raffinose and Fibrosis

Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation.. Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI.. Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft.. Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Butadienes; Cell Hypoxia; Cell Line; Cold Ischemia; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Glutathione; Heart Transplantation; Insulin; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitriles; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Raffinose; Rats; Signal Transduction; Ventricular Function, Left

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cells, Cultured; Chemistry, Pharmaceutical; Curcumin; Cyclodextrins; Disease Models, Animal; Fibrosis; Flow Cytometry; Glutathione; Graft Rejection; Humans; Inflammation; Insulin; Kidney Transplantation; Kidney Tubules; Male; Organ Preservation Solutions; Oxidative Stress; Prostate; Raffinose; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine

Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury. Coagulation represents a key mechanism involved in this process. Melagatran (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-beta (TGF-beta) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

Topics: Adenosine; Allopurinol; Animals; Anticoagulants; Azetidines; Base Sequence; Benzylamines; Chronic Disease; DNA Primers; Fibrosis; Glutathione; Humans; Insulin; Kidney; Kidney Transplantation; Male; Models, Animal; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Plasminogen Activator Inhibitor 1; Raffinose; RNA, Messenger; Signal Transduction; Swine; Temperature; Tissue Donors; Tissue Plasminogen Activator; Transforming Growth Factor beta; Transplantation, Autologous

To assess the function of arterial grafts after prolonged preservation in the University of Wisconsin solution (UW), in vitro and in vivo.. Carotid arteries were harvested from dogs and stored for 1-21 days at 4 degrees C in UW (n = 10) or in PBS (0.9% NaCl, pH 7.4), (PBS) (n = 10). Slices were examined by lightmicroscopy (LM) and scanning electron microscopy (SEM). For viability testing, specimens were connected to an isometric force transducer (2 x n = 9). Contractile and relaxation responses were examined by adding phenylephrine (200 microM) and metacholine (200 microM), respectively. For in vivo studies (n = 41), 2.5cm carotid artery segments were implanted or orthotopically, as autografts and allografts, after 14 days of storage in UW or in PBS. Autologous veins were used as controls. After 28 days or 56 days, arteriography was performed and the grafts were excised for LM and SEM.. The arterial endothelial layer remained intact after up to 14 days of storage in UW. In PBS, the endothelium was lost after 3 days. The functional response after 14 days storage in UW was approximately 50% vs. 0% after 14 days in PBS. In the autografts, total patencies (28 days + 56 days) were 100% (8/8) and 63% (5/8) for UW and PBS stored grafts, respectively. In the allografts, the UW and PBS preserved grafts showed total patencies of 86% (12/14) and 83% (5/6), respectively. Microscopically, the allografts showed fibrotic degeneration.. Arteries are well preserved in UW up to 14 days of storage. Arterial autografts preserved in UW showed good patency and better integrity of the vessel wall after implantation, than grafts stored in PBS or allografts (without immunosuppressive therapy).

Topics: Adenosine; Allopurinol; Animals; Carotid Arteries; Cold Temperature; Dogs; Endothelium, Vascular; Female; Fibrosis; Glutathione; Insulin; Methacholine Chloride; Microscopy, Electron, Scanning; Muscarinic Agonists; Muscle, Smooth, Vascular; Organ Preservation Solutions; Phenylephrine; Raffinose; Sodium Chloride; Time Factors; Tissue Preservation; Tissue Survival; Transplantation, Autologous; Transplantation, Homologous; Vascular Patency; Vasoconstrictor Agents; Veins

Frozen section examination was performed on 385 donor livers before transplantation. Exclusion criteria were applied to the donor livers examined to exclude potentially dysfunctional livers. The exclusion criteria included the following: severe macrovesicular steatosis, ischemic necrosis, prominent chronic portal inflammation, prominent periductular fibrosis, granulomatous inflammation, bridging fibrosis, and malignancy. Twenty-seven of the 385 donor livers examined were excluded before transplantation. The following histologic features were present in the excluded livers: severe steatosis (22), ischemic necrosis (2), portal inflammation (1), and periductular fibrosis (2). Steatosis was present in 51 of the 385 (13.25%) organs examined, including 22 of the donor organs excluded before transplantation. Twenty-nine livers with mild to moderate steatosis were implanted into size and blood type-matched recipients. Indicators of allograft function (prothrombin time and bilirubin) and damage (aspartate aminotransferase and alanine aminotransferase) were measured daily for the first 10 days after transplant. There was no statistically significant difference between the group of nonfat livers and donor livers containing mild steatosis. Statistically significant higher posttransplant serum alanine aminotransferase and prothrombin time levels were present in the patients with livers implanted with mild versus moderate steatosis. The 1-year survival rate for patients receiving fatty versus nonfatty donor livers was not statistically different (Kaplan-Meier, P = 0.592). No significant differences were found in the clinical and laboratory characteristics of donors whose organs were implanted compared with the clinical and laboratory characteristics of donors whose organs were excluded. The primary nonfunction rate after applying the exclusion criteria was 1.4%, which is a significant decrease compared with our primary nonfunction rate of 8.5% before using frozen section examination. Frozen section examination is useful in excluding donor organs which may become dysfunctional after transplantation.

Topics: Adenosine; Adult; Allopurinol; Azo Compounds; Child; Fatty Liver; Female; Fibrosis; Frozen Sections; Glutathione; Hepatitis; Humans; Hypertonic Solutions; Insulin; Ischemia; Liver; Liver Transplantation; Male; Necrosis; Organ Preservation; Organ Preservation Solutions; Raffinose; Staining and Labeling; Survival Rate; Tissue Donors