 28 days. Cyclosporine was associated with increased intimal thickening compared to controls ( P < 0.004). Tacrolimus had no effect on intimal thickening, whilst rapamycin significantly inhibited intimal thickening at both 14 and 28 days ( P < 0.004 and P < 0.026, respectively). All groups significantly inhibited matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, transforming growth factor (TGF)-beta and collagen III expression at 14 days ( P < 0.001), but increased ECM deposition. However, rapamycin marginally reduced ECM deposition compared to cyclosporine ( P < 0.06). Treatment with cyclosporine was associated with worsening of vascular narrowing, whilst rapamycin showed a beneficial reduction in intimal thickening. Treatment with all immunosuppressive agents resulted in increased ECM deposition. Rapamycin may halt the progression of vascular narrowing compared to both cyclosporine and tacrolimus.

Topics: Angioplasty, Balloon; Animals; Carotid Artery, Common; Collagen Type III; Cyclosporine; Extracellular Matrix Proteins; Fibrosis; Gene Expression; Hyperplasia; Immunosuppressive Agents; Male; Matrix Metalloproteinase Inhibitors; Rats; Rats, Sprague-Dawley; Sirolimus; Tacrolimus; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Tunica Intima

Epithelial cell injury, inflammation, progressive fibrosis, and airway obliteration are histological features of post-transplant obliterative bronchiolitis (OB). Cyclooxygenase (COX)-2 is expressed in acute and chronic inflammatory responses. Our aim was to elucidate the possible role of COX-2 in post-transplant OB by using a heterotopic bronchial porcine model. Bronchial allografts from non-related donors were transplanted subcutaneously into 24 random-bred domestic pigs, each weighing about 20 kg. Groups studied had grafts, non-treated allografts, allografts given cyclosporine A (CsA), methylprednisolone (MP), and azathioprine (Aza), and allografts given CsA, MP, and everolimus. Grafts were serially harvested during a follow-up period of 21 days for histology (H&E) and immunohistochemistry. Immunostaining was performed with monoclonal IgG against human COX-2 peptide, and histological alterations and immunohistochemical positivity were graded on a scale from 0 to 5. Epithelial COX-2 index was calculated by multiplying the percentage of positive cells by grade of epithelial COX-2 intensity. Ischaemic epithelial loss, evident in all implants, recovered rapidly in autografts, and bronchi remained patent. Epithelial loss in non-treated allografts preceded fibroblast proliferation, resulting in total luminal obliteration. In CsA-, MP-, and Aza-treated allografts epithelial destruction and luminal obliteration were delayed, and these were prevented in CsA-, MP-, and everolimus-treated allografts. COX-2 expression due to operative ischaemia was evident in all implants on day 2. Thereafter, the epithelial COX-2 index preceded epithelial injury and obliteration. During the inflammatory response and fibroblast proliferation, COX-2 expression occurred in macrophages and fibroblasts. In conclusion, in the early stage of OB development, COX-2 induction occurred in airway epithelial cells prior to luminal obliteration. In addition, the observation that COX-2 expression in macrophages and fibroblasts paralleled the onset of inflammation and fibroblast proliferation indicates a role in OB development, but the causal relationships need further study.

Topics: Animals; Azathioprine; Bronchi; Bronchiolitis Obliterans; Chondrocytes; Cyclooxygenase 2; Cyclosporine; Disease Models, Animal; Epithelial Cells; Everolimus; Fibroblasts; Fibrosis; Immunohistochemistry; Immunosuppressive Agents; Isoenzymes; Macrophages; Methylprednisolone; Postoperative Period; Prostaglandin-Endoperoxide Synthases; Sirolimus; Swine

Rapamycin inhibits extracellular matrix (ECM) accumulation (fibrosis) and vascular remodeling in experimental models of chronic allograft dysfunction (CAD) by poorly understood mechanisms. The aim of this study was to assess the effect of rapamycin on the expression of fibrosis-associated genes and correlate this with observed changes in ECM remodeling in an experimental of model allograft vasculopathy.. Vascular remodeling and ECM accumulation (picrosirius red) were measured by computerized histomorphometry of F344-to-Lewis rat aortic allograft sections harvested at serial timepoints. Expression of fibrosis associated genes was studied by means of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).. Rapamycin (0.5 mg/kg/day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodeling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intragraft gelatinase, collagen III and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA levels. At a lower dosage (0.25 mg/kg/day), rapamycin inhibited intimal hyperplasia and medial ECM accumulation, but there was a lesser effect on vascular remodeling. Lower dose allografts were also seen to have a more severe inflammatory infiltrate and larger amounts of intragraft matrix metalloproteinase 9 (MMP 9) mRNA than those treated with the higher dose.. These data suggest that, in addition to the tissue response to injury, the alloimmune injury itself may contribute directly to the vascular remodeling that occurs in allograft vasculopathy. Rapamycin at higher but not lower doses inhibited both of these pathologic processes.

Topics: Animals; Aorta; Fibrosis; Gene Expression; Immunosuppressive Agents; Models, Animal; Organ Transplantation; Rats; Rats, Inbred Lew; Sirolimus; Vascular Diseases

Chronic allograft dysfunction, the leading cause of solid-organ transplant failure, is characterised by histological evidence of extracellular matrix (ECM) accumulation (fibrosis). The aim of this study was to compare the effect of combined rapamycin and cyclosporine therapy on fibrosis-associated gene expression and ECM turnover during the development of allograft vasculopathy, compared with either agent alone. Lewis recipients of F344 rat thoracic-to-abdominal aorta transplants were administered rapamycin, cyclosporine, combined rapamycin and cyclosporine or no treatment. F344-to-F344 isografts served as controls. Six grafts in each group were harvested at 16 weeks. Vascular remodelling and ECM accumulation (Sirius red) were measured by computerised histomorphometry of aortic sections. Messenger RNA was extracted from frozen tissue, and expression of fibrosis-associated genes was studied by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Rapamycin (0.5 mg/kg per day) or cyclosporine (5 mg/kg per day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodelling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intra-graft gelatinase, collagen III and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA levels. Combined rapamycin and cyclosporine inhibited intimal hyperplasia; however, there was a lesser effect on vascular remodelling and medial ECM accumulation. Combined-treatment aortic allografts were also seen to have a more-severe inflammatory infiltrate and larger amounts of intra-graft matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-beta and TIMP-1 mRNA than those treated with monotherapy. Rapamycin and cyclosporine act synergistically to inhibit intimal hyperplasia but not the inflammatory infiltrate, allograft fibrosis or vessel remodelling. In the high-responder F344-to-Lewis rat model, effective immunosuppression is required to reduce graft fibrosis.

Topics: Animals; Aorta; Aortic Diseases; Azo Compounds; Cyclosporine; Drug Therapy, Combination; Fibrosis; Gene Expression; Graft Rejection; Immunosuppressive Agents; Matrix Metalloproteinase 9; Rats; Rats, Inbred F344; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Transforming Growth Factor beta; Transplantation, Homologous

Most patients with functioning heart transplants have established and progressive chronic allograft vasculopathy, a fibroproliferative process for which there is no effective treatment. Coronary artery disease is characterized by histologic evidence of extracellular matrix accumulation (fibrosis). This study compares the effect of rapamycin administered to rats with established allograft vasculopathy on histologic indices of disease progression, extracellular matrix accumulation (fibrosis), and the expression of genes known to regulate extracellular matrix turnover in this model.. Lewis recipients of Fisher 344 rat thoracic to abdominal aorta transplants were administered rapamycin starting at 8, 12, and 16 weeks posttransplant or no treatment. Six grafts in each group were harvested at 24 weeks. Vascular remodeling and collagen accumulation (Sirius red) were measured by computerized histomorphometry of aortic sections. mRNA was extracted from frozen tissue, and the expression of fibrosis-associated genes was studied by means of semiquantitative reverse transcriptase-polymerase chain reaction.. Rapamycin had no effect on the progression of early or established allograft vasculopathy with regard to intimal thickening, remodeling, extracellular matrix accumulation, or profibrotic gene expression, regardless of the time commenced.. The attenuation of the fibroproliferative response in rodents by rapamycin is not seen if the onset of rapamycin therapy is delayed.

Topics: Animals; Aorta, Thoracic; Extracellular Matrix; Fibrosis; Gene Expression; Inflammation; Rats; Rats, Inbred F344; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; Transplantation, Homologous; Transplantation, Isogeneic