Activation Of Collagen Genes Research Articles

Thrombospondin 2 is a key determinant of fibrogenesis in non-alcoholic fatty liver disease.

Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. This study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 upregulation. Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analysed using a combination of computational biology, genetic, immunological, and pharmacological approaches. Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction (qPCR) examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r = .349, p < .001). In support of this, scRNA-seq data and insitu hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGFβ) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGFβ increased THBS2/TSP2 and collagen expression independently of the TGFβ-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD.

Vitamin C Loaded Albumin Nanoparticles Treatment and Its Effect on Collagen I and III and mir-133 Genes Expression

Wound healing is a complex biological process in which many molecules, including microRNA molecules, play an important role in its regulation. It is well established that reducing mir-155 expression can accelerate wound healing. In this study, the effect of using nanoparticle loaded by vitamin C in wound healing and mir-133b, collagen I and III expression changes was investigated. In this study, first nanoparticles of albumin protein were produced and then loaded with vitamin C. to evaluate the toxicity and determine the appropriate dose, these nanoparticles were affected on fibroblast cell and cell behavior was investigated. Also in the molecular study, changes in the expression of collagen I and III genes were studied. The results show that nanoparticles containing vitamin C had a positive effect on the expression of collagen I and III compared to the control group. Also we observed a decrease in the expression of mir-133 compared to the control group. Therefore, the results of this study clearly indicate the role of nanoparticles in improving the expression of collagen I and III genes, also the expression of mir-133 gene is reduced, which leads to the activation of collagen genes I and III, which these findings favor the high expression of collagen by fibroblasts, which ultimately have a positive effect on wound healing.

Role of PGE-2 and Other Inflammatory Mediators in Skin Aging and Their Inhibition by Topical Natural Anti-Inflammatories

Human skin aging is due to two types of aging processes, “intrinsic” (chronological) aging and “extrinsic” (external factor mediated) aging. While inflammatory events, triggered mainly by sun exposure, but also by pollutants, smoking and stress, are the principle cause of rapid extrinsic aging, inflammation also plays a key role in intrinsic aging. Inflammatory events in the skin lead to a reduction in collagen gene activity but an increase in activity of the genes for matrix metalloproteinases. Inflammation also alters proliferation rates of cells in all skin layers, causes thinning of the epidermis, a flattening of the dermo-epidermal junction, an increase in irregular pigment production, and, finally, an increased incidence of skin cancer. While a large number of inflammatory mediators, including IL-1, TNF-alpha and PGE-2, are responsible for many of these damaging effects, this review will focus primarily on the role of PGE-2 in aging. Levels of this hormone-like mediator increase quickly when skin is exposed to ultraviolet radiation (UVR), causing changes in genes needed for normal skin structure and function. Further, PGE-2 levels in the skin gradually increase with age, regardless of whether or not the skin is protected from UVR, and this smoldering inflammation causes continuous damage to the dermal matrix. Finally, and perhaps most importantly, PGE-2 is strongly linked to skin cancer. This review will focus on: (1) the role of inflammation, and particularly the role of PGE-2, in accelerating skin aging, and (2) current research on natural compounds that inhibit PGE-2 production and how these can be developed into topical products to retard or even reverse the aging process, and to prevent skin cancer.

Secreted Klotho Attenuates Inflammation-Associated Aortic Valve Fibrosis in Senescence-Accelerated Mice P1.

Senescence-accelerated mice P1 (SAMP1) is an aging model characterized by shortened lifespan and early signs of senescence. Klotho is an aging-suppressor gene. The purpose of this study is to investigate whether in vivo expression of secreted klotho (Skl) gene attenuates aortic valve fibrosis in SAMP1 mice. SAMP1 mice and age-matched (AKR/J) control mice were used. SAMP1 mice developed obvious fibrosis in aortic valves, namely fibrotic aortic valve disease. Serum level of Skl was decreased drastically in SAMP1 mice. Expression of MCP-1 (monocyte chemoattractant protein 1), ICAM-1 (intercellular adhesion molecule 1), F4/80, and CD68 was increased in aortic valves of SAMP1 mice, indicating inflammation. An increase in expression of α-smooth muscle actin (myofibroblast marker), transforming growth factorβ-1, and scleraxis (a transcription factor of collagen synthesis) was also found in aortic valves of SAMP1 mice, suggesting that accelerated aging is associated with myofibroblast transition and collagen gene activation. We constructed adeno-associated virus 2 carrying mouse Skl cDNA for in vivo expression of Skl. Skl gene delivery effectively increased serum Skl of SAMP1 mice to the control level. Skl gene delivery inhibited inflammation and myofibroblastic transition in aortic valves and attenuated fibrotic aortic valve disease in SAMP1 mice. It is concluded that senescence-related fibrotic aortic valve disease in SAMP1 mice is associated with a decrease in serum klotho leading to inflammation, including macrophage infiltration and transforming growth factorβ-1/scleraxis-driven myofibroblast differentiation in aortic valves. Restoration of serum Skl levels by adeno-associated virus 2 carrying mouse Skl cDNA effectively suppresses inflammation and myofibroblastic transition and attenuates aortic valve fibrosis. Skl may be a potential therapeutic target for fibrotic aortic valve disease.

Abstract 12745: Interleukin-4 Promotes Procollagen Type-I Alpha 1 Gene Expression via Activator Protein 1 Activation in Cardiac Fibroblasts

Interleukin-4 (IL-4) activates the signal transducer and activator of transcription 6 signaling pathway, resulting in the upregulation of procollagen genes and promotion of collagen production in cardiac fibroblasts (CFs). In addition, transcription factors may participate in the transcriptional activation of collagen genes. We hypothesize that IL-4 activates activator protein 1 (AP-1) via c-Jun phosphorylation, promoting transcriptional activation of the procollagen type-I alpha 1 (Col1α1) gene and collagen type-I expression in CFs. Primary mouse CFs were used. Western blot analyses revealed that IL-4 (10 ng/ml) increased phosphorylation of c-Jun, a subunit of AP-1. Additionally, our experiments utilizing electrophoretic mobility shift assay (EMSA) revealed a significant increase in DNA binding activity for AP-1 in IL-4-treated CFs compared to control cells (1.00±0.07 to 2.14±0.38 fold change versus control, p &lt;0.05). Here we aimed to determine whether IL-4-activated AP-1 participates in transcriptional activation of the Col1α1 gene leading to increased collagen type-I expression in CFs. To that end, we blocked AP-1 with shRNA lentiviral particles specific for c-Jun in CFs, detected Col1α1 mRNA by qRT-PCR, and measured collagen type-I by Western blot. We found that IL-4 significantly induced Col1α1 mRNA expression after incubation with control shRNA-transduced cells for 6 hours (1.01±0.01 to 1.52±0.07 fold change versus control, p &lt;0.005), and it increased collagen type-I expression after 24 hours of incubation (1.00±0.01 to 1.95±0.12 fold change versus control, p &lt;0.005). In contrast, IL-4 did not increase mRNA levels of Col1α1 (1.09±0.02 to 0.98±0.03, p &gt;0.05) and the collagen type-I (0.95±0.05 to 1.01±0.10, p &gt;0.05) in c-Jun shRNA-transfected cells. Our data demonstrate that IL-4-activated AP-1, an ubiquitous transcription factor containing phosphorylated c-Jun, enhanced Col1α1 gene expression and collagen type-I expression in CFs. Data generated from this study provides a novel molecular basis for potential treatment of cardiac fibrosis by targeting IL-4-induced signaling in CFs.

Angiotensin II induces type I collagen gene expression in human dermal fibroblasts through an AP-1/TGF-β1-dependent pathway

Angiotensin II is critically involved in skin wound healing, but the underlying mechanism remains unclear. This study investigated the effect of angiotensin II on type I collagen gene activation in human dermal fibroblasts and the possible mechanism involved. Angiotensin II stimulated the mRNA and protein expression of type I collagen and TGF-β1. Effects were abolished by the angiotensin AT1 receptor antagonist ZD7155 but not by the AT2 blocker PD123319. Blockade of TGF-β1 markedly inhibited angiotensin II-induced type I collagen gene expression. Activator protein-1 (AP-1) decoy ODNs transfection suppressed angiotensin II-induced TGF-β1 expression, and also, diminished type I collagen expression. These data indicated that angiotensin II induces collagen gene activation in human dermal fibroblasts through an AT1-mediated AP-1/TGF-β1 signaling pathway.

CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of the Fibrotic Pathway in an in Vitro Model of Renal Disease

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Cross-talk between MCP-3 and TGFβ promotes fibroblast collagen biosynthesis

Recent studies have demonstrated upregulation of monocyte chemoattractant protein-3 (MCP-3/CCL7) in fibrosis and have suggested that in addition to a major role in regulating leucocyte recruitment this chemokine may also promote extracellular matrix (ECM) overproduction by fibroblasts. In the present study we explore interplay between MCP-3 and transforming growth factor beta (TGFβ), a potent profibrotic cytokine. We demonstrate that MCP-3 promotes activation of TGFβ signalling pathways leading to increased type I collagen secretion. In addition we show that MCP-3 gene expression is stimulated by recombinant TGFβ1, raising the possibility for synergy between these two mediators in the fibrotic microenvironment. Comparison of downstream signalling pathways that regulate collagen gene activation by both cytokines confirms the central role of MAPK pathway activation in mediating the effects of both factors. An additive effect of these two agonists was demonstrated by comparative microarray analysis for key TGFβ regulated transcripts including PAI-1, OSF2 and IGFBP6. Together, our results confirm cross-talk between MCP-3 and TGFβ that may be critical in the development of fibrosis.

Expression of types I, III and IV collagen genes in fibrotic skin and nerve lesions of toxic oil syndrome patients.

We have studied the skin and nerve fibrosis in toxic oil syndrome by in situ hybridization using specific cDNA probes for types I, III, and IV collagens. Fibroblasts with high levels of type I and III collagen mRNA were observed in biopsies from fibrotic skin areas. Similarly, type IV collagen mRNA was abundant in cells within the fibrotic process of the nerves. These results suggest that the excessive accumulation of collagen in toxic oil syndrome results from transcriptional activation of collagen genes in a subpopulation of fibroblasts.

IL-4 and IL-13 induce type I collagen gene activity via the JNK signaling pathway in human fibroblasts. (94.28)

Abstract Little is understood of the molecular mechanisms by which fibrotic diseases such as in Scleroderma, are manifested by interleukins (IL) 4 and 13. Studies support a role for the JNK signaling pathway via transcription factors c-Jun and ATF2, being involved in activating the collagen promoter and translation of the collagen gene. To examine the role of JNK signaling in IL-4/IL-13-induced collagen gene activation, we transfected human fibroblasts with a luciferase reporter plasmid under control of the α2(I) collagen promoter to assess JNK signaling on type I collagen promoter activity. We reveal a critical role for JNK 1 in IL-4/IL-13-induced collagen promoter activity subsequent to ectopic expression of c-Jun and ATF2. In contrast, introduction of a dominant negative (dn) mutant reduces basal and cytokine-induced promoter activity. We demonstrate that the JNK signaling pathway leading to the activation of the collagen gene in fibroblasts is effective not only for TGF-β signaling but also for IL-4/IL-13 signaling.

Acetaldehyde Inhibits PPARγ via H 2O 2-Mediated c-Abl Activation in Human Hepatic Stellate Cells

Accumulating evidence indicates that acetaldehyde (AcCHO) is one of the main mediators of fibrogenesis in alcoholic liver disease. AcCHO stimulates synthesis of fibrillar collagens in hepatic stellate cells, but the molecular events directly involved in the activation of collagen genes are debatable. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that is expressed in stellate cells, and its activation by specific ligands inhibits collagen synthesis. In this study, we evaluated the effects of AcCHO on PPARgamma transcriptional activity and its correlation with the AcCHO-induced collagen synthesis in hepatic stellate cells. AcCHO treatment inhibited ligand-dependent and -independent PPARgamma transcriptional activity, and this effect was correlated with an increased phosphorylation of a mitogen-activated protein kinase site at serine 84 of the human PPARgamma. Transfection of the PPARgammaSer84Ala mutant completely prevented the effect of AcCHO on PPARgamma activity and in parallel abrogated the induction of collagen gene expression by AcCHO. The effect of AcCHO on PPARgamma activity and phosphorylation was blocked by extracellular signal-regulated kinase (ERK) 1/2 and protein kinase C (PKC)delta inhibitors as well as by catalase, suggesting that hydrogen peroxide is involved in the molecular cascade responsible for PPARgamma phosphorylation via activation of the PKCdelta/ERK pathway. Furthermore, inhibition of c-Abl completely abrogated the effect of AcCHO on either PPARgamma function or collagen synthesis; in addition, expression of the PPARgammaSer84Ala mutant prevented the profibrogenic signals mediated by c-Abl activation. Our results showed that the induction of collagen expression by AcCHO in stellate cells is dependent on PPARgamma phosphorylation induced by a hydrogen peroxide-mediated activation of the profibrogenic c-Abl signaling pathway.

Tumor Necrosis Factor (TNF) α Increases Collagen Accumulation and Proliferation in Intestinal Myofibroblasts via TNF Receptor 2

Intestinal fibrosis is an incurable complication of Crohn's disease involving increased numbers of collagen-producing myofibroblasts. Tumor necrosis factor (TNF) alpha has defined proinflammatory roles in Crohn's disease but its role in fibrosis is unclear. We tested the hypothesis that TNFalpha increases collagen accumulation and proliferation in intestinal myofibroblasts and has additive effects in combination with insulin-like growth factor (IGF) I. The mechanisms, TNF receptor isoform, and downstream signaling pathways were examined. Intestinal myofibroblasts from wild-type (WT) mice or mice homozygous for disruption of genes encoding TNFR1 (TNFR1-/-), TNFR2 (TNFR2-/-), or both (TNFR1/2-/-), were treated with TNFalpha, IGF-I, or both. In WT cells, TNFalpha and IGF-I stimulated type I collagen accumulation and DNA synthesis in an additive manner. IGF-I, but not TNFalpha, stimulated type I collagen gene activation. TNFalpha, but not IGF-I, induced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and reduced matrix metalloproteinases-2 activity and collagen degradation. TNFalpha also activated ERK1/2. These responses to TNFalpha were absent in TNFR2-/- and TNFR1/2-/- myofibroblasts, whereas TNFR1-/- cells showed similar responses to WT. Inhibition of ERK1/2 diminished TNFalpha induced DNA synthesis in WT and TNFR1-/- cells. Differences in TNFalpha-induced STAT3/DNA binding activity and not NFkappaB and AP-1 transcriptional activation correlated with impaired collagen accumulation/TIMP-1 induction in TNFR2(-/-) cells. Constitutively active STAT3 rescued TIMP-1 expression in TNFR2-/- cells. We conclude that TNFalpha and IGF-I may additively contribute to fibrosis during intestinal inflammation. TNFR2 is a primary mediator of fibrogenic actions of TNFalpha acting through ERK1/2 to stimulate proliferation and through STAT3 to stimulate TIMP-1 and inhibit collagen degradation.

Leptin enhances α1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.

Basement membrane protein and matrix metalloproteinase deregulation in engineered human oral mucosa following infection with Candida albicans

A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10(5) C. albicans/cm2 for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.

Aldehydes potentiate α 2(I) collagen gene activity by JNK in hepatic stellate cells

Hepatic stellate cells (HSCs) are responsible for type I collagen deposition in liver fibrosis that leads to cirrhosis. The purpose of this study was to examine potential molecular signals that lead to increased α 2(I) collagen gene expression by acetaldehyde, the primary metabolite of alcohol and malondialdehyde (MDA), a lipid peroxidation product known to be associated with chronic liver injury. MDA and the combination of MDA and acetaldehyde were employed to determine the effect on α 2(I) collagen gene expression as assessed by transient transfection analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Immunoblot and subsequent immunoprecipitation analysis examined stress-activated protein kinase (SAPK) activity. Cotransfection with a dominant negative mutant for c-jun nuclear kinase (dnJNK1) was also employed with the α 2(I) collagen promoter. MDA increased α 2(I) collagen gene expression nearly 2.5- to 3-fold, however there was no synergistic effect of the combination of acetaldehyde and MDA on α 2(I) collagen gene activation and expression. Acetaldehyde, MDA, or both significantly increased JNK activity when compared to untreated stellate cells. The dnJNK1 expression vector abrogated α 2(I) collagen transgene activity. In conclusion, JNK activation appears to be critical in the signaling cascade of oxidative metabolites of chronic alcohol-related liver injury and collagen gene activation.

Alterations in the regulation of expression of the alpha 1(I) collagen gene (COL1A1) in systemic sclerosis (scleroderma).

At present, the mechanisms that regulate the expression of collagen genes in normal and pathologic fibroblasts are not known. Thus, the detailed study of transcriptional regulation of COL1A1 in SSc cells will increase our current understanding of the pathophysiology of fibrotic diseases. These studies will yield valuable information regarding the important biological process of regulation of collagen gene expression under normal and pathologic conditions, a process that has remained elusive despite intense recent investigations. It is now evident that persistent overproduction of collagen is responsible for the progressive nature of tissue fibrosis in SSc. Up-regulation of collagen gene expression in SSc fibroblasts appears to be a critical event in this process. The coordinate transcriptional activation of numerous collagen genes suggests a fundamental alteration in the regulatory control of gene expression in SSc fibroblasts. Trans-acting nuclear factors which bind to cis-acting elements in enhancer (intronic) and promoter regions of the genes modulate the basal and inducible transcriptional activity of the collagen genes. The identification of the nuclear transcription factors that regulate normal collagen gene expression may provide promising approaches to the therapy of this incurable disease.

Transforming Growth Factor-β and Receptor Tyrosine Kinase–Activating Growth Factors Negatively Regulate Collagen Genes in Smooth Muscle of Hypertensive Rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.

Doppler echocardiographic assessment and cardiac gene expression analysis of the left ventricle in myocardial infarcted rats.

The purpose of this study was to examine cardiac geometry and function by Doppler echocardiography and to analyze mRNA expression of cardiac phenotype and extracellular matrix in myocardial infarcted rats. Doppler echocardiograms and hemodynamics were measured 2 weeks after myocardial infarction (MI). mRNA levels in the non-infarcted left ventricle (LV) and infarct site were measured by Northern blot analysis. LV internal diastolic dimension was greater in infarcted (MI) than in sham-operated rats (control) (MI 7.2+/-0.3 mm vs control 4.6+0.3 mm, p<0.01). The fractional shortening decreased in MI rats (MI 32+4% vs control 61+/-3%, p<0.01). Peak early filling velocity increased in MI rats (MI 91+/-5 cm/sec vs control 72+/-4 cm/sec, p<0.05), and deceleration rate of the early filling wave was more rapid in rats with MI (MI 25.1+/-2.8 m/sec2 vs control 12.4+/-1.7 m/sec2, p < 0.01). Late filling velocity decreased (MI 16+/-3 cm/sec vs control 35+/-6 cm/sec, p <0.05), resulting in a marked increase in the ratio of early filling to late filling (MI 7.1+/-1.2 vs control 2.5+/-0.4, p<0.01). mRNA levels for beta-myosin heavy chain (beta-MHC), a-skeletal actin, atrial natriuretic polypeptide (ANP), collagen types I and III, and matrix metalloproteinase 2 (MMP-2) in the non-infarcted LV increased significantly by 1.8-, 2.4-, 4.7-, 2.6-, 2.1- (all p<0.01) and 1.4-fold (p<0.05), respectively, compared with sham-operated myocardium. In the infarct site, mRNA levels for transforming growth factor (TGF)-beta1, collagen types I and III, and MMP-2 significantly increased by 3.2-, 11.0-, 9.7-, and 6.3-fold (all p<0.01), respectively, compared with sham-operated myocardium. Myocardial infarcted rat was characterized by cavity dilation and marked abnormalities of systolic and diastolic function, accompanied by a shift of myocytes to fetal phenotype and activation of collagen genes in the non-infarcted myocardium.

Inhibition by angiotensin II type 1 receptor antagonist of cardiac phenotypic modulation after myocardial infarction

The purpose of this study was to examine the cardiac phenotype and remodeling after myocardial infarction and the effect of the angiotensin II type 1 (AT1) receptor antagonist (TCV-116) on the gene expression. Myocardial infarction in rats was produced by ligation of the coronary artery. TCV-116 (10 mg/kg/day) was administered orally to rats from 1 day after myocardial infarction. At 1, 2 and 3 weeks after myocardial infarction, blood pressure and heart rate were measured, and the heart was removed. The left ventricle was measured for infarct size and weight, and then the total RNA from the non-ischemic left ventricle was extracted. mRNAs in the non-ischemic left ventricle were measured by Northern blot analysis. The weight of the non-ischemic left ventricle was significantly increased 3 weeks after infarction. This was completely prevented by TCV 116 treatment. mRNA levels for β-myosin heavy chain (β-MHC), atrial natriuretic polypeptide (ANP), collagen types I and III and transforming growth factor-β1 (TGF-β1) in the non-ischemic left ventricle were increased by a factor of 3.0, 6.7, 7.9, 4.0 and 1.4 ( P<0.01), respectively, 1 week after infarction. There was no increase in a-skeletal actin mRNA at 1 and 2 weeks, but it was increased by a factor of 2.9 ( P<0.05) at 3 weeks. On the other hand, there was no change in α-MHC mRNA during the 3 weeks. TCV-116 significantly suppressed the increased gene expression of β-MHC and α-skeletal actin in the non-ischemic myocardium at all time points, and also suppressed the expression of ANP at 2 and 3 weeks. However, TCV-116 failed to inhibit the expression of collagen I and III mRNAS at 1 and 3 weeks. These results show that myocardial infarction causes a rapid shift of myocytes to fetal phenotype and a rapid activation of collagen genes in the non-ischemic myocardium. AT 1 receptor may be responsible for the phenotypic modulation of myocotes following myocardial infarction.

Parenchymal Fe and collagen gene expression: an iron-clad association?

Background/Aims: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity. Methods: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situhybridization analyses on liver samples were performed. Results: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and II. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis. Conclusions: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during longterm iron overload in rodents.