Fibrillar Collagen Gene Expression Research Articles

The transcriptional regulation of periostin by scleraxis in cardiac myofibroblasts

Extracellular matrix (ECM) remodeling resulting in cardiac fibrosis is a crucial factor in determining the severity of myocardial dysfunction during heart failure. In response to stress or damage, cardiac fibroblasts undergo phenotype conversion to myofibroblasts, the primary cell type responsible for ECM synthesis and remodeling in the heart. Periostin, a marker of the myofibroblast phenotype, is a matricellular secreted protein that contributes to cardiac remodeling. Periostin plays important roles in mediating cell–matrix signal transduction and in promoting pro‐fibrotic TGF‐β signaling via coupling with matrix associated lysyl oxidase; it is highly expressed in myofibroblasts, but is not expressed in fibroblasts. We have shown that the transcription factor scleraxis induces the phenotype conversion of cardiac fibroblasts to myofibroblasts by directly transactivating the expression of proteins highly up‐regulated in myofibroblasts, including collagen 1α2, fibronectin and α‐smooth muscle actin. Here we report that the treatment of primary adult rat cardiac myofibroblasts with angiotensin II significantly increases periostin gene expression in parallel with increased scleraxis expression. Over‐expression of scleraxis in myofibroblasts increased periostin expression nearly 19‐fold at the mRNA level. TGF‐b treatment induced periostin expression in cardiac myofibroblasts isolated from wild type mice, but failed to show a similar effect in cells isolated from scleraxis null mice. Using in silico analysis, we identified putative E‐box sites within the periostin gene promoter to which scleraxis may bind to potentially regulate promoter transactivation. Our data suggests that scleraxis is necessary and sufficient for TGF‐β mediated periostin expression. Our findings are consistent with a potential role for scleraxis in cardiac fibrosis, where periostin and fibrillar collagen gene expression are elevated in parallel with scleraxis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes

The mechanism involved in the onset of aortic valve (AoV) disease remains unclear despite its poor prognosis and frequency. Recently, we reported that Krox20 (EGR2 in humans) is involved in AoV development and dysfunction. Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases. Transcriptional levels of Col1a2/COL1A2 and Krox20/EGR2 in AoVs from Krox20(+/-) mice and human patients operated on for severe aortic regurgitation were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Human control valves were obtained from three transplanted patients without AoV disease. Twenty-one heterozygous Krox20(+/-) mice were compared with 35 controls at different ages. Three independent measurements of valve thickness were performed on magnified tissue sections using Image J software. In vivo valve structure and function were evaluated using the high-frequency Vevo(®) 2100 echocardiogram. qRT-PCR analysis using AoVs from patients with severe aortic regurgitation showed a decrease in EGR2 expression associated with significant downregulation of COL1A2 expression (P<0.05). Similar results were observed in the AoVs of Krox20(+/-) mice. Anatomical examination revealed that incomplete invalidation of Krox20 caused significant thickening of the aortic leaflet compared with controls (145±22 vs. 75±24μm; P=0.01). Within the mutant group, this thickening worsened significantly over time (Krox20(+/-) mice aged>7 vs.<7months: 136±48 vs. 102±41μm; P<0.001). Moreover, the aortic leaflets of embryonic day 18.5 Krox20(+/-) embryos were significantly more thickened than those from controls, suggesting that this disease begins during embryonic development. Echo-Doppler analysis showed a significant increase in AoV dysfunction in heterozygous versus control mice (53% vs. 17%; P<0.001), suggesting a tight relationship between valve architecture and function. Morphometric analysis revealed that the most severe AoV dysfunction was always associated with the most thickened valves. Classic histological analysis revealed that mutant AoVs had extracellular matrix disorganization, with features of human myxomatous degeneration, including excess of proteoglycan deposition in spongiosa and reduction of collagen fibre in fibrosa, but no calcification. Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.

Molecular characterization and expression analysis of the first Porifera tumor necrosis factor superfamily member and of its putative receptor in the marine sponge Chondrosia reniformis

Here we report the molecular cloning and characterization of the first Tumor Necrosis Factor homologous and of its putative receptor in the marine sponge Chondrosia reniformis: chTNF and chTNFR, respectively. The deduced chTNF amino acid sequence is a type II transmembrane protein containing the typical TNFSF domain. Phylogenetic analysis reveals that chTNF is more related to Chordata TNFs rather than to other invertebrates. chTNF and chTNFR are constitutively expressed both in the ectosome and in the choanosome of the sponge, with higher levels in the ectosome. chTNF and chTNFR mRNAs were monitored in sponge fragmorphs treated with Gram+ or Gram− bacteria. chTNF was significantly upregulated in Gram+-treated fragmorphs as compared to controls, while chTNFR was upregulated by both treatments. Finally, the possible chTNF fibrogenic role in sponge fragmorphs was studied by TNF inhibitor treatment measuring fibrillar and non fibrillar collagen gene expression; results indicate that the cytokine is involved in sponge collagen deposition and homeostasis.

Abstract 192: Scleraxis is a Required Component of the Cardiac Extracellular Matrix Gene Expression Program

The collagenous cardiac extracellular matrix (ECM) reinforces the myocardium and facilitates intercellular communication, but excessive ECM production in fibrosis results in dysfunction and heart failure. The transcription factor scleraxis directly governs expression of type I fibrillar collagen in ECM-rich tissues including tendons and the heart. We have also shown that scleraxis is required for collagen 1α2 gene expression induced by the potent pro-fibrotic TGFβ-Smad signaling pathway. We therefore examined the broader role of scleraxis in myocardial ECM production, including mechanisms regulating the expression of scleraxis itself. Scleraxis knockout in mice resulted in a dramatic ~50% loss of cardiac ECM components including fibrillar collagens, proteoglycans and matrix metalloproteinases, concomitant with a significant decrease in fibroblast number. Scleraxis knockdown in primary cardiac proto-myofibroblasts recapitulated these changes without increasing cell death, suggesting a reduction in fibroblast precursors in knockout mice in vivo. Conversely, over-expression of scleraxis had the opposite effect, up-regulating expression of ECM genes and numerous markers indicative of increased commitment to a myofibroblast cell fate. Scleraxis increased proto-myofibroblast contractility via direct transactivation of the α-smooth muscle actin promoter. Similar to TGFβ, pro-fibrotic angiotensin II and Connective Tissue Growth Factor induced scleraxis expression, suggesting that scleraxis behaves as a common transcriptional effector for multiple fibrotic pathways. Fibrillar collagen gene expression induced by these factors was significantly attenuated by scleraxis knockdown, further implicating scleraxis in fibrotic ECM synthesis. Intriguingly, the histone deacetylase inhibitor trichostatin A, which has been reported to exert anti-fibrotic effects in the heart, significantly reduced scleraxis expression in cardiac myofibroblasts. These data collectively identify scleraxis as a central and requisite transcriptional regulator of fibroblast phenotype and the ECM gene expression program in the heart, and provide rationale for the investigation of anti-scleraxis strategies to attenuate fibrosis in patients.

Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.

Regulation of fibrillar collagen gene expression and protein accumulation in volume-overloaded cardiac hypertrophy.

Interstitial collagen accumulation has been extensively demonstrated to be increased at both mRNA and protein levels in pressure-overloaded cardiac hypertrophy. However, few data are available regarding the effects of volume overload on myocardial collagens. To determine whether the alterations of collagens may occur in volume-overloaded cardiac hypertrophy, we measured collagen types I and III mRNA levels and protein accumulation in left ventricular (LV) myocardium of rats at 3, 7, and 28 days after the creation of an aortocaval (AC) shunt. Eccentric LV hypertrophy was produced in rats with AC shunting. Northern blot analysis on RNA extracted from LV tissue indicated that the steady state mRNA levels for both type I and III collagen were persistently upregulated in AC shunt rats compared with sham-operated operated control rats. In contrast, the biochemical collagen protein concentration and morphometric collagen volume fraction were comparable between sham-operated control and AC shunt rats at any study time point. Furthermore, the immunohistochemical staining of types I and III collagen and Sirius red staining on myocardial tissue sections revealed no significant alterations in the distribution or density of fibrillar collagens in AC shunt rats. Tissue collagenase activity was not different between control and AC shunt rats after 28 days. Cardiac volume overload increases LV collagen mRNA as does pressure overload. However, in contrast to pressure-overloaded hypertrophy, the upregulation of collagen transcriptional activity does not result in subsequent myocardial fibrosis in volume-overloaded hypertrophy due to AC shunting. Therefore, the upregulation of collagen gene expression and protein accumulation might be different in pressure-overloaded and volume overloaded hypertrophy.

968-27 Basic Fibroblast Growth Factor has Potent Selective Effects on Collagen Gene Expression by Human Vascular Smooth Muscle Cells

Basic fibroblast growth factor (bFGF) is a smooth muscle cell (SMC) mitogen known to playa role in the early response of the vessel wall to balloon injury. During this initial stage, remodeling of the extracellular matrix (ECM), including collagen, is necessary for cell proliferation, migration and lesion expansion. We therefore determined the effect of bFGF on fibrillar collagen gene expression by human vascular SMCs. SMCs, cultivated from internal mammary artery, were serum-deprived and incubated with bFGF (50 ng/ml) or vehicle. Collagen synthesis rate was determined by labeling cultures with [ 3 H]proline, separating proteins byelectrophoresis, and visualizing and quantifying bands by fluorography and laser densitometry, respectively. This revealed a significant, net decrease in collagen synthesis induced by bFGF. To determine the mechanism of this response, the effect of bFGF on collagen mRNA was determined by Northern blot analysis using probes specific for α-chains of collagen types I, III, and V, accounting for the major and minor fibrillar collagens found in human atherosclerotic plaque. Cell proliferation was assessed by probing for histone 3.2. Despite a consistent increase in SMC proliferation, bFGF reduced expression of α 1 (I) collagen mRNA by upto 90%. In contrast, bFGF increased expression of α 1 (III) and α 2 (V) collagen mRNA by approximately 20%. Because type III and V collagen fibers are finer than type I, the divergent results may reflect a shift to a less mature collagen lattice induced by bFGF. Total fibrillar collagen synthesis by human SMCs is reduced by bFGF, due to a potent selective inhibition of type I collagen expression. This effect of bFGF on the ECM may be a prerequisite for SMC accumulation and lesion formation following balloon angioplasty.

Organization and characterization of fibrillar collagens in fish scales in situ and in vitro

ABSTRACT The characterization of the fibrillar collagens and the cellular control of their spatial deposition were studied in fish scales using immunofluorescence, electron microscopy, electrophoretic and HPLC analyses, immunoprecipitation and hybridization with cDNA probes. This study was carried out on undisturbed and regenerating scales in situ and in organ and cell cultures from regenerating scales. The hyposquamal scleroblasts forming a pseudoepithelium show an apico-basal polarization and synthesize thick collagen fibrils (100 nm) organized in a plywood pattern as long as the integrity of the cell-cell and cell-collagenous matrix contacts are preserved. In culture, scleroblasts become fibroblast-like and produce an unordered meshwork of thin collagen fibrils (30 nm). Comparison of the synthesized collagens in culture with those extracted from the scales indicates that culture conditions modify fibrillogenesis but do not change the expression of fibrillar collagen genes. Type I collagen, the prédominent component, is associated with the minor type V collagen. Type HI collagen was not present. In type I collagen, a third chain, α3 chain, was identified. The ratio between the 3 chains suggests the coexistence of two heterotrimers (αl(I))2 α2(1) and αl(I) α2(1) α3(I). Analysis by HPLC and electrophoresis of the cyanogen bromide-derived peptides obtained from the purified α3 chain support the hypothesis that αl(I) and α3(I) chains are encoded by two different genes. The presence of the two types of heterotrimers in vivo as well as in vitro could correspond to an innate property of the goldfish scleroblasts. Despite the fact that teleost cyanogen bromide-derived peptides differ from those of higher vertebrates, homologies with the mammalian collagen genes (human, for example) are sufficient to allow the detection of mRNA transcripts for al(I), α2(I) and α2(V) from confluent scleroblast cultures with human probes.

Molecular pathobiology of human collagens.

The fibril-forming collagens (types I-III, V and XI) represent a homogeneous and evolutionary related group of proteins and genes. In addition to serving as supportive elements, these macromolecules influence the spatial and ontogenic diversity of extracellular matrices, for they regulate a number of developmental programs and cellular activities, such as adhesion, proliferation and migration. Deranged expression of fibrillar collagen genes results in a number of inherited and acquired disorders which greatly affect the structural integrity of the organism. An understanding of collagen biosynthesis and regulation in normal and diseased states provides an opportunity to dissect biological problems which relate to a wide variety of subjects, including morphogenesis, relationships between structure and function of proteins, gene expression and human mutations.

Differential expression of fibrillar collagen genes during callus formation

An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of messenger RNAs for types I, II and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue at various time points. The total RNAs were analyzed in Northern hybridization for their contents of procollagen mRNAs using specific cDNA clones. The results show that during the first week of fracture repair type III collagen mRNA is increased to the greatest extent, followed by type II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or III collagen mRNA.