Fibroblast Extracellular Matrix Protein Research Articles

Pentaerythritol Tetranitrate Targeting Myocardial Reactive Oxygen Species Production Improves Left Ventricular Remodeling and Function in Rats With Ischemic Heart Failure.

Reduced nitric oxide bioavailability contributes to progression of cardiac dysfunction and remodeling in ischemic heart failure. Clinical use of organic nitrates as nitric oxide donors is limited by development of nitrate tolerance and reactive oxygen species formation. We investigated the effects of long-term therapy with pentaerythritol tetranitrate (PETN), an organic nitrate devoid of tolerance, in rats with congestive heart failure after extensive myocardial infarction. Seven days after coronary artery ligation, rats were randomly allocated to treatment with PETN (80 mg/kg BID) or placebo for 9 weeks. Long-term PETN therapy prevented the progressive left ventricular dilatation and improved left ventricular contractile function and relaxation in rats with congestive heart failure. Mitochondrial superoxide anion production was markedly increased in the failing left ventricular myocardium and nearly normalized by PETN treatment. Gene set enrichment analysis revealed that PETN beneficially modulated the dysregulation of mitochondrial genes involved in energy metabolism, paralleled by prevention of uncoupling protein-3, thioredoxin-2, and superoxide dismutase-2 downregulation. Moreover, PETN provided a remarkable protective effect against reactive fibrosis in chronically failing hearts. Mechanistically, induction of heme oxygenase-1 by PETN prevented mitochondrial superoxide generation, NOX4 upregulation, and ensuing formation of extracellular matrix proteins in fibroblasts from failing hearts. In summary, PETN targeting reactive oxygen species generation prevented the changes of mitochondrial antioxidant enzymes and progressive fibrotic remodeling, leading to amelioration of cardiac functional performance. Therefore, PETN might be a promising therapeutic option in the treatment of ischemic heart diseases involving oxidative stress and impairment in nitric oxide bioactivity.

The -2518 Promotor Polymorphism in the MCP-1 Gene Is Associated with Systemic Sclerosis

Factors influencing the initiation or progression of sclerosis in patients with systemic sclerosis (SSc) are poorly understood. Monocyte chemotactic protein-1 (MCP-1) is a potent chemokine, which is upregulated in fibroblasts during development of sclerosis. In this study, we investigated the frequency of the functional -2518G MCP-1 promoter polymorphism in 18 patients with SSc and 139 healthy controls. In the lesional skin of the same SSc patients, expression of MCP-1 protein was examined by immunohistochemistry. To investigate a genotype/phenotype correlation, basal as well as tumor necrosis factor (TNF)-induced MCP-1 expression was analyzed in fibroblasts isolated from the skin of SSc patients with different MCP-1 genotypes by quantitative RT-PCR and ELISA. Genotyping for the -2518 (A/G) MCP-1 promotor polymorphism showed that GG homozygotes were significantly more frequent in patients with SSc than in controls (28%vs 6%). Results of immunohistochemistry revealed that MCP-1 was expressed in keratinocytes, infiltrating inflammatory cells, fibroblasts, and endothelial cells in scleroderma skin, whereas normal control skin showed no MCP-1 expression. MCP-1 expression in fibroblasts from GG-homozygote individuals tended to be stronger as compared to AG or AA genotypes. Furthermore, basal as well as TNF-induced MCP-1 expression of fibroblasts isolated from a GG-homozygote SSc patient was significantly higher than MCP-1 expression of fibroblasts isolated from heterozygote or AA-homozygote donors. The A -2518G polymorphism of the MCP-1 gene appears to affect MCP-1 expression of skin fibroblasts of patients with SSc. In accordance, the G/G genotype may predispose patients to SSc.

Stimulation of alpha 1 (I) procollagen gene expression in NIH-3T3 cells by the human T cell leukemia virus type 1 (HTLV-1) Tax gene.

The mechanisms that regulate the expression of genes encoding extracellular matrix proteins in fibroblasts and other mesenchymal cells have remained elusive. Studies from several laboratories have indicated that Tax, a trans-regulatory protein from the human T cell leukemia virus type I not only augments viral gene expression but also triggers the expression of various cellular genes. Here, we examined the hypothesis that the expression of collagen genes may also be modulated by Tax. NIH-3T3 cells were simultaneously transfected with a Tax expressor plasmid and a chimeric construct containing regulatory sequences (-804 to +42 bp) of the alpha 1(I) procollagen gene (COL1A1) promoter. The results indicated that the promoter activity of the -804 to bp COL1A1 fragment increased up to 12-fold in cells expressing Tax. Deletion analysis revealed that the region of COL1A1 encompassing nucleotides -174 to -84 contained the Tax-responsive elements. A gene segment encompassing nucleotides -187 to -67, which contained this region, proved sufficient to confer Tax inducibility (2.5-fold) to a herpes simplex virus thymidine kinase promoter. Stably transfected NIH-3T3 cell clones that constitutively produce Tax displayed elevated levels of alpha 1(I) procollagen and fibronectin transcripts and increased production and accelerated processing of type I procollagen. These findings suggest that retroviral proteins may be involved in the pathogenesis of idiopathic diseases accompanied by collagen overproduction.

Expression and Accumulation of Lysyl Oxidase, Elastin, and Type I Procollagen in Human Menkes and Mottled Mouse Fibroblasts

Menkes syndrome in humans is an X-linked disorder characterized in part by abnormal copper transport, cellular copper sequestration, and defective crosslinking of collagen and elastin. A decrease in the functional activity of lysyl oxidase, a cuproenzyme, is thought in part to be responsible for the decreased crosslinking of collagen and elastin. It has also been suggested that low levels of lysyl oxidase activity may occur secondarily to disturbances in intracellular copper translocation and consequently impaired incorporation of copper into lysyl oxidase. Herein, we examine the expression and accumulation of selected extracellular matrix proteins in fibroblasts from a Menkes patient, as well as fibroblasts from the tortoiseshell (Mo To/y) mouse. The Mo To mutation is an allele of the mottled (Mo) locus, which is considered to be a murine analog of the human Menkes locus. In both Menkes and tortoiseshell fibroblasts, levels of lysyl oxidase mRNA transcripts were less than 15% of levels for corresponding controls. The level of elastin mRNA transcripts was also markedly lower in both cell lines in comparison to controls. In contrast, the levels of procollagen Type I mRNA were similar or enhanced in Menkes and Mo To/y fibroblasts compared to their respective controls. Consequently, we conclude that the connective tissue defects associated with Menkes syndrome and those occurring in mottled mouse mutants involve more than abnormal copper utilization in the formation of lysyl oxidase holoenzyme. Based on the present studies in cell culture, the production of essential enzymes and matrix proteins, such as lysyl oxidase and elastin, appear to be altered at the level of transcription or mRNA turnover.

Transcriptional regulation of osteocalcin production by transforming growth factor-beta in rat osteoblast-like cells.

Osteocalcin (OC) is one of the abundant non-collagenous bone matrix proteins produced exclusively by osteoblasts, and its serum level is used as an indicator of bone metabolism in patients. Transforming growth factor-beta (TGF beta) is abundant in bones and platelets, promotes wound healing in vivo, and is a potent stimulator of the production of extracellular matrix proteins in fibroblasts and osteoblasts. The effects of TGF beta on OC gene expression were examined in rat osteoblast-like cells, ROS17/2.8. TGF beta 1 decreased OC levels in the culture media 2- to 3-fold. TGF beta 1 also decreased the level of osteocalcin mRNA about 3-fold in a dose-dependent manner. TGF beta 2 and TGF beta 1,2 a heterodimeric form, showed similar effects on OC mRNA levels as TGF beta 1. The suppression of the OC message level was detectable at 24 h and lasted for up to 72 h. This effect on OC mRNA was blocked by cycloheximide. The stability of OC mRNA was not changed by TGF beta 1. On the other hand, the rate of OC gene transcription was reduced 4- to 5-fold, as estimated by in vitro nuclear transcription (run-on) assay. TGF beta 1 blocked the increase in the OC mRNA level induced by PTH or 1,25-dihydroxyvitamin D3. These results indicate that TGF beta inhibits osteocalcin gene expression at least in part through transcriptional control.