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Gunter Wolf - One of the best experts on this subject based on the ideXlab platform.
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Lack of Type VIII Collagen in Mice Ameliorates Diabetic Nephropathy
Diabetes, 2009Co-Authors: Ulrich Hopfer, Helmut Hopfer, Catherine Meyer-schwesinger, Ivonne Loeffler, Naomi Fukai, Bjorn R. Olsen, Rolf A.k. Stahl, Gunter WolfAbstract:OBJECTIVE Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen–dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by STZ injections in collagen VIII–deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal–regulated kinase (ERK1/2) and p27Kip1 was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells. RESULTS Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1 −/ COL8A2 − animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1 −/ COL8A2 − mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27Kip1 expression. Overexpression of Col8a1 in mesangial cells induced proliferation. CONCLUSIONS Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.
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Collagen type VIII expression in human diabetic nephropathy
European Journal of Clinical Investigation, 2007Co-Authors: John Gerth, Ulrich Hopfer, Maja T Lindenmeyer, Clemens D Cohen, Hermann Josef Grone, M. Sommer, Gunter WolfAbstract:Background Collagen type VIII is a non-fibrillar short-chain collagen that may modulate migration, proliferation and adherence of various cells. Only very sparse information exists on collagen type VIII expression in human diabetic nephropathy. Material and methods We retrospectively studied mRNA expression for the two collagen type VIII chains (COL8A1 and COL8A2) in 20 biopsies with histologically confirmed diabetic nephropathy by real-time PCR, and compared glomerular and tubular expression with normal kidney [pre-transplant biopsies (n = 10)]. Expression of collagen type VIII was also studied in biopsies from patients with benign nephrosclerosis (BNS; n=16) and focal-segmental glomerulosclerosis (FSGS; n = 9). Results A strong specific induction of COL8A1 mRNA was found in diabetic nephropathy in both glomerular and tubular compartments. There was also a robust induction of COL8A2 in diabetic nephropathy, but overall expression was lower than that of COL8A1 transcripts. No significant increase in COL8A1 and COL8A2 mRNAs expression was found in biopsies from patients with BNS and FSGS compared with normal kidneys. The cross-reactivity of the used anti-al (VIII) antibody with human tissue was confirmed by Western blots. Immunohistological analysis revealed only little staining for collagen type VIII in the normal kidney, localized to vessels. There was an up-regulation of collagen type VIII protein expression as shown by immunohistochemistry in the diabetic nephropathy biopsies mainly localized to mesangial cells, tubules and the interstitium. Proteinuria and serum creatinine did not correlate with glomerular or tubular COL8A1 and COL8A2 mRNA expression in diabetic patients. Conclusion Our study systemically investigates collagen type VIII expression in human biopsies. Induction of collagen type VIII was specific for diabetic nephropathy and did not occur in the other renal diseases studied. More specific factors of the diabetic environment are likely involved in the stimulated expression because there was no correlation of collagen type VIII mRNA expression with proteinuria. Since collagen type VIII may influence proliferation and migration of cells, it is possible that an increase in renal expression of collagen type VIII initiates other pathophysiological processes (e.g. proliferation of renal fibroblasts) involved in diabetic nephropathy.
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Collagen type VIII expression in human diabetic nephropathy
European Journal of Clinical Investigation, 2007Co-Authors: John Gerth, Ulrich Hopfer, Maja T Lindenmeyer, Clemens D Cohen, Hermann Josef Grone, M. Sommer, Gunter WolfAbstract:BACKGROUND: Collagen type VIII is a non-fibrillar short-chain collagen that may modulate migration, proliferation and adherence of various cells. Only very sparse information exists on collagen type VIII expression in human diabetic nephropathy. MATERIAL AND METHODS: We retrospectively studied mRNA expression for the two collagen type VIII chains (COL8A1 and COL8A2) in 20 biopsies with histologically confirmed diabetic nephropathy by real-time PCR, and compared glomerular and tubular expression with normal kidney [pre-transplant biopsies (n = 10)]. Expression of collagen type VIII was also studied in biopsies from patients with benign nephrosclerosis (BNS; n = 16) and focal-segmental glomerulosclerosis (FSGS; n = 9). RESULTS: A strong specific induction of COL8A1 mRNA was found in diabetic nephropathy in both glomerular and tubular compartments. There was also a robust induction of COL8A2 in diabetic nephropathy, but overall expression was lower than that of COL8A1 transcripts. No significant increase in COL8A1 and COL8A2 mRNAs expression was found in biopsies from patients with BNS and FSGS compared with normal kidneys. The cross-reactivity of the used anti-alpha1(VIII) antibody with human tissue was confirmed by Western blots. Immunohistological analysis revealed only little staining for collagen type VIII in the normal kidney, localized to vessels. There was an up-regulation of collagen type VIII protein expression as shown by immunohistochemistry in the diabetic nephropathy biopsies mainly localized to mesangial cells, tubules and the interstitium. Proteinuria and serum creatinine did not correlate with glomerular or tubular COL8A1 and COL8A2 mRNA expression in diabetic patients. CONCLUSION: Our study systemically investigates collagen type VIII expression in human biopsies. Induction of collagen type VIII was specific for diabetic nephropathy and did not occur in the other renal diseases studied. More specific factors of the diabetic environment are likely involved in the stimulated expression because there was no correlation of collagen type VIII mRNA expression with proteinuria. Since collagen type VIII may influence proliferation and migration of cells, it is possible that an increase in renal expression of collagen type VIII initiates other pathophysiological processes (e.g. proliferation of renal fibroblasts) involved in diabetic nephropathy.
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targeted disruption of col8a1 and COL8A2 genes in mice leads to anterior segment abnormalities in the eye
The FASEB Journal, 2005Co-Authors: Gunter Wolf, Helmut Hopfer, Naomi Fukai, Ulrike Hopfer, Nancy C Joyce, En Li, Bjorn R. OlsenAbstract:Collagen VIII is localized in subendothelial and subepithelial extracellular matrices. It is a major component of Descemet’s membrane, a thick basement membrane under the corneal endothelium, where it forms a hexagonal lattice structure; a similar structure, albeit less extensive, may be formed in other basement membranes. We have examined the function of collagen VIII in mice by targeted inactivation of the genes encoding the two polypeptide subunits, Col8a1 and COL8A2. Analysis of these mice reveals no major structural defects in most organs, but demonstrates that type VIII collagen is required for normal anterior eye development, particularly the formation of a corneal stroma with the appropriate number of fibroblastic cell layers and Descemet’s membrane of appropriate thickness. Complete lack of type VIII collagen leads to dysgenesis of the anterior segment of the eye: a globoid, keratoglobus-like protrusion of the anterior chamber with a thin corneal stroma. Descemet’s membrane is markedly thinned. T...
Douglas B Gould - One of the best experts on this subject based on the ideXlab platform.
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use of sodium 4 phenylbutyrate to define therapeutic parameters for reducing intracerebral hemorrhage and myopathy in col4a1 mutant mice
Disease Models & Mechanisms, 2018Co-Authors: Genki Hayashi, Cassandre Labelledumais, Douglas B GouldAbstract:Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers that constitute a major component of nearly all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that includes variable cerebrovascular and skeletal muscle manifestations. The pathogenicity of COL4A1 and COL4A2 mutations is generally attributed to impaired secretion into basement membranes. Sodium 4-phenylbutyrate (4PBA) is an FDA-approved drug that promotes mutant heterotrimer secretion in vitro and in vivo . Here, we use different 4PBA treatment paradigms to define therapeutic parameters for preventing cerebrovascular and muscular pathologies in Col4a1 mutant mice. We show efficacy of long-term 4PBA treatment in reducing the severity of intracerebral hemorrhages (ICH) in Col4a1 mutant mice aged up to 8 months. In addition, we demonstrate that maximal efficacy of 4PBA on ICH and myopathy was achieved when treatment was initiated prenatally, whereby even transient 4PBA administration had lasting benefits after being discontinued. Importantly, postnatal treatment with 4PBA also reduced ICH and skeletal myopathy severities in Col4a1 mutant mice, which has significant clinical implications for patients with COL4A1 and COL4A2 mutations.
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Molecular and Genetic Analyses of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention
Circulation, 2015Co-Authors: Marion Jeanne, Jeff Jorgensen, Douglas B GouldAbstract:Background—Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases, including cerebral microbleeds, porencephaly, and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown, and there is a lack of effective treatment. Methods and Results—Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small-vessel disease, recurrent hemorrhagic strokes, and age-related macroangiopathy. We showed that allelic heterogeneity, genetic context, and environmental factors such as intense exercise or anticoagulant medication modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endo...
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allelic heterogeneity contributes to variability in ocular dysgenesis myopathy and brain malformations caused by col4a1 and col4a2 mutations
Human Molecular Genetics, 2014Co-Authors: Cassandre Labelledumais, Marion Jeanne, William Kauffman, Jennifer M Allen, Jack Favor, Douglas B GouldAbstract:Collagen type IV alpha 1 and 2 (COL4A1 and COL4A2) are present in nearly all basement membranes. COL4A1 and COL4A2 mutations are pleiotropic, affecting multiple organ systems to differing degrees, and both genetic-context andenvironmentalfactorsinfluencethisvariableexpressivity.Here,we reportimportant phenotypic and molecular differences in an allelic series of Col4a1 and Col4a2 mutant mice that are on a uniform genetic background.WeevaluatedthreeorganscommonlyaffectedbyCOL4A1andCOL4A2mutationsanddiscovered allelic heterogeneity in the penetrance and severity of ocular dysgenesis, myopathy and brain malformations. Similarly, we show allelic heterogeneity in COL4A1 and COL4A2 biosynthesis. While most mutations that we examined caused increased intracellular and decreased extracellular COL4A1 and COL4A2, we identified three mutations with distinct biosynthetic signatures. Reduced temperature or presence of 4-phenylbutyrate amelioratedbiosyntheticdefectsinprimarycelllinesderivedfrommutantmice.Together,ourdatademonstrate the effects and clinical implications of allelic heterogeneity in Col4a1 -a ndCol4a2-related diseases. Understanding allelic differences will be valuable for increasing prognostic accuracy and for the development of therapeutic interventions that consider the nature of the molecular cause in patients with COL4A1 and COL4A2 mutations.
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col4a1 and col4a2 mutations and disease insights into pathogenic mechanisms and potential therapeutic targets
Human Molecular Genetics, 2012Co-Authors: Cassandre Labelledumais, Douglas B GouldAbstract:Heterotrimers composed of collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) constitute one of the most abundant components of nearly all basement membranes. Accordingly, mutations in COL4A1 or COL4A2 are pleiotropic and contribute to a broad spectrum of disorders, including myopathy, glaucoma and hemorrhagic stroke. Here, we summarize the contributions of COL4A1 and COL4A2 mutations in human disease, integrate knowledge gained from model organisms and evaluate the implications for pathogenic mechanisms and therapeutic approaches.
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abstract 3665 col4a1 and col4a2 mutations cause genetically modifiable cerebrovascular diseases
Stroke, 2012Co-Authors: Marion Jeanne, Cassandre Labelledumais, Berkeley W Kauffman, Steven M Greenberg, Jonathan Rosand, Yichinn Weng, Michelle De Leau, Douglas B GouldAbstract:Mutations in the type IV collagen alpha 1 gene (COL4A1) cause Cerebrovascular Diseases (CVDs) in mice and human patients. Patients with COL4A1 mutations suffer from a broad range of CVDs, from infantile porencephaly to debilitating or fatal intracerebral hemorrhage (ICH), to subclinical cerebral microbleeds, suggesting that environmental and other genetic factors may influence their phenotypes. COL4A1 is one of the most abundant proteins in basement membranes and forms heterotrimers with COL4A2. Among possible pathogenic mechanisms are cellular stress due to the toxic intracellular aggregation of the COL4A1 and COL4A2 proteins and/or their absence in the basement membrane. Our first goal is to identify the relative contributions of COL4A1 and COL4A2 mutations to sporadic ICH and to understand the cellular mechanisms and genetic complexity underlying the disease. We identified novel COL4A1 mutations and for the first time, we discovered COL4A2 mutations in a cohort of 96 patients with sporadic ICH. Using a cell-based assay we determined that the mutations impair COL4A1 and COL4A2 secretion. We showed that mutant COL4A1 or COL4A2 proteins accumulate within the cell where they titrate normal COL4A1 and COL4A2 proteins. Interestingly, we found that some of the mutations can ultimately result in endoplasmic reticulum (ER) stress and activation of the Unfolded Protein Response. Our second goal was to test the hypothesis that differences in genetic context could contribute to phenotypic variability in human patients. Thus, we characterized CVD in Col4a1 mutant mice with two different genetic backgrounds. Using cerebral magnetic resonance imaging and histological analysis, we show that one or more genetic modifiers from the CAST/EiJ strain significantly reduce the size and frequency of ICHs detected in Col4a1 mutant mice on a C57BL/6J background. In conclusion, we found that both COL4A1 and COL4A2 mutations cause ICH in human patients, our results support that ER stress could be involved in the pathogenesis and we showed that genetic context is crucial for expressivity and severity of the CVD. We predict that ongoing experiments to better understand the cell biology of COL4A1 and COL4A2 mutations and the mechanisms of genetic modification could lead to targeted therapeutics to reduce the risk of CVD in patients with COL4A1 or COL4A2 mutations.
John D Gottsch - One of the best experts on this subject based on the ideXlab platform.
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distinct clinical phenotype of corneal dystrophy predicts the p leu450trp substitution in COL8A2
Cornea, 2016Co-Authors: Allen O Eghrari, Amer S Riazuddin, John D GottschAbstract:PURPOSE: The p.(Leu450Trp) substitution in Collagen, Type VIII, Alpha 2 (COL8A2) is associated with an early-onset corneal dystrophy. Here we identify distinct anterior corneal and keratorefractive changes associated with this disease and replicate its distinguishing endothelial characteristics in a new family. METHODS: We reviewed clinical data from a large family associated with the p.(Leu450Trp) COL8A2 mutation. We compared clinical photographs and keratometry over an 11-year period. We sought to replicate these findings, and after a 40-year-old male subject presented similarly, we obtained a peripheral blood sample and sequenced COL8A2. RESULTS: Of 10 individuals with the p.(Leu450Trp) substitution, clinical records noted corneal edema in 6, of which 4 showed epithelial microcystic edema. Eleven-year progression data reveal a marked increase in subepithelial corneal edema and gradual, profound increase in anterior corneal astigmatism. Sequencing of genomic DNA from the unrelated individual predictably identified a c.1349T>G [p.(Leu450Trp)] heterozygous variation in COL8A2. Confocal microscopy confirmed attenuated endothelium, and histopathology revealed no guttae, consistent with findings from a previously identified family. CONCLUSIONS: Peripheral, anterior microcystic corneal edema represents a characteristic aspect of the phenotype associated with the p.(Leu450Trp) substitution in COL8A2, in at least 2 of 3 known affected families worldwide. We describe long-term progression and Descemet stripping endothelial keratoplasty for this disease for the first time.
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immunohistochemistry and electron microscopy of early onset fuchs corneal dystrophy in three cases with the same l450w COL8A2 mutation
Transactions of the American Ophthalmological Society, 2006Co-Authors: Cheng Zhang, Olof H Sundin, Robert W Bell, Walter J Stark, Richard W Green, Zenaida De La Cruz, John D GottschAbstract:PURPOSE: A rare, familial early-onset form of Fuchs corneal dystrophy (FCD) is caused by mutation in the COL8A2 gene. This study describes the aberrant pattern of distribution of collagen type VIII and basement membrane components in Descemet's membrane (DM) and endothelium of three individuals with the same L450W mutation that represent different stages of early-onset FCD. METHODS: Immunohistochemical studies with bright field, fluorescence, and confocal microscopy characterized the pathology of sectioned corneal buttons with antibodies against COL8A1, COL8A2, COL4, laminin, and fibronectin. A portion of each was processed for electron microscopy. RESULTS: Histologic examination of pathologic changes in case 1 demonstrated relative preservation of the endothelium, whereas in case 2 much of this layer was atrophic and in case 3 there was complete loss of the endothelium. DM also increased in thickness to 25 mum for case 1, to 31 mum for case 2, and to 38 mum for case 3. Case 1 was the only specimen to reveal shallow warts along the posterior surface of DM, whereas the most advanced specimen, case 3, showed evidence of earlier nodularity that had been buried by the accretion of further extracellular matrix material. The posterior aspect of DM in this specimen had the unusual property of lighter staining relative to the anterior region of DM, laid down earlier in life. Immunocytochemistry revealed increased expression and complex, sharply defined patterns of deposition of collagen VIII, collagen IV, laminin, and fibronectin. Ultrastructurally, the posterior nonbanded layer of DM was intermixed with banded collagen, and the posterior region of DM showed a high density of foci of spindle-shaped structures with intense-staining bands, spaced at approximately 120 nm. Finally, ultrastructural studies of the endothelium in case 1 revealed unusual accumulation of swelling mitochondria. The endothelial cells also had large amounts of abnormal prominent rough endoplasmic reticulum. Type VIII collagen alpha 2 immunogold signal was associated with the highly granular ribosomes of the rough endoplasmic reticulum of these patients. CONCLUSIONS: Microscopic and electron microscopic examination revealed pathological changes in DM of L450W COL8A2 mutants that were consistent with several-fold increased growth of the extracellular matrix and progressive deposition and synthesis of extracellular material by endothelial cells. As with late-onset FCD, this is accompanied by attenuation and eventual loss of the endothelium itself. Whether the abnormal deposition of collagen, laminin, and fibronectin contributes to the dysfunction and death of the endothelium remains to be determined.
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fuchs corneal dystrophy aberrant collagen distribution in an l450w mutant of the COL8A2 gene
Investigative Ophthalmology & Visual Science, 2005Co-Authors: John D Gottsch, Cheng Zhang, Olof H Sundin, Robert W Bell, Walter J Stark, Richard W GreenAbstract:PURPOSE: To characterize histologically Descemet's membrane in an early-onset Fuchs corneal dystrophy (FCD) COL8A2 mutant and compare these findings with corneas from late-onset FCD and normal corneas. METHODS: A corneal explant from a patient with the L450W COL8A2 mutation and others with late-onset disease were studied with antibodies to collagens IV, VIIIA1, VIIIA2, fibronectin, and laminin. Transmission electron microscopy was performed on a portion of the explant. Control explants included eye bank corneas without known disease and surgical explants from unrelated conditions. RESULTS: In normal corneas, a regular array of colocalized COL8A1 and COL8A2 was observed in the anterior half of Descemet's membrane. In the L450W mutant, Descemet's membrane was several times thicker than normal and traversed by refractile strands and blebs that stained intensely for COL8A2, a feature also observed in late-onset FCD. Both the alpha1 and alpha2 subtypes of collagen VIII were observed at high levels along the anterior edge of Descemet's, another abnormal feature also found in late-onset FCD. Ultrastructure of the L450W cornea revealed a well-formed anterior banded layer more than three times thicker than normal. An unusual, thin internal layer was rich in patches of wide-spaced collagen. The layer is a distinctive pathologic structure that is associated with FCD and is characterized by approximately 120 nm periodicity and the presence of collagen VIII. Depositions of collagen IV, fibronectin, and laminin were also greatly increased in the of posterior Descemet's membrane, yet another general feature shared between early- and late-onset disease. CONCLUSIONS: Early-onset COL8A2 L450W disease involves massive accumulation and abnormal assembly of collagen VIII within Descemet's membrane, a process that is presumed to begin during fetal development. Both early- and late-onset subtypes of FCD appear to be the result of abnormal basement membrane assembly rather than a primary defect in endothelial metabolism.
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inheritance of a novel COL8A2 mutation defines a distinct early onset subtype of fuchs corneal dystrophy
Investigative Ophthalmology & Visual Science, 2005Co-Authors: John D Gottsch, Olof H Sundin, Walter J Stark, Karl W Broman, Elizabeth C L Vito, Amol K Narang, John M Thompson, Malcolm MagovernAbstract:PURPOSE: To characterize the genetic basis and phenotype of inherited Fuchs corneal dystrophy (FCD). METHODS: DNA from blood was used for genome-wide linkage scans with tandem repeat polymorphisms. Mutation detection involved sequencing PCR-amplified exons. Families with FCD were clinically evaluated and graded on the Krachmer severity scale. Confocal specular microscopy visualized the morphology of endothelial guttae, small protrusions of Descemet's membrane that are characteristic of FCD. RESULTS: Linkage was obtained to 1p34.3-p32 for the autosomal dominant kindred originally reported by Magovern in 1979. All 21 cases with FCD and one with posterior polymorphous dystrophy were heterozygous for L450W, a novel point mutation in the COL8A2 gene. Of 62 independent cases of familial FCD, none had the previously reported mutations in COL8A2. Corneal guttae in COL8A2 patients were small, rounded, and associated with the endothelial cell center. This contrasts with common FCD, in which guttae were larger, sharply peaked, and initially positioned at edges of endothelial cells. The profile of age and disease severity for the L450W FCD kindred suggested that disease onset occurred in infancy, compared with an average age of onset of 50 years estimated for 201 familial FCD patients in 62 other families. CONCLUSIONS: A novel pathogenic L450W COL8A2 mutation was identified and its highly distinctive pathology characterized. This indicates that COL8A2 mutations give rise to a rare subtype of FCD. This study also provides the first direct evidence that COL8A2-FCD progresses from early to late stages in 25 years, a rate similar to that estimated for late-onset FCD.
John Varga - One of the best experts on this subject based on the ideXlab platform.
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Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators
Oncogene, 2000Co-Authors: Asish K Ghosh, Weihua Yuan, Yasuji Mori, John VargaAbstract:Transforming growth factor-β (TGF-β) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-β- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected fibroblasts, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-β to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-β. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-β stimulation of collagen gene expression in fibroblasts.
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stimulation of type i collagen transcription in human skin fibroblasts by tgf β involvement of smad 3
Journal of Investigative Dermatology, 1999Co-Authors: Shu Jen Chen, Weihua Yuan, Yasuji Mori, Anait S Levenson, Maria Trojanowska, John VargaAbstract:Transforming growth factor-β (TGF-β) stimulates the transcription of the α2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-β. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-β. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans -activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-β response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-β. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-β-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-β-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-β treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-β-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-β signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-β; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-β.
Huan Meng - One of the best experts on this subject based on the ideXlab platform.
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l450w and q455k COL8A2 knock in mouse models of fuchs endothelial corneal dystrophy show distinct phenotypes and evidence for altered autophagy
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Huan Meng, Mario Matthaei, Shukti Chakravarti, Rhonda Grebe, Martha Kimos, Narendrakumar Ramanan, Caroline L Speck, Charles G EberhartAbstract:Purpose. We compared the cellular phenotypes and studied the role of autophagy in the pathogenesis of Fuchs endothelial corneal dystrophy (FECD) using two α2 collagen VIII (COL8A2) knock-in mouse models and human FECD tissues.
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Endothelial cell whole genome expression analysis in a mouse model of early-onset Fuchs' endothelial corneal dystrophy.
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Mario Matthaei, Huan Meng, Charles G Eberhart, Jianfei Hu, Eva Maria Lackner, Jiang QianAbstract:PURPOSE: To investigate the endothelial gene expression profile in a COL8A2 Q455K mutant knock-in mouse model of early-onset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD. METHODS: Diseased or normal endothelial phenotypes were verified in 12-month-old homozygous COL8A2(Q455K/Q455K) mutant and wild-type mice by clinical confocal microscopy. An endothelial whole genome expression profile was generated by microarray-based analysis. Result validation was performed by real-time PCR. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples. RESULTS: Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Real-time PCR assessment confirmed increased Cox2 (P = 0.001) and Jun mRNA (P = 0.03) levels in COL8A2(Q455K/Q455K) mutant compared to wild-type mice. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA (P < 0.0001) and JUN mRNA (P = 0.002) and tissue microarray analysis showed increased endothelial COX2 (P = 0.02) and JUN protein (P = 0.04). CONCLUSIONS: The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD.
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Endothelial Cdkn1a (p21) Overexpression and Accelerated Senescence in a Mouse Model of Fuchs Endothelial Corneal Dystrophy
Investigative Ophthalmology & Visual Science, 2012Co-Authors: Mario Matthaei, Huan Meng, Alan K. Meeker, Charles G EberhartAbstract:PURPOSE: Stress of the endoplasmic reticulum and oxidative stress play critical roles in the pathogenesis of Fuchs Endothelial Corneal Dystrophy (FECD). In the normal aging cornea, cellular stress has been associated with a loss in proliferative capacity (premature senescence) of corneal endothelial cells (CECs). The present study used a transgenic COL8A2(Q455K/Q455K) knock-in mouse model of early-onset FECD to identify the endothelial expression profile of specific cellular stress response-related targets, which may be relevant to late-onset FECD. METHODS: The differential endothelial mRNA levels of cellular stress response-related genes were determined in 12-month-old homozygous COL8A2(Q455K/Q455K) mutant and wild-type mice using customized PCR arrays. Result validation and analysis of additional senescence-related transcripts was performed by real-time PCR. Expression of p53 and p21 was assessed by immunofluorescence. Senescence-associated β-galactosidase (SA-β-Gal) activity was investigated by histochemical labeling. Human FECD samples and normal controls were examined for p21 expression by immunohistochemistry. RESULTS: PCR-array analysis showed greater than 2-fold and/or significantly altered endothelial regulation of 19 cellular stress response-related transcripts in COL8A2(Q455K/Q455K) mutant mice; real-time PCR documented statistically significant upregulation of senescence-associated targets Cdkn1a (p21), Serpine1 (PAI-1), Tagln (Sm22), Fn1 and Clu (ApoJ). Immunofluorescence revealed increased expression of nuclear p53 and p21 in mutant animals. SA-β-Gal staining detected increased proportions of senescent CECs in mutant mice. Human FECD endothelium exhibited increased levels of nuclear p21 protein. CONCLUSIONS: Our results identify endothelial Cdkn1a (p21) upregulation in a mouse model of early-onset FECD, confirm overexpression of p21 in late-onset human FECD endothelium, and suggest a role for premature senescence in FECD.
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an alpha 2 collagen viii transgenic knock in mouse model of fuchs endothelial corneal dystrophy shows early endothelial cell unfolded protein response and apoptosis
Human Molecular Genetics, 2012Co-Authors: Huan Meng, Naren Ramanan, Mario Matthaei, Shukti Chakravarti, Richard Bonshek, Graeme C M Black, Rhonda Grebe, Martha KimosAbstract:Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (COL8A2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the COL8A2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.
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Cryopreservation and long-term culture of transformed murine corneal endothelial cells
Graefe's Archive for Clinical and Experimental Ophthalmology, 2012Co-Authors: Christoph Engler, Clare Kelliher, Sungdong Chang, Huan MengAbstract:Purpose To characterize the morphology and gene expression of transformed murine corneal endothelial cells. Methods Primary immortomouse corneal endothelial cells were continuously cultured before and after cryopreservation. Morphologic assessment, real time-reverse transcriptase polymerase chain reaction ((RT)-PCR) and immunofluorescence studies were performed using newly cultured cells, cells that had been continuously in culture for 1 year, and cryopreserved cells, to assess for structural and functional integrity. The expression of corneal endothelial markers zonula occludens-1 (ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and myofibroblast markers Desmin, alpha smooth muscle actin (αSMA), and Vimentin was assessed and compared by both RT-PCR and immunofluorescence. Results Cells in culture formed a monolayer, and exhibited a polygonal shape after reaching confluence. Cells retained this morphology during the full observation time of 12 months and when reused after cryopreservation. Immunofluorescence experiments exhibited positive staining for NaK-ATPase and COL8A2 with low variability between the three groups. In RT-PCR experiments, ZO1, COL8A2 and Desmin were increased in fresh and thawed cells, αSMA was decreased, and NaK-ATPase and Vimentin remained unchanged, compared to 12-month-old cells. Comparing fresh and thawed cells, COL8A2 was increased in thawed cells, while Desmin was increased in fresh cells. Conclusions Using the immortomouse strain, murine corneal endothelial cells can be propagated over a long time period and be used after cryopreservation. Cells retain the expression of NaK-ATPase, but show some decline in ZO1 and COL8A2 over time and after cryopreservation. The expression of myofibroblast markers suggests an endothelial-to-mesenchymal transformation process in culture.