tretinoin and Fibrosis

The vitamin A metabolite, retinoic acid, is an important signaling molecule during embryonic development serving critical roles in morphogenesis, organ patterning and skeletal and neural development. Retinoic acid is also important in postnatal life in the maintenance of tissue homeostasis, while retinoid-based therapies have long been used in the treatment of a variety of cancers and skin disorders. As the number of people living with chronic disorders continues to increase, there is great interest in extending the use of retinoid therapies in promoting the maintenance and repair of adult tissues. However, there are still many conflicting results as we struggle to understand the role of retinoic acid in the multitude of processes that contribute to tissue injury and repair. This review will assess our current knowledge of the role retinoic acid signaling in the development of fibroblasts, and their transformation to myofibroblasts, and of the potential use of retinoid therapies in the treatment of organ fibrosis.

Topics: Adult; Animals; Fibroblasts; Fibrosis; Humans; Myofibroblasts; Retinoids; Signal Transduction; Tretinoin

Fibrotic diseases, such as liver, pulmonary and renal fibrosis, are common end-stage conditions and represent a major global health problem. Furthermore, effective therapeutic measures are presently unavailable. Extracellular matrix accumulation is the most prominent characteristic in the pathogenesis of fibrotic disease. Retinoic acid, including all-trans retinoic acid, 9-cis and 13-cis retinoic acid, play important roles in various physiological processes, such as in embryonic development, reproduction, vision, cell growth, differentiation, apoptosis and inflammation. Present studies report that retinoic acid treatment may affect various processes involved in the onset and progression of fibrotic disease. However, the therapeutic effects of retinoic acid in such diseases remain controversial. Several reports indicate that retinoic acid positively affects the progression of fibrosis and alleviates the accumulation of the extracellular matrix, whereas other studies report the opposite; that retinoic acid exacerbates fibrosis and induces extracellular matrix accumulation. Signaling pathways might be an important influencing factor and differences in signaling events might be responsible for the contradictory role of retinoic acid in fibrotic diseases. Since there was no review available that investigated the role of retinoic acid and the signaling pathways involved, we retrospectively studied the literature and provide a comprehensive analysis of retinoic acid's role in fibrotic diseases, and provide an overview of the signal transduction pathways involved in its pathogenesis.

Topics: Animals; Extracellular Matrix; Fibrosis; Humans; Organ Specificity; Signal Transduction; Tretinoin

Pancreatic fibrosis is mainly manifested by imbalance in extracellular matrix (ECM) homeostasis due to excessive deposition of collagen in pancreas by activated pancreatic stellate cells (PSCs). Recently, some drugs have exhibited therapeutic potentials for the treatment of pancreatic fibrosis; however, currently, no effective clinical strategy is available to remodel imbalanced ECM homeostasis because of inferior targeting abilities of drugs and collagen barriers that hinder the efficient delivery of drugs. Herein, we design and prepare collagen-binding peptide (CBP) and collagenase I co-decorated dual drug-loaded lipid nanoparticles (named AT-CC) for pancreatic fibrosis therapy. Specifically, AT-CC can target fibrotic pancreas via the CBP and degrade excess collagen by the grafted collagenase I, thereby effectively delivering all-trans-retinoic acid (ATRA) and ammonium tetrathiomolybdate (TM) into pancreas. The released ATRA can reduce collagen overproduction by inhibiting the activation of PSCs. Moreover, the released TM can restrain lysyloxidase activation, consequently reducing collagen cross-linking. The combination of ATRA and TM represses collagen synthesis and reduces collagen cross linkages to restore ECM homeostasis. The results of this research suggest that AT-CC is a safe and efficient collagen-targeted degradation drug-delivery system for reversing pancreatic fibrosis. Furthermore, the strategy proposed herein will offer an innovative platform for the treatment of chronic pancreatitis.

Topics: Cells, Cultured; Collagen; Collagenases; Extracellular Matrix; Fibrosis; Homeostasis; Humans; Pancreas; Tretinoin

The aim of this study was to investigate the effects of all-trans retinoic acid (atRA) on oxidative stress in renal tubular epithelial cells induced by high glucose (HG) and its potential mechanism. We investigated the effects of atRA in HG-induced renal epithelial cell line HK-2. Seven groups were designed for this experiment: negative control, mannitol, high-glucose (HG), HG combined with a low concentration of atRA, HG combined with a middle concentration of atRA, HG combined with a high concentration of atRA, and HG combined with captopril. After 48 h of incubation, oxidative stress factor expression in the supernatant was detected by enzyme-linked immunosorbent assay. Reactive oxygen species and cell apoptosis expression were assessed by flow cytometry. NADPH oxidase, fibrosis factor, and angiotensin-converting enzyme 2/angiotensin (1-7)/mas receptor (ACE2/Ang (1-7)/MasR) pathway-related protein expressions were determined by western blot analysis. The expressions of oxidative stress factors, NADPH oxidase components, and fibrosis factors were significantly higher after HG treatment. Apoptosis of HK2 cells in the HG group was also significantly higher. AtRA could reverse the above abnormal changes in a concentration-dependent manner. HG significantly promoted the expression of ACE, Ang II, and Ang II type 1 receptor (AT1R), whereas it inhibited the expression of ACE2, Ang (1-7), and MasR. With the elevation of concentration, atRA could gradually suppress the expression of ACE, Ang II, and AT1R, but facilitate ACE2, Ang (1-7), and MasR. These results were statistically significant. AtRA could significantly inhibit oxidative stress and apoptosis of renal tubular epithelial cells induced by HG. The mechanism may inhibit the ACE/Ang II/AT1R pathway and/or activate ACE2/Ang (1-7)/MasR pathway.

Topics: Angiotensin II; Angiotensin-Converting Enzyme 2; Epithelial Cells; Fibrosis; Glucose; Humans; NADPH Oxidases; Oxidative Stress; Peptide Fragments; Peptidyl-Dipeptidase A; Tretinoin

Systemic sclerosis (SSc) is a chronic and systemic autoimmune disease marked by the skin and visceral fibrosis. Metabolic alterations have been found in SSc patients; however, serum metabolomic profiling has not been thoroughly conducted. Our study aimed to identify alterations in the metabolic profile in both SSc patients before and during treatment, as well as in mouse models of fibrosis. Furthermore, the associations between metabolites and clinical parameters and disease progression were explored.. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS)/MS was performed in the serum of 326 human samples and 33 mouse samples. Human samples were collected from 142 healthy controls (HC), 127 newly diagnosed SSc patients without treatment (SSc baseline), and 57 treated SSc patients (SSc treatment). Mouse serum samples were collected from 11 control mice (NaCl), 11 mice with bleomycin (BLM)-induced fibrosis and 11 mice with hypochlorous acid (HOCl)-induced fibrosis. Both univariate analysis and multivariate analysis (orthogonal partial least-squares discriminate analysis (OPLS-DA)) were conducted to unravel differently expressed metabolites. KEGG pathway enrichment analysis was performed to characterize the dysregulated metabolic pathways in SSc. Associations between metabolites and clinical parameters of SSc patients were identified by Pearson's or Spearman's correlation analysis. Machine learning (ML) algorithms were applied to identify the important metabolites that have the potential to predict the progression of skin fibrosis.. The newly diagnosed SSc patients without treatment showed a unique serum metabolic profile compared to HC. Treatment partially corrected the metabolic changes in SSc. Some metabolites (phloretin 2'-O-glucuronide, retinoyl b-glucuronide, all-trans-retinoic acid, and betaine) and metabolic pathways (starch and sucrose metabolism, proline metabolism, androgen and estrogen metabolism, and tryptophan metabolism) were dysregulated in new-onset SSc, but restored upon treatment. Some metabolic changes were associated with treatment response in SSc patients. Metabolic changes observed in SSc patients were mimicked in murine models of SSc, indicating that they may reflect general metabolic changes associated with fibrotic tissue remodeling. Several metabolic changes were associated with SSc clinical parameters. The levels of allysine and all-trans-retinoic acid were negatively correlated, while D-glucuronic acid and hexanoyl carnitine were positively correlated with modified Rodnan skin score (mRSS). In addition, a panel of metabolites including proline betaine, phloretin 2'-O-glucuronide, gamma-linolenic acid and L-cystathionine were associated with the presence of interstitial lung disease (ILD) in SSc. Specific metabolites identified by ML algorithms, such as medicagenic acid 3-O-b-D-glucuronide, 4'-O-methyl-(-)-epicatechin-3'-O-beta-glucuronide, valproic acid glucuronide, have the potential to predict the progression of skin fibrosis.. Serum of SSc patients demonstrates profound metabolic changes. Treatment partially restored the metabolic changes in SSc. Moreover, certain metabolic changes were associated with clinical manifestations such as skin fibrosis and ILD, and could predict the progression of skin fibrosis.

Topics: Animals; Biomarkers; Fibrosis; Glucuronides; Humans; Lung Diseases, Interstitial; Mice; Scleroderma, Systemic; Tretinoin

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to irreversible fibrosis of the skin and the internal organs. The etiology of SSc is complex, its pathophysiology is poorly understood, and clinical therapeutic options are restricted. Thus, research into medications and targets for treating fibrosis is essential and urgent. Fos-related antigen 2 (Fra2) is a transcription factor that is a member of the activator protein-1 family. Fra2 transgenic mice were shown to have spontaneous fibrosis. All-trans retinoic acid (ATRA) is a vitamin A intermediate metabolite and ligand for the retinoic acid receptor (RAR), which possesses anti-inflammatory and anti-proliferative properties. Recent research has demonstrated that ATRA also has an anti-fibrotic effect. However, the exact mechanism is not fully understood. Interestingly, we identified potential binding sites for the transcription factor RARα to the promoter region of the FRA2 gene through JASPAR and PROMO databases. In this study, the pro-fibrotic effect of Fra2 in SSc is confirmed. SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc animals exhibit increased levels of Fra2. Inhibition of Fra2 expression in SSc dermal fibroblasts with Fra2 siRNA markedly decreased collagen I expression. ATRA reduced the expressions of Fra2, collagen I, and α-smooth muscle actin(α-SMA) in SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc mice. In addition, chromatin immunoprecipitation and dual-luciferase assays demonstrated that retinoic acid receptor RARα binds to the FRA2 promoter and modulates its transcriptional activity. ATRA decreases collagen I expression both in vivo and in vitro via the reduction of Fra2 expression. This work establishes the rationale for expanding the use of ATRA in the treatment of SSc and indicates that Fra2 can be used as an anti-fibrotic target.

Topics: Animals; Bleomycin; Collagen Type I; Disease Models, Animal; Fibroblasts; Fibrosis; Mice; Mice, Transgenic; Receptors, Retinoic Acid; Scleroderma, Systemic; Skin; Transcription Factor AP-1; Tretinoin

All-trans retinoic acid (ATRA) is one of essentially active metabolite of vitamin A, and plays an important role in diverse physiological processes, such as cellular growth and function. Renal interstitial fibrosis (RIF) is a common pathological characteristic of chronic renal disease causing end-stage renal disease currently lacking effective treatment. Low level of Angiopoietins-1 (Angpt-1) is associated with extracellular matrix accumulation and fibrosis diseases. This study was performed to assess the association of ATRA with Angpt-1 in RIF disease. Rats were divided into three groups: group of sham (SHO group), group of unilateral ureteral obstruction group (UUO group), UUO mice administrated daily at the dose of ATRA (ATRA group). Masson-staining was used to detect the histologic lesion. Immunohistochemistry and Western-blot were applied to determine the targeted proteins. RIF score was significantly increased in UUO rats when compared with that of SHO group, and the fibrosis score was notably reduced in ATRA group. Transforming growth factor-β1 (TGF-β1), collagen IV (Col-IV) and fibronectin (FN) expressions in UUO group were significantly up-regulated, whereas Angpt-1 expression was significantly down-regulated compared with the SHO group. ATRA treatment reduced TGF-β1, Col-IV and FN expressions and improved Angpt-1 expression compared with the UUO group. The protein expression of Angpt-1 in kidney tissue of UUO group was negatively correlated with RIF index and protein expressions of Col-IV, FN and TGF-β1. In conclusion, low expression of Angpt-1 was associated with the RIF disease and ATRA treatment can increase the Angpt-1 and alleviate the RIF lesion in UUO rats.

Topics: Angiopoietin-1; Animals; Collagen Type IV; Disease Models, Animal; Disease Progression; Down-Regulation; Extracellular Matrix; Fibronectins; Fibrosis; Male; Nephritis, Interstitial; Rats; Transforming Growth Factor beta1; Tretinoin

During muscle regeneration, excessive formation of adipogenic and fibrogenic tissues, from their respective fibro/adipogenic progenitors (FAPs), impairs functional recovery. Intrinsic mechanisms controlling the proliferation and differentiation of FAPs remain largely unexplored.. Here, we investigated the role of retinoic acid (RA) signalling in regulating FAPs and the subsequent effects on muscle restoration from a cardiotoxin-induced injury. Blockage of retinoic acid receptor (RAR) signalling was achieved through dominant negative retinoic acid receptor α (RARα403) expression specific in PDGFRα+ FAPs in vivo and by BMS493 treatment in vitro. Effects of RAR-signalling on FAP cellularity and muscle regeneration were also investigated in a high-fat diet-induced obese mice model.. Supplementation of RA increased the proliferation of FAPs during the early stages of regeneration while suppressing FAP differentiation and promoting apoptosis during the remodelling stage. Loss of RAR-signalling caused ectopic adipogenic differentiation of FAPs and impaired muscle regeneration. Furthermore, obesity disrupted the cellular transition of FAPs and attenuated muscle regeneration. Supplementation of RA to obese mice not only rescued impaired muscle fibre regeneration, but also inhibited infiltration of fat and fibrotic tissues during muscle repair. These beneficial effects were abolished after blocking RAR-signalling in FAPs of obese mice.. These data suggest that RAR-signalling in FAPs is a critical therapeutic target for suppressing differentiation of FAPs and facilitating the regeneration of muscle and other tissues.. This study was supported by grants from the National Institutes of Health (R01-HD067449 and R21-AG049976) to M.D.

Topics: Adipogenesis; Animals; Cell Differentiation; Fibrosis; Immunohistochemistry; Male; Mesenchymal Stem Cells; Mice; Mice, Transgenic; Muscle, Skeletal; Obesity; Regeneration; Signal Transduction; Tretinoin

Tubular injury and interstitial fibrosis are the hallmarks of chronic kidney disease. While recent studies have verified that proximal tubular injury triggers interstitial fibrosis, the impact of fibrosis on tubular injury and regeneration remains poorly understood. We generated a novel mouse model expressing diphtheria toxin receptor on renal fibroblasts to allow for the selective disruption of renal fibroblast function. Administration of diphtheria toxin induced upregulation of the tubular injury marker Ngal and caused tubular proliferation in healthy kidneys, whereas administration of diphtheria toxin attenuated tubular regeneration in fibrotic kidneys. Microarray analysis revealed down-regulation of the retinol biosynthesis pathway in diphtheria toxin-treated kidneys. Healthy proximal tubules expressed retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in retinoic acid biosynthesis. After injury, proximal tubules lost RALDH2 expression, whereas renal fibroblasts acquired strong expression of RALDH2 during the transition to myofibroblasts in several models of kidney injury. The retinoic acid receptor (RAR) RARγ was expressed in proximal tubules both with and without injury, and αB-crystallin, the product of an RAR target gene, was strongly expressed in proximal tubules after injury. Furthermore, BMS493, an inverse agonist of RARs, significantly attenuated tubular proliferation in vitro. In human biopsy tissue from patients with IgA nephropathy, detection of RALDH2 in the interstitium correlated with older age and lower kidney function. These results suggest a role of retinoic acid signaling and cross-talk between fibroblasts and tubular epithelial cells during tubular injury and regeneration, and may suggest a beneficial effect of fibrosis in the early response to injury.

Topics: Aldehyde Dehydrogenase 1 Family; Aldehyde Oxidoreductases; Animals; Benzoates; Biomarkers; Biopsy; Cell Line; Cell Proliferation; Diphtheria Toxin; Disease Models, Animal; Epithelial Cells; Fibrosis; Glomerulonephritis, IGA; Humans; Kidney Tubules, Proximal; Lipocalin-2; Mice; Myofibroblasts; Receptors, Retinoic Acid; Regeneration; Renal Insufficiency, Chronic; Retinal Dehydrogenase; Retinoic Acid Receptor gamma; Stilbenes; Tretinoin; Up-Regulation

Tubulointerstitial fibrosis is considered a hallmark of maladaptive repair processes after tubular injury leading to chronic kidney disease. Nakamura and colleagues show that, upon injury, myofibroblasts promote epithelial repair by producing retinoic acid in place of injured tubular cells. These results suggest that resident fibroblasts turning into myofibroblasts maintain a cross-talk that protects tubular epithelial cells from injury and can restore tissue integrity and functionality, challenging the concept that fibrosis is only detrimental in nature.

Topics: Fibrosis; Friends; Humans; Kidney; Myofibroblasts; Tretinoin

Airway smooth muscle (ASM) is a dynamic and complex tissue involved in regulation of bronchomotor tone, but the molecular events essential for the maintenance of ASM homeostasis are not well understood. Observational and genome-wide association studies in humans have linked airway function to the nutritional status of vitamin A and its bioactive metabolite retinoic acid (RA). Here, we provide evidence that ongoing RA signaling is critical for the regulation of adult ASM phenotype. By using dietary, pharmacologic, and genetic models in mice and humans, we show that (a) RA signaling is active in adult ASM in the normal lung, (b) RA-deficient ASM cells are hypertrophic, hypercontractile, profibrotic, but not hyperproliferative, (c) TGF-β signaling, known to cause ASM hypertrophy and airway fibrosis in human obstructive lung diseases, is hyperactivated in RA-deficient ASM, (d) pharmacologic and genetic inhibition of the TGF-β activity in ASM prevents the development of the aberrant phenotype induced by RA deficiency, and (e) the consequences of transient RA deficiency in ASM are long-lasting. These results indicate that RA signaling actively maintains adult ASM homeostasis, and disruption of RA signaling leads to aberrant ASM phenotypes similar to those seen in human chronic airway diseases such as asthma.

Topics: Adult; Animals; Benzoates; Cells, Cultured; Disease Models, Animal; Female; Fibrosis; Humans; Hypertrophy; Lung; Lung Diseases, Obstructive; Male; Mice; Mice, Transgenic; Muscle, Smooth; Myocytes, Smooth Muscle; Primary Cell Culture; Receptors, Retinoic Acid; Signal Transduction; Stilbenes; Tretinoin

Methylglyoxal (MG), a metabolic intermediate of glycolysis is a precursor for endogeneous production of advanced glycation end-products. The increased production of MG have negative influence over the structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic cardiac complications. Retinoic acid (RA), an active metabolite of vitamin A, has a major role in preventing cardiac remodeling and ventricular fibrosis. Hence, the objective of the present study was to determine whether rats administered with all-trans retinoic acid (RA) could attenuate MG induced pathological effects. Wistar rats were divided into 4 groups. Group 1 rats were kept as control; Group 2 rats were administrated with MG (75 mg/kg/day) for 8 weeks. Group 3 rats were given RA (Orally, 1.0 mg/kg/day) along with MG; Group 4 rats received RA alone. Cardiac antioxidant status, induction of fibrosis, AGE receptor (RAGE) and cytokines expression was evaluated in the heart tissues. Administration of MG led to depletion of antioxidant enzymes, induction of fibrosis (p < 0.001), up-regulated expression of RAGE (3.5 fold), TGF-β (4.4 fold), SMAD2 (3.7 fold), SMAD3 (6.0 fold), IL-6 (4.3 fold) and TNF-α (5.5 fold) in the heart tissues compared to control rats. Moreover, the exogenous administration of MG caused significant (p < 0.001) increase in the circulating CML levels. Whereas, RA treatment prevented the induction of fibrosis and restored the levels of cytokines and RAGE expression. Methylglyoxal-induced fibrosis can lead to pathological effects in the heart tissues. RA attenuates the effects of MG in the heart, suggesting that it can be of added value to usual diabetic therapy.

Topics: Animals; Cytokines; Dietary Supplements; Fibrosis; Gene Expression Regulation; Male; Pyruvaldehyde; Rats; Rats, Wistar; Receptor for Advanced Glycation End Products; Smad2 Protein; Smad3 Protein; Tretinoin; Ventricular Dysfunction; Ventricular Remodeling

We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp. Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis.. The proteomic remodeling was largely prevented in MSCp. Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair.. Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart.

Topics: Animals; Biological Phenomena; Down-Regulation; Fibrosis; Humans; Keto Acids; Male; Mesenchymal Stem Cells; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Neovascularization, Pathologic; Proteomics; Signal Transduction; Swine; Tretinoin; Up-Regulation; Ventricular Function, Left; Ventricular Remodeling

Keloid disease is a recurrent fibroproliferative cutaneous tumor of unknown pathogenesis for which clinical management remains unsatisfactory. To obtain new insights into hitherto underappreciated aspects of keloid pathobiology, we took a laser capture microdissection-based, whole-genome microarray analysis approach to identify distinct keloid disease-associated gene expression patterns within defined keloid regions. Identification of the aldo-keto reductase enzyme AKR1B10 as highly up-regulated in keloid epidermis suggested that an imbalance of retinoic acid metabolism is likely associated with keloid disease. Here, we show that AKR1B10 transfection into normal human keratinocytes reproduced the abnormal retinoic acid pathway expression pattern we had identified in keloid epidermis. Cotransfection of AKR1B10 with a luciferase reporter plasmid showed reduced retinoic acid response element activity, supporting the hypothesis of retinoic acid synthesis deficiency in keloid epidermis. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in up-regulation of transforming growth factor-β1, transforming growth factor-β2, and collagens I and III in both keloid and normal skin fibroblasts, mimicking the typical profibrotic keloid profile. Our study results suggest that insufficient retinoic acid synthesis by keloid epidermal keratinocytes may contribute to the pathogenesis of keloid disease. We refocus attention on the role of injured epithelium in keloid disease and identify AKR1B10 as a potential new target in future management of keloid disease.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Culture Media, Conditioned; Epidermis; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Keloid; Keratinocytes; Oligonucleotide Array Sequence Analysis; Plasmids; Response Elements; Signal Transduction; Transcriptional Activation; Tretinoin; Up-Regulation

Recently a mouse skin carcinogenesis study reported that a β-blocker carvedilol displayed antitumor-properties via antihyperplastic effects. However, the antihyperplastic mechanism is unclear as the β-blocker is characterized with multiple pleiotropic effects including stimulation of endothelial NO release and verapamil-like calcium channel blocking activity. To investigate the nature and the origin of the antihyperplastic effects, we tested topical pretreatment with pindolol, heptaminol, ATRA or verapamil against Balb/c mouse ear skin hyperplasia that was induced by TPA. We found that pindolol, heptaminol or ATRA, but not verapamil, inhibited the TPA-induced immunoinflammatory skin changes in an NO-dependent manner, which included epidermal hyperplasia, skin edema and fibrosis. Furthermore, we also observed NO-dependent alleviation of the TPA-induced NK cell depletion in the ear tissues by heptaminol pretreatment. Together our results suggest that stimulation of NO generation from constitutive synthases may be primarily responsible for the reported antihyperplastic and NK cell-preserving effects of the β-blockers, and that similar effects may be observed in other immunity normalizing compounds that also promote endothelial NO synthesis.

Topics: Animals; Female; Fibrosis; Heptaminol; Hyperplasia; Killer Cells, Natural; Mice; Mice, Inbred BALB C; NG-Nitroarginine Methyl Ester; Nitric Oxide; Pindolol; Skin; Tetradecanoylphorbol Acetate; Tretinoin; Verapamil

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.

Topics: Adult; Aged; Aged, 80 and over; Aldehyde Dehydrogenase; Animals; Cells, Cultured; Cicatrix; Conjunctiva; Disulfiram; Female; Fibroblasts; Fibrosis; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Mucous Membrane; Pemphigoid, Benign Mucous Membrane; Tretinoin

Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of β-catenin, platelet-derived growth factor (PDGF)-Rβ transforming growth factor (TGF)-βRII and collagen 1α1 in vivo. Wnt 2 and β-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of β-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFβRII, PDGFRβ and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/β-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice.

Topics: Actins; Active Transport, Cell Nucleus; Animals; Apoptosis; Axin Protein; Cells, Cultured; Ceruletide; Collagen Type I; Disease Progression; Drug Evaluation, Preclinical; Fibrosis; Gene Expression Regulation; Lipase; Male; Mice; Mice, Inbred BALB C; Organ Size; Pancreas; Pancreatic alpha-Amylases; Pancreatic Stellate Cells; Pancreatitis, Chronic; Proteoglycans; Random Allocation; Receptor, Platelet-Derived Growth Factor beta; Receptors, Transforming Growth Factor beta; RNA, Messenger; Tretinoin; Wnt Signaling Pathway

Laminectomy is a widely accepted treatment for lumbar disorders, and epidural fibrosis (EF) is a common complication. EF is thought to cause post-operative pain recurrence after laminectomy or discectomy. All-trans retinoic acid (ATRA) has shown anti-fibrotic, anti-inflammatory, and anti-proliferative functions. The object of this study was to investigate the effects of ATRA on the prevention of EF in post-laminectomy rats. In vitro, the anti-fibrotic effect of ATRA was demonstrated with cultured fibroblasts count, which comprised of those that were cultured with/without ATRA. In vivo, rats underwent laminectomy at the L1-L2 levels. We first demonstrated the beneficial effects using 0.05% ATRA compared to vehicle (control group). We found that a higher concentration of ATRA (0.1%) achieved dose-dependent results. Hydroxyproline content, Rydell score, vimentin-positive cell density, fibroblast density, inflammatory cell density and inflammatory factor expression levels all suggested better outcomes in the 0.1% ATRA rats compared to the other three groups. Presumably, these effects involved ATRA's ability to suppress transforming growth factor (TGF-β1) and interleukin (IL)-6 which was confirmed with reverse-transcriptase polymerase chain reaction (RT-PCR). Finally we demonstrated that ATRA down-regulated nuclear factor (NF)-κB by immunohistochemistry and western blotting for p65 and inhibition of κB (IκBα), respectively. Our findings indicate that topical application of ATRA can inhibit fibroblast proliferation, decrease TGF-β1 and IL-6 expression level, and prevent epidural scar adhesion in rats. The highest concentration employed in this study (0.1%) was the most effective. ATRA suppressed EF through down-regulating NF-κB signaling, whose specific mechanism is suppression of IκB phosphorylation and proteolytic degradation.

Topics: Animals; Cell Count; Cells, Cultured; Cicatrix; Dose-Response Relationship, Drug; Dura Mater; Epidural Space; Fibroblasts; Fibrosis; Keratolytic Agents; Laminectomy; Lumbar Vertebrae; Male; NF-kappa B; Rats; Rats, Wistar; Tretinoin

All-trans retinoic acid (ATRA) has been used for the treatment of acute promyelocytic leukemia. It remains unclear, however, whether ATRA affects cyclooxygenase-2 (COX-2; an enzyme involved in prostaglandin production), PGE2, and thromboxane A2 (TXA2) (metabolic products of COX-2) by a transforming growth factor-β/Smad-signaling pathway, which plays important roles in mesangial-cell proliferation and renal fibrosis. In this study, the mRNA and protein of Smad3, Smad7, and COX-2 were detected by reverse transcription-polymerase chain reaction and Western blot, respectively, in mesangial cells stimulated by transforming growth factor-β (TGF-β) and treated with ATRA at various concentrations and times. The protein level of PGE2 and TXA2 was also measured by enzyme-linked immunosorbent assay. The localization of Smad3 and Smand7 was observed by confocal microscope. Cell proliferation was detected by MTT assay, while apoptosis was determined using Hoechest staining. The expression of Smad3, Smad7, and COX-2 mRNA and protein was increased by exogenous TGF-β, but inhibited by pretreatment of ATRA, in dose and time-dependent manners. In addition, the expression of Smad3 and Smad7 was significantly reduced not only by staurosporine, an inhibitor of threonine/serine protein kinases as well as smad, but also by NS-398, an inhibitor of COX-2. PGE2 and TXA2 were raised by TGF-β, but also decreased by ATRA, staurosporine, and NS-398. Moreover, ATRA reversed the translocation of Smad3 and Smad7 induced by TGF-β. Compared with the control, TGF-β also significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently inhibited TGF-β-induced cell proliferation, but had no significant effect on apoptosis in rat mesangial cells. Therefore, ATRA repressed COX-2, PGE2, and TXA2 via the TGF-β/Smad-signaling pathway and inhibited mesangial-cell proliferation, which might subsequently prevent renal fibrosis.

Topics: Animals; Apoptosis; Cell Line; Cell Proliferation; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Fibrosis; Glomerular Mesangium; Mesangial Cells; Nitrobenzenes; Protein Transport; Rats; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Staurosporine; Sulfonamides; Thromboxane A2; Transforming Growth Factor beta; Tretinoin

In hypertensive rodents, retinoic acid (RA) prevents adverse cardiac remodelling and improves myocardial infarction outcome, but its role in obesity-related changes of cardiac tissue are unclear. We hypothesized that all-trans RA (ATRA) treatment will improve the cardioprotective oxytocin-natriuretic peptides (OT-NP) system, preventing apoptosis and collagen accumulation in hearts of ob/ob mice, a mouse model of obesity and insulin resistance. Female 9-week-old B6.V-Lep/J ob/ob mice (n = 16) were divided into 2 groups: 1 group (n = 8) treated with 100 μg of ATRA dissolved in 100 μL of corn oil (vehicle) delivered daily (∼2 μg·g body weight(-1)·day(-1)) by stomach intubation for 16 days, and 1 group (n = 8) that received the vehicle alone. A group of nonobese littermate mice (n = 9) served as controls. Ob/ob mice exhibited obesity, hyperglycaemia, and downregulation of the cardiac OT-NP system, including the mRNA for the transcription factor GATA4, OT receptor and brain NP, and the protein expression for endothelial nitric oxide synthase. Hearts from ob/ob mice also demonstrated increased apoptosis and collagen accumulation. ATRA treatment induced weight loss and decreased adipocytes diameter in the visceral fat, thus reducing visceral obesity, which is associated with a high risk for cardiovascular disease. RA treatment was associated with a reduction in hyperglycemia and a normalization of the OT-NP system's expression in the hearts of ob/ob mice. Furthermore, ATRA treatment prevented apoptosis and collagen accumulation in hearts of ob/ob mice. The present study indicates that ATRA treatment was effective in restoring the cardioprotective OT-NP system and in preventing abnormal cardiac remodelling in the ob/ob mice.

Topics: Animals; Apoptosis; Female; Fibrosis; Gene Expression Regulation; Mice; Mice, Obese; Myocytes, Cardiac; Natriuretic Peptides; Obesity; Tretinoin

End-stage renal disease is a cause for death worldwide. Renal transplant is a therapeutic alternative, restricted by the scant number of donors. Function of the donor kidney is under risk of adverse circumstances such as fibrosis, where profibrotic effect of transforming growth factor beta 1 (TGF-β1) plays a key role. Efforts to diminish risks of damage in the remnant kidney of the donor are required. Vitamin A represents one alternative. It has beneficial effects on some nephropathies, mainly those related to oxidative stress. It also participates in normal intrauterine renal development. We studied the effect of all-trans retinoic acid (ATRA), active form of vitamin A, on postnephrectomy compensatory growth, in male or female rats. Compensatory growth and renal function were evaluated on four experimental groups: Control without treatment (CTL), ATRA-treated intact rats (CTL + RA), nephrectomized rats (NFX), and ATRA-treated nephrectomized rats (NFX + RA). We evaluated glomerular function (inulin clearance), tubular function (fractional excretions of sodium and potassium), and urinary flow. Renal mass was also estimated. In ATRA-treated animals, compensatory growth was higher than in nephrectomized rats without treatment. Hyperfiltration after nephrectomy was less intense in ATRA-treated female than in male rats. In tubular functions, effect of ATRA was more evident in female than in male rats. Glomerular expression of TGF-β1 was lower in ATRA-treated animals than in controls. ATRA reduced intensity and duration of compensatory changes after nephrectomy, improving recovery.

Topics: Animals; Female; Fibrosis; Fluorescent Antibody Technique; Glomerular Filtration Rate; Kidney; Male; Nephrectomy; Organ Size; Oxidative Stress; Rats; Rats, Wistar; Sex Characteristics; Transforming Growth Factor beta1; Tretinoin; Urination

Retinoids, including all-trans retinoic acid (tRA), have dose-dependent pro-fibrotic effects in experimental kidney diseases. To understand and eventually prevent such adverse effects, it is important to establish relevant in vitro models and unravel their mechanisms.. Fibrogenic effects of retinoids were assessed in NRK-49F renal fibroblasts using picro-Sirius red staining for collagens and quantified by spectrophotometric analysis of the eluted stain. Other methods included RT-qPCR, immunoassays and matrix metalloproteinase (MMP) activity assays.. With or without TGF-β1, tRA was dose-dependently pro-fibrotic, notably increasing collagen accumulation. tRA and TGF-β1 additively suppressed expression of mRNA for MMP2, 3 and 13 and suppressed MMP activity. tRA, in the presence of TGF-β1, induced plasminogen activator inhibitor-1 (PAI-1) mRNA and they additively induced PAI-1 protein expression. A PAI-1 inhibitor, a pan-retinoic acid receptor (RAR) antagonist and a pan-retinoid X receptor (RXR) antagonist each partially prevented the pro-fibrotic effect of tRA. The dose-dependent pro-fibrotic effects of a pan-RXR agonist were similar to those of tRA. A pan-RAR agonist showed weaker, less dose-dependent pro-fibrotic effects and the pro-fibrotic effects of RARα and RARβ-selective agonists were even smaller. An RARγ-selective agonist did not affect fibrogenesis.. An in vitro model for the pro-fibrotic effects of retinoids was established in NRK-49F cells. It was associated with reduced MMP activity and increased PAI-1 expression, and was probably mediated by RXR and RAR. To avoid or antagonize the pro-fibrotic activity of tRA, further studies on RAR isotype-selective agonists and PAI-1 inhibitors might be of value.

Topics: Animals; Cell Line; Cells, Cultured; Collagen; Fibroblasts; Fibrosis; Humans; Kidney; Matrix Metalloproteinases; Models, Biological; Plasminogen Activator Inhibitor 1; Rats; Receptors, Retinoic Acid; Retinoid X Receptors; Transforming Growth Factor beta1; Tretinoin

All-trans retinoic acid (ATRA) is an important therapeutic agent for prevention of the renal diseases. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway is a key signaling pathway which takes part in the progression of renal interstitial fibrosis (RIF). This investigation was performed to study the effect of ATRA in RIF rats and its effect on the TGF-β1/Smad3 signaling pathway. Sixty Wistar male rats were divided into three groups at random: sham operation group (SHO), model group subjected to unilateral ureteral obstruction (GU), model group treated with ATRA (GA), n = 20, respectively. RIF index, protein expression of TGF-β1, collagen-IV (Col-IV) and fibronectin (FN) in renal interstitium, and mRNA and protein expressions of Smad3 in renal tissue were detected at 14-day and 28-day after surgery. The RIF index was markedly elevated in group GU than in SHO group (p < 0.01), and the RIF index of GA group was alleviated when compared with that in GU group (p < 0.01). Compared with in group SHO, the mRNA/protein expression of Smad3 in renal tissue was significantly increased in group GU (p < 0.01). However, the mRNA and protein expressions of Smad3 in renal tissue in GA group were not markedly alleviated by ATRA treatment when compared with those in GU (each p > 0.05). Protein expressions of TGF-β1, Col-IV, and FN in GU group were markedly increased than those in SHO group (each p < 0.01), and their expressions in GA group were markedly down-regulated by ATRA treatment than those of GU group (all p < 0.01). The protein expression of Smad3 was positively correlated with RIF index, protein expression of TGF-β1, Col-IV or FN (each p < 0.01). In conclusion, ATRA treatment can alleviate the RIF progression in UUO rats. However, ATRA cannot affect the signaling pathway of TGF-β1/Smad3 in the progression of RIF.

Topics: Analysis of Variance; Animals; Biopsy, Needle; Blotting, Western; Disease Models, Animal; Disease Progression; Down-Regulation; Fibrosis; Gene Expression Regulation; Immunohistochemistry; Male; Nephritis, Interstitial; Random Allocation; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reference Values; Risk Factors; Signal Transduction; Smad3 Protein; Statistics, Nonparametric; Transforming Growth Factor beta1; Treatment Outcome; Tretinoin

It has been reported that the all-trans retinoic acid (atRA)-mediated protective effects in various cells are related to the inhibition of nuclear factor (NF)-κB activities. There exists some evidence that an increase in vascular endothelial growth factor (VEGF), which is expressed by proximal tubular epithelial cells and regulated by NFκB, may play a critical role in maintaining peritubular capillary endothelium in renal disease. By stimulating the production of VEGF, hypoxia is involved in tubulointerstitial fibrosis processes in various renal diseases.. NRK52E cells survival rate was proportional to absorbance in dimethyl-thiazol-diphenyltetrazoliumbromide tests. Quantitative real-time polymerase chain reaction and Western blot were performed to assay the expression of VEGF, p65, and Scpep1. The activation of NFκB was determined by electrophoretic mobility shift assay. Co-immunoprecipitation analysis demonstrates that whether the Scpep1 and NFκB protein interacted.. We demonstrated that the hypoxia-mimicking agent CoCl2 triggered hypoxia injury of rat proximal tubular epithelial cells and significantly reduced cell viability. Addition of atRA increased the cell survival rate. Under CoCl(2)-mimicking hypoxic conditions, the expression of VEGF and p65 increased. The addition of atRA significantly attenuated the expression of VEGF and p65. There was a similar variation of NFκB/DNA binding activities. atRA not only activated distinct pathways to stimulate the expression of Scpep1, a retinoid-inducible gene, under normoxic conditions, but also acted as a CoCl(2)-mimicking hypoxia.. The protective effects of atRA against hypoxia-induced injury might be involved in suppression of VEGF expression via stimulating Scpep1 distinct pathways and inhibiting the NFκB pathway.

Topics: Animals; Blotting, Western; Carboxypeptidases; Cell Hypoxia; Cell Line; Cell Survival; Cobalt; Cytoprotection; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Epithelial Cells; Fibrosis; Gene Expression Regulation; Immunoprecipitation; Kidney Tubules, Proximal; Rats; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factor RelA; Tretinoin; Vascular Endothelial Growth Factor A

Topics: Actins; Animals; Collagen Type IV; Disease Models, Animal; Fibronectins; Fibrosis; Kidney; Kidney Diseases; Rats; Transforming Growth Factor beta1; Tretinoin; Ureteral Obstruction

This study was performed to investigate the association of prohibitin with renal interstitial fibrosis (RIF) lesion and to explore the association of all-trans retinoic acid (ATRA) treatment with prohibitin expression in RIF rats. Rats were divided into three groups: the sham operation group (SHO), the model group subjected to unilateral ureteral obstruction (UUO), and the model group treated with ATRA (GA). Renal tissues were collected at 14 and 28 days after surgery, and the relevant indicators were detected. In comparison with the SHO group, the RIF index in the UUO group was markedly elevated (p < 0.01), and the RIF index in the GA group was alleviated compared with that in the UUO group (p < 0.01). Compared with the SHO group, the expression of prohibitin (protein or mRNA) in the UUO group was significantly reduced (each p < 0.01). Prohibitin expression in the GA group was markedly increased when compared with that in the UUO (p < 0.01). The expression of TGF-β1 (protein and mRNA), protein expressions of Col-IV, fibronectin, α-SMA and cleaved Caspase-3, ROS generation and cell apoptosis index in the UUO group were markedly higher than those in the SHO group (all p < 0.01), and their expressions in the GA group were markedly down-regulated compared to those in the UUO group (all p < 0.01, respectively). The protein expression of prohibitin was negatively correlated with the RIF index, protein expression of TGF-β1, Col-IV, fibronectin, α-SMA or cleaved Caspase-3, ROS generation and the cell apoptosis index (each p < 0.01). In conclusion, lower expression of prohibitin is associated with the RIF, and ATRA treatment is associated with increased prohibitin, which can prevent the progression of RIF.

Topics: Actins; Animals; Apoptosis; Caspase 3; Collagen Type IV; Fibronectins; Fibrosis; Gene Expression Regulation; Kidney Diseases; Male; Prohibitins; Rats, Wistar; Reactive Oxygen Species; Repressor Proteins; RNA, Messenger; Transforming Growth Factor beta1; Tretinoin

The authors wish to change Figure 2 of the paper published in IJMS [1]. The positions of H(1) and H(2) in the previous article were reversed. These errors have been amended in an amended version of the manuscript, which is available from the International Journal of Molecular Sciences website. The authors and publisher apologize for the inconvenience. [...].

Topics: Animals; Antineoplastic Agents; Fibrosis; Gene Expression Regulation; Kidney; Kidney Diseases; Prohibitins; Rats; Repressor Proteins; Tretinoin

Retinoic acids, a group of natural and synthetic vitamin A derivatives, have potent anti-proliferative, anti-inflammatory and anti-fibrotic properties. We investigated the therapeutic effect of all-trans-retinoic acid (ATRA) on unilateral ureteral obstruction (UUO) model mice.. First, to evaluate the prophylactic effect, we administered 0.5 mg of ATRA for 3 days before UUO (UUO ATRA). Then, to evaluate the therapeutic effects, we administered 0.5 mg of ATRA 3 days after UUO (Day 3 ATRA). We compared the histological changes and immunostaining of macrophages, α-smooth muscle actin (α-SMA) and collagen I, and mRNA expression of monocyte chemotactic protein-1 (MCP-1), transforming growth factor (TGF)-β(1) and TGF-β R-II by RT-PCR 7 days after UUO.. In the UUO ATRA and Day 3 ATRA groups, we observed a significant improvement in histological and immunological findings, including macrophage infiltration and improved expression of MCP-1, TGF-β(1), α-SMA and collagen I compared with the UUO Day 7 group.. ATRA treatment is not only an effective prophylactic strategy, but also a therapeutic strategy for the treatment of progressive renal fibrosis in diseased kidneys.

Topics: Actins; Animals; Chemokine CCL2; Collagen Type I; Disease Models, Animal; Female; Fibrosis; Kidney Diseases; Mice; Mice, Inbred C57BL; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tretinoin; Ureteral Obstruction

Alb/TGF-beta(1) transgenic mice overexpress active transforming growth factor-beta(1) (TGF-beta(1)) in the liver, leading to increased circulating levels of the cytokine and progressive renal fibrosis. This study was designed to explore if exogenous all-trans retinoic acid (tRA) prevents renal fibrosis in this animal model.. The retinoid profile in kidney and liver of wild-type and Alb/TGF-beta(1) transgenic mice was examined by high-performance liquid chromatography and slow-release pellets containing different amounts of tRA were implanted subcutaneously to treat the Alb/TGF-beta(1) transgenic mice, starting at 1 week of age; mice were sacrificed 2 weeks later.. Kidneys of 3-week-old wild-type mice had abundant tRA, which was completely absent in kidneys of the transgenic mice. Low doses of tRA (6-10.7 mg/kg/day) failed to affect renal fibrosis although it tended to suppress the mRNA expression of some molecular markers of fibrosis and retinal dehydrogenase 2 (RALDH2), a gene encoding a key tRA-synthesising enzyme. These tendencies disappeared, mortality tended to increase and RALDH2 and connective tissue growth factor (CTGF) mRNAs significantly increased in the medium-dose group (12.7-18.8 mg/kg/day). High doses (20.1-27.4 mg/kg/day) showed even higher toxicity with increased renal fibrosis and significant mortality.. Alb/TGF-beta(1) transgenic mice are characterised by depletion of endogenous renal tRA. Exogenous tRA dose-dependently increases mortality and kidney fibrosis, which is associated with dose-dependent regulation of renal RALDH2 and CTGF mRNA expression.

Topics: Animals; Connective Tissue Growth Factor; Disease Models, Animal; Fibrosis; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Transforming Growth Factor beta1; Tretinoin

To determine the usefulness of slow-releasing all-trans-retinoic acid (ATRA) in polytetrafluoroethylene (PTFE)/polylactide-co-glycolide (PLGA) for delayed adjustable strabismus surgery.. Animal study.. A prospective, masked-observer, controlled study was performed in 25 rabbits. Fifty rabbit eyes were divided randomly into three groups. After a recession of the superior rectus muscle, a PTFE/PLGA laminate containing ATRA, PTFE alone, or balanced salt solution was applied beneath and over the superior rectus muscle in the PTFE/PLGA/all-trans-retinoic acid group (ATRA group), the polytetrafluoroethylene group (PTFE group), and the control group, respectively. Delayed adjustment was performed once on each superior rectus muscle at 3 or 5 weeks after surgery by a masked observer.. In the control group, adjustment was possible in 2 of 5 eyes at 3 weeks after surgery and impossible in any eye at 5 weeks after surgery. In the PTFE and ATRA groups, adjustment was possible in all 10 eyes at 3 and 5 weeks after surgery. On comparing adjustability, a significant difference was observed between the PTFE group and the control group or between the ATRA group and the control group 5 weeks after surgery (P = .0003 and P = .0003, respectively). A significant difference was observed between the ATRA group and the control group in terms of adhesion between superior rectus muscles and sclerae at 5 weeks after surgery (P = .006).. Slow-releasing ATRA in PTFE/PLGA was found to reduce adhesion and to allow delayed adjustment in most eyes for up to 5 weeks after surgery.

Topics: Absorbable Implants; Animals; Delayed-Action Preparations; Drug Delivery Systems; Fibrosis; Keratolytic Agents; Lactic Acid; Oculomotor Muscles; Ophthalmologic Surgical Procedures; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polytetrafluoroethylene; Prospective Studies; Rabbits; Strabismus; Suture Techniques; Tretinoin

To investigate the effects of all-trans retinoic acid (ATRA) on the expression of transforming growth factor-beta 1 (TGF-beta 1) and collagen I (COL-I )in rat model of peritoneal dialysis, which may relate to the prevention peritoneal fibrosis.. Peritoneal dialysis model was established in rats, and then the rats were given ATRA 2 mg/kg (small dose group) or 5 mg/kg (large dose group) by the way of intraperitoneal injection once a day. All the rats were sacrificed on day 28. TGF-beta 1 and COL-I protein expression of peritoneum were measured by immunohistochemistry. TGF-beta 1 mRNA expression were examined with real time polymerase chain reaction (RT-PCR).. Masson stain showed that the peritoneum thickness was significantly increased in the rats model, and collagen deposition was evident in the thickened submesothelial compact zone. With the treatment of ATRA, either in small or large dose, pathological changes were significantly lessened. The expression of TGF-beta 1 and COL-I of peritoneum was increased significantly in the rats model, but the levels in the two ATRA treated groups were lower than those of the untreated group.. ATRA could decrease the experession of TGF-beta 1 and COL-I in peritoneum and delay the progression of peritoneal fibrosis.

Topics: Animals; Collagen Type I; Fibrosis; Gene Expression; Immunohistochemistry; Male; Models, Animal; Peritoneal Dialysis; Peritoneum; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta1; Tretinoin

The mouse model of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal fibrosis allows for detailed study of the extracellular matrix changes that complicate Crohn's disease. Indomethacin induces intestinal fibrosis, while retinoic acid (RA) reduces liver fibrosis. Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix-modifying agent, may potentially link these opposing effects. Our aim was to determine the effects of indomethacin and RA and to evaluate their correlation to SPARC expression in the TNBS mouse model. CD-1 mice were randomised to TNBS enemas weekly for 2 or 8 weeks with or without indomethacin (0.2 mg/kg per day) or RA (100 microg/kg per day). At 2 weeks, indomethacin/TNBS enhanced and RA reduced inflammation, tissue destruction and fibrosis. The expression of SPARC was inversely related to fibrosis, but not to inflammation, in the TNBS-alone groups at 2 weeks; these differences were lost by 8 weeks. The results demonstrate that indomethacin increases TNBS-induced fibrosis in mice, while RA reduces it, and that SPARC may link these opposing effects.

Topics: Animals; Crohn Disease; Disease Models, Animal; Female; Fibrosis; Immunoenzyme Techniques; Indomethacin; Mice; Osteonectin; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA; Statistics, Nonparametric; Tretinoin; Trinitrobenzenesulfonic Acid

This study was designed to determine the effect of all-trans retinoic acid (RA) on the development of cardiac remodeling in a pressure overload rat model. Male Sprague-Dawley rats were subjected to sham operation and the aortic constriction procedure. A subgroup of sham control and aortic constricted rats were treated with RA for 5 mo after surgery. Pressure-overloaded rats showed significantly increased interstitial and perivascular fibrosis, heart weight-to-body weight ratio, and gene expression of atrial natriuretic peptide and brain natriuretic peptide. Echocardiographic analysis showed that pressure overload induced systolic and diastolic dysfunction, as evidenced by decreased fractional shortening, ejection fraction, stroke volume, and increased E-to-E(a) ratio and isovolumic relaxation time. RA treatment prevented the above changes in cardiac structure and function and hypertrophic gene expression in pressure-overloaded rats. RA restored the ratio of Bcl-2 to Bax, inhibited cleavage of caspase-3 and -9, and prevented the decreases in the levels of SOD-1 and SOD-2. Pressure overload-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by RA, via upregulation of mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-2. The pressure overload-induced production of angiotensin II was inhibited by RA via upregulation of expression of angiotensin-converting enzyme (ACE)2 and through inhibition of the expression of cardiac and renal renin, angiotensinogen, ACE, and angiotensin type 1 receptor. Similar results were observed in cultured neonatal cardiomyocytes in response to static stretch. These results demonstrate that RA has a significant inhibitory effect on pressure overload-induced cardiac remodeling, through inhibition of the expression of renin-angiotensin system components.

Topics: Animals; Animals, Newborn; Aorta, Thoracic; Apoptosis; Atrial Natriuretic Factor; Blood Pressure; Blotting, Western; Cells, Cultured; Fibrosis; Heart; Heart Rate; Hypertrophy, Left Ventricular; Ligation; Male; Mitogen-Activated Protein Kinases; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Physical Stimulation; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Ultrasonography

Intestinal fibrosis leading to severe bowel dysmobility or obstruction is a troublesome adverse effect of abdominal or pelvic radiation therapy. We have recently reported that all-trans-retinoic acid (ATRA) prevents radiation- or bleomycin-induced lung fibrosis. Here, we examined the impact of ATRA on the mouse model of radiation-induced intestinal fibrosis.. We evaluated the histology of late radiation fibrosis in surgical samples. We then performed histological examinations and quantitative measurements of mRNA of interleukin-6 and transforming growth factor-beta(1) in intestinal tissues of irradiated mice with or without intraperitoneal administration of ATRA and investigated the effect of ATRA on the transdifferentiation and the production of collagen of irradiated human intestinal fibroblasts.. Human samples of late radiation enteritis showed thickened submucosa and serosa, consistent with mouse model. Administration of ATRA attenuated irradiation-induced intestinal fibrosis and reduced mRNA of interleukin-6 and transforming growth factor-beta(1). In vitro studies disclosed that ATRA suppressed the transdifferentiation of irradiated intestinal fibroblasts and diminished the production of collagen from the cells.. Our findings indicate that ATRA ameliorates irradiation-induced intestinal fibrosis. ATRA could be a novel approach in the treatment of fibrosis associated with chronic radiation enteritis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Cell Line; Cell Transdifferentiation; Disease Models, Animal; Enteritis; Female; Fibroblasts; Fibrosis; Humans; Interleukin-6; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Middle Aged; Radiotherapy; RNA, Messenger; Transforming Growth Factor beta1; Tretinoin

Activated pancreatic stellate cells (PSCs) are implicated in the production of alcohol induced pancreatic fibrosis. PSC activation is invariably associated with loss of cytoplasmic vitamin A (retinol) stores. Furthermore, retinol and ethanol are known to be metabolised by similar pathways. Our group and others have demonstrated that ethanol induced PSC activation is mediated by the mitogen activated protein kinase (MAPK) pathway but the specific role of retinol and its metabolites all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-RA) in PSC quiescence/activation, or its influence on ethanol induced PSC activation is not known. Therefore, the aims of this study were to (i) examine the effects of retinol, ATRA, and 9-RA on PSC activation; (ii) determine whether retinol, ATRA, and 9-RA influence MAPK signalling in PSCs; and (iii) assess the effect of retinol supplementation on PSCs activated by ethanol.. Cultured rat PSCs were incubated with retinol, ATRA, or 9-RA for varying time periods and assessed for: (i) proliferation; (ii) expression of alpha smooth muscle actin (alpha-SMA), collagen I, fibronectin, and laminin; and (iii) activation of MAPKs (extracellular regulated kinases 1 and 2, p38 kinase, and c-Jun N terminal kinase). The effect of retinol on PSCs treated with ethanol was also examined by incubating cells with ethanol in the presence or absence of retinol for five days, followed by assessment of alpha-SMA, collagen I, fibronectin, and laminin expression.. Retinol, ATRA, and 9-RA significantly inhibited: (i) cell proliferation, (ii) expression of alpha-SMA, collagen I, fibronectin, and laminin, and (iii) activation of all three classes of MAPKs. Furthermore, retinol prevented ethanol induced PSC activation, as indicated by inhibition of the ethanol induced increase in alpha-SMA, collagen I, fibronectin, and laminin expression.. Retinol and its metabolites ATRA and 9-RA induce quiescence in culture activated PSCs associated with a significant decrease in the activation of all three classes of MAPKs in PSCs. Ethanol induced PSC activation is prevented by retinol supplementation.

Topics: Alitretinoin; Animals; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Dual Specificity Phosphatase 1; Enzyme Activation; Enzyme Inhibitors; Ethanol; Extracellular Matrix Proteins; Fibrosis; Immediate-Early Proteins; Mitogen-Activated Protein Kinases; Pancreas; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Rats; Receptors, Retinoic Acid; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Vanadates; Vitamin A

We investigated the preventive effects of a synthetic acyclic retinoid, NIK-333, on the early and late events of hepatocarcinogenesis in male F344 rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB). NIK-333 was administered once a day on consecutive days at a dose of 10, 40, or 80 mg/kg body weight along with the supplementation with 3'-MeDAB-containing diet for 16 wk. Animals from each group were sacrificed at 4 and 16 wk after the commencement of the experiment to determine the effect of NIK-333 on the early and late stages of carcinogenesis, respectively. NIK-333 suppressed the emergence of both oval-like cells expressing transforming growth factor (TGF)-alpha, putative progenitors of hepatocellular carcinoma (HCC), and activated hepatic stellate cells, major matrix-producing cells of the liver, in the early stage and inhibited the incidence of HCC in the late phase. These results suggest that NIK-333 is a promising drug for the chemoprevention of HCC by uniquely suppressing the early events of hepatocarcinogenesis, that is, development of both oval-like cells and fibrogenesis.

Topics: Actins; Adenoma; Animals; Antineoplastic Agents; Carcinoma; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Fibrosis; Liver; Liver Neoplasms, Experimental; Male; Methyldimethylaminoazobenzene; Rats; Rats, Inbred F344; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor alpha; Tretinoin

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.

Topics: Acyltransferases; Adipose Tissue; Animals; Blotting, Western; Cell Line; Chromatography, High Pressure Liquid; Chylomicrons; Cytosol; Female; Fibrosis; Genotype; Humans; Lipids; Liver; Male; Mice; Mice, Transgenic; Microscopy, Electron; Microsomes, Liver; Radioimmunoassay; Sex Factors; Time Factors; Tissue Distribution; Tretinoin; Vitamin A

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primarily mediated through its receptors, ligand-dependent transcription factors of the steroid/thyroid/vitamin D nuclear receptor superfamily. Recent studies indicate that administration of RA mitigates progressive kidney disease, underscoring its renoprotective potential. In this study, we investigated the effects of 9-cis-RA on glomerular mesangial cell activation induced by transforming growth factor (TGF)-beta1 using an in vitro cell culture system. In human mesangial cells 9-cis-RA suppressed TGF-beta1-induced alpha-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression, but it did not significantly affect cell proliferation and survival. Interestingly, 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, and activated c-met receptor phosphorylation. Similar to HGF, 9-cis-RA induced expression of the Smad transcriptional co-repressor TGIF in mesangial cells. Overexpression of exogenous TGIF by transfection or 9-cis-RA treatment suppressed trans-activation of the TGF-beta-responsive promoter. Moreover, conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, these results indicate that 9-cis-RA possesses anti-fibrotic ability by antagonizing TGF-beta1 in mesangial cells and that 9-cis-RA activity is likely mediated through a mechanism dependent on HGF/c-met receptor signaling.

Topics: Actins; Adenoviridae; Alitretinoin; Animals; Blotting, Western; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Fibronectins; Fibrosis; Fluorescent Antibody Technique, Indirect; Glomerular Mesangium; Humans; Luciferases; Mice; Phosphorylation; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Proto-Oncogene Proteins c-met; Rats; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Up-Regulation

Livers of aryl hydrocarbon receptor (AHR)-null mice have high levels of retinoic acid (RA), retinol, and retinyl palmitate. Hepatic accumulation of RA in these mice may be responsible in part for the hepatic phenotype characterized by small liver size and fibrosis. The increased levels of hepatic RA may be due to decreased metabolism of RA to 4-hydroxyretinoic acid. To identify the P450 isoform(s) involved in RA metabolism, liver microsomes from AHR-null and wild-type mice were subjected to Western blotting and probed with antibodies to rat P450s that cross-react with murine forms. Signal intensity in Western blots probed with anti-rat CYP2C6 antibodies correlated with levels of RA 4-hydroxylation. Furthermore, this anti-rat CYP2C6 antibody inhibited RA 4-hydroxylase activity of wild-type mouse liver microsomes to the levels of AHR-null mouse liver. When used to screen a mouse liver cDNA expression library, this antibody exclusively recognized the murine P450 CYP2C39. Catalytic assays of five recombinant mouse CYP2Cs expressed in Escherichia coli revealed that only CYP2C39 was competent for RA 4-hydroxylation (K(m) = 812.3 nm and V(max) 47.85 (fmol/min/pmol P450)). Real time reverse transcriptase-PCR used to assess the Cyp2C39 mRNA expression showed decreased levels (30%) of this transcript in AHR-null compared with wild-type liver, consistent with decreased protein levels observed by Western blot analysis using an antibody to a CYP2C39-specific peptide. These data show that CYP2C39 catalyzes RA catabolism and thus possibly controls RA levels in mouse liver. Down-regulation of Cyp2C39 is hypothesized to be responsible for the liver phenotype in the AHR-null mouse.

Topics: Animals; Blotting, Western; Catalysis; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; DNA Primers; DNA, Complementary; Down-Regulation; Fibrosis; Kinetics; Liver; Male; Mice; Mice, Transgenic; Microsomes, Liver; Mixed Function Oxygenases; Models, Chemical; Peptides; Phenotype; Polychlorinated Dibenzodioxins; Polymerase Chain Reaction; Protein Isoforms; Rats; Receptors, Aryl Hydrocarbon; Recombinant Proteins; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

There are in vitro data linking all-trans retinoic acid (atRA) with inhibition of hypertrophy and hyperplasia in cardiomyocytes, vascular smooth muscle cells, and fibroblasts. In the present study, we tested the hypothesis that chronic treatment with atRA may blunt the process of myocardial remodeling in spontaneously hypertensive rats (SHR). Four-week-old male SHR were treated with atRA (5 or 10 mg.kg-1.day-1) given daily for 3 mo by gavage; age- and sex-matched Wistar-Kyoto rats (WKY) and placebo-treated SHR served as controls. At the end of the treatment period, cardiac geometry and function were assessed by Doppler echocardiography. Histological examination and RIA were performed to evaluate medial thickening of intramyocardial and renal arteries, perivascular and interstitial collagen content, and atrial natriuretic peptide (ANP) and IGF-I in the heart, respectively. The novel finding of the present study is that atRA prevented hypertrophy of intramyocardial and intrarenal arteries and ventricular fibrosis. However, atRA treatment did not lower blood pressure or left ventricular weight and left ventricular weight-to-body weight ratio in SHR. atRA did not change cardiac geometry and function as assessed by Doppler echocardiography. atRA showed no influence on either ANP or IGF-I levels. In conclusion, the present study suggests that chronic atRA treatment prevents medial thickening of intramyocardial and intrarenal arteries and ventricular fibrosis during the development of hypertension. Left ventricular hypertrophy and cardiac geometry and function are not changed by atRA treatment.

Topics: Animals; Body Weight; Coronary Vessels; Drug Administration Schedule; Fibrosis; Heart; Heart Ventricles; Hemodynamics; Hypertension; Hypertrophy; Male; Myocardium; Organ Size; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Renal Artery; Tretinoin; Tunica Media

Studies have shown that angiotensin-converting enzyme inhibitors and an angiotensin II receptor blocker can delay, but cannot reverse, the progression of experimentally induced radiation nephropathy. In an effort to find a method for reversing injury, three agents were tested in a rat model of radiation nephropathy. Pirfenidone (a phenyl-pyridone antifibrotic) and thiaproline (an inhibitor of collagen deposition) were not capable of retarding the development of radiation nephropathy. However, all-trans retinoic acid (an anti-inflammatory agent) exacerbated radiation nephropathy. We speculated that the detrimental effects of retinoic acid might be the result of stimulation of renal cell proliferation. However, retinoic acid had no effect on tubular or glomerular cell proliferation in normal animals and did not enhance radiation-induced proliferation. A recent report that retinoic acids inhibit nitric oxide production suggested an alternative mechanism, since inhibition of production of nitric oxide is known to exacerbate radiation nephropathy. Experiments demonstrated that retinoic acid exacerbated the radiation-induced drop in renal production of nitric oxide, suggesting that the detrimental effect of all-trans retinoic acid might be explained by inhibition of renal nitric oxide activity. Particularly in view of the recent clinical report of enhancement of radiation nephropathy by retinoic acid in patients receiving bone marrow transplantation, the combination of retinoic acid and renal irradiation should be carried out with great caution.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Extracellular Matrix; Fibrosis; Hypertension, Renal; Kidney; Kidney Diseases; Kidney Failure, Chronic; Proteinuria; Pyridones; Radiation Injuries, Experimental; Rats; Rats, Inbred Strains; Thiazoles; Thiazolidines; Tretinoin; Uremia; Whole-Body Irradiation

Biocompatibility studies of all-trans-retinoic acid (RA)-loaded microspheres were carried out after they were subcutaneously injected into rats. To characterize the inflammatory response to these microspheres, tissue reactions at the implantation site and cell types in the interstices of the microspheres were evaluated for 180 days. On the 15th day, the cross-sectional area of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres was four times larger than that of the control microspheres. The size of the fibrous capsules surrounding the implantation site of the RA-loaded microspheres decreased significantly over a period of 75 days, while the size of the fibrous capsules surrounding the implantation site of the control microspheres remained almost constant throughout the entire course of 180 days. The tissue response to the RA-loaded microspheres was more intensified by the increased extensive cellular infiltration of macrophages, granulation tissue, and fibrosis than that to the control microspheres. The difference in the inflammatory response between the RA-loaded microspheres and the control microspheres was significant for 75 days after implantation. It was suggested that the released RA from the microspheres stimulated inflammatory responses. However, no further enhanced inflammation reactions were detected after RA had been completely released from the microspheres.

Topics: Animals; Biocompatible Materials; Drug Delivery Systems; Female; Fibrosis; Granulation Tissue; Inflammation; Injections, Subcutaneous; Macrophages; Materials Testing; Microspheres; Polyesters; Polyethylene Glycols; Rats; Tretinoin

The inhibitory effect of tazarotene, an acetylenic retinoid, on human dermal fibroblast in vitro was compared to that of all-trans-retinoic acid. The proliferation of fibroblasts was inhibited by both retinoids at the concentration of 1 microM after 5 days of culture. Synthesis of DNA and collagen was inhibited by both retinoids concentration-dependently up to 10 microM, although tazarotene was weaker in the inhibition of collagen synthesis. These results suggest the possible usefulness of tazarotene in the treatment of fibrotic diseases.

Topics: Bromodeoxyuridine; Cell Division; Cells, Cultured; Collagen; Dermatologic Agents; DNA; Fibroblasts; Fibrosis; Humans; Nicotinic Acids; Receptors, Retinoic Acid; Retinoids; Skin; Tretinoin

Expression of heat-shock protein 47 in intact and fibrotic liver and in hepatic constituent cells was investigated in mice. Immunohistochemical study of intact liver and Western blot analysis of the protein from isolated liver cells revealed that stellate cells and smooth muscle cells of interlobular vessels, but not hepatocytes, Kupffer cells, or endothelial cells, expressed heat-shock protein 47. The protein was found in both vitamin-A-storing stellate cells and myofibroblast-like cells. The amount of the protein in cultured stellate cells was reduced by dexamethasone but was not regulated by quercetin, transforming growth factor beta, interferon gamma, or retinoic acid. In CCl4-treated or bile-duct-ligated mouse liver, the number of cells positive for heat-shock protein 47 markedly increased in the centrilobular area or around the periportal area, respectively, and the level of heat-shock protein 47 also increased.

Topics: Animals; Blotting, Western; Cells, Cultured; Dexamethasone; Fibrosis; Heat-Shock Proteins; HSP47 Heat-Shock Proteins; Immunohistochemistry; Interferon-gamma; Liver; Male; Mice; Mice, Inbred ICR; Quercetin; Transformation, Genetic; Transforming Growth Factor beta; Tretinoin