Extracellular Matrix Proteins In Response Research Articles

Potential Efficacy of Proteasome Inhibitor, Delanzomib, for the Treatment of Renal Fibrosis.

Renal fibrosis is scarring and tissue hardening caused by the excess deposition of extracellular matrix proteins in response to chronic inflammation. Renal fibrosis is the primary cause of a progressive loss of renal function, and is an important therapeutic target because it ultimately leads to end-stage renal failure, which can be treated only by either dialysis or kidney transplantation. There is no effective treatment that specifically targets renal fibrosis. Myofibroblasts are known to evade apoptosis by activating molecular mechanisms in response to pro-survival biomechanical and growth factor signals from the fibrotic microenvironment. In this study, we screened and selected compounds that selectively cause cell death in myofibroblasts in vitro and studied their possible potency against renal fibrosis in a mouse model. Several proteasome inhibitors induced selective cell death in myofibroblasts differentiated from the human fibroblast cell line (MRC5). The in vivo antifibrotic effect of Delanzomib (Dz), one of the proteasome inhibitors most sensitive to myofibroblasts in vitro, was investigated in a Unilateral Ureteric Obstruction (UUO) mouse model. Treatment with Dz decreased the expression levels of the actin-alpha-2 (ACTA2) and collagen-type-1-alpha-1 (COL1A1) genes in the kidney, which are common fibrosis markers. These results suggest that Dz might be a compound that suppresses renal fibrosis by inducing selective cell death of myofibroblasts, although further investigation is required.

SPONTANEOUSLY HYPERTENSIVE RATS EXHIBIT ENHANCED CARDIAC CONNEXIN-43 AND REDUCED PRO-FIBROTIC MARKERS IN RESPONSE TO COLD ACCLIMATION

Objective: We have previously shown that down-regulation of myocardial gap junction channel protein, connexin-43 (Cx43), and up-regulation of extracellular matrix (ECM) proteins in hearts of spontaneously hypertensive rats (SHR) promote occurrence of lethal arrhythmia. Cold acclimation has a potential for reducing cardiovascular risk, especially in setting of various pathological conditions, but there is a gap of knowledge concern the heart response to adaptive thermogenesis. The purpose of the study is to explore myocardial Cx43 and ECM proteins in genetically hairless strain of SHR (SHRM) that represents rewarding model of increased adaptive thermogenesis. Design and method: Adult hairless males and females SHRM strain that harbors mutated desmoglein-4 gene and their wild type SHR were housed at standard 22 C (that is below thermo-neutrality for these animals) and 12:12 hour light: dark cycle. WKY control rats were used as reference normotensive strain. Heart samples of the left ventricular tissue from euthanatized rats were used for the proteomics of Cx43, protein kinases, which phosphorylates Cx43, and for ECM markers TGF-, SMAD2/3, MMP-2, collagen-1. Immunofluorescence detection of Cx43 was performed to examine its myocardial topology. Results: Cx43 protein and its phosphorylated status was reduced in males and females SHR vs WKY rat hearts. In contrast, adaptive thermogenesis resulted in increased both parameters in SHRM regardless the sex. Moreover, pathological topology of Cx43 attributed to its enhanced localization at lateral sides of the cardiomyocytes in SHR was attenuated in SHRM. In addition, there were gentle differences in protein levels of PKA, PKG, PKC, MAPK, Akt between SHR and SHRM. Protein level of TGF- and SMAD 2/3 were increased in SHR compare to WKY rat hearts but decreased in SHRM unlike collagen-1 that was not altered in SHRM compare to SHR. Conclusions: Findings revealed beneficial up-regulation of myocardial Cx43 along with down-regulation of extracellular matrix proteins in response of hairless SHR to increased thermogenesis.

Pirfenidone Has Anti-fibrotic Effects in a Tissue-Engineered Model of Human Cardiac Fibrosis.

A fundamental process in the development and progression of heart failure is fibrotic remodeling, characterized by excessive deposition of extracellular matrix proteins in response to injury. Currently, therapies that effectively target and reverse cardiac fibrosis are lacking, warranting novel therapeutic strategies and reliable methods to study their effect. Using a gelatin methacryloyl hydrogel, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and human fetal cardiac fibroblasts (hfCF), we developed a multi-cellular mechanically tunable 3D in vitro model of human cardiac fibrosis. This model was used to evaluate the effects of a promising anti-fibrotic drug—pirfenidone—and yields proof-of-concept of the drug testing potential of this platform. Our study demonstrates that pirfenidone has anti-fibrotic effects but does not reverse all TGF-β1 induced pro-fibrotic changes, which provides new insights into its mechanism of action.

CXCL6‐EGFR‐induced Kupffer cells secrete TGF‐β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C‐MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.

Cattle Tick Rhipicephalus microplus-Host Interface: A Review of Resistant and Susceptible Host Responses.

Ticks are able to transmit tick-borne infectious agents to vertebrate hosts which cause major constraints to public and livestock health. The costs associated with mortality, relapse, treatments, and decreased production yields are economically significant. Ticks adapted to a hematophagous existence after the vertebrate hemostatic system evolved into a multi-layered defense system against foreign invasion (pathogens and ectoparasites), blood loss, and immune responses. Subsequently, ticks evolved by developing an ability to suppress the vertebrate host immune system with a devastating impact particularly for exotic and crossbred cattle. Host genetics defines the immune responsiveness against ticks and tick-borne pathogens. To gain an insight into the naturally acquired resistant and susceptible cattle breed against ticks, studies have been conducted comparing the incidence of tick infestation on bovine hosts from divergent genetic backgrounds. It is well-documented that purebred and crossbred Bos taurus indicus cattle are more resistant to ticks and tick-borne pathogens compared to purebred European Bos taurus taurus cattle. Genetic studies identifying Quantitative Trait Loci markers using microsatellites and SNPs have been inconsistent with very low percentages relating phenotypic variation with tick infestation. Several skin gene expression and immunological studies have been undertaken using different breeds, different samples (peripheral blood, skin with tick feeding), infestation protocols and geographic environments. Susceptible breeds were commonly found to be associated with the increased expression of toll like receptors, MHC Class II, calcium binding proteins, and complement factors with an increased presence of neutrophils in the skin following tick feeding. Resistant breeds had higher levels of T cells present in the skin prior to tick infestation and thus seem to respond to ticks more efficiently. The skin of resistant breeds also contained higher numbers of eosinophils, mast cells and basophils with up-regulated proteases, cathepsins, keratins, collagens and extracellular matrix proteins in response to feeding ticks. Here we review immunological and molecular determinants that explore the cattle tick Rhipicephalus microplus-host resistance phenomenon as well as contemplating new insights and future directions to study tick resistance and susceptibility, in order to facilitate interventions for tick control.

Urotensin II-induced store-operated Ca2+ entry contributes to glomerular mesangial cell proliferation and extracellular matrix protein production under high glucose conditions

Glomerular mesangial cell (GMC) proliferation and matrix expansion are pathological hallmarks of a wide range of kidney diseases, including diabetic nephropathy. Although the circulating level of peptide hormone urotensin II (UII) and kidney tissue expression of UII and UII receptors (UTR) are increased in diabetic nephropathy, it remains unclear whether UII regulates GMC growth and extracellular matrix (ECM) accumulation. In this study, we tested the hypothesis that UII-induced Ca2+ signaling controls GMC proliferation and ECM production under normal and high glucose conditions. Mouse GMCs cultured under normal glucose conditions proliferated and synthesized ECM proteins in response to stimulation by mouse UII. UII-induced GMC proliferation and ECM protein synthesis were dependent on TRPC4 channel-mediated store-operated Ca2+ entry (SOCE) and sequential activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Ca2+/cAMP response element-binding protein (CREB) transcription factor. Under high glucose conditions, GMCs synthesized UII. Moreover, proliferation and ECM production in high glucose-challenged GMCs were attenuated by selective UTR antagonist, TRPC4 channel blocker, and CaMKII and CREB-binding protein/p300 inhibitors. These findings indicate that UII-induced SOCE via TRPC4 channels stimulates CaMKII/CREB-dependent GMC proliferation and ECM protein production. Our data also suggest that UII synthesis contributes to GMC proliferation and ECM accumulation under high glucose conditions.

MXRA5 is a TGF-β1-regulated human protein with anti-inflammatory and anti-fibrotic properties.

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling‐associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor‐β1 (TGFβ1) up‐regulated MXRA5 mRNA and protein expression. TGFβ1‐induced MXRA5 up‐regulation was prevented by either interference with TGFβ1 activation of the TGFβ receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro‐inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA‐induced down‐regulation of constitutive MXRA5 expression resulted in higher TWEAK‐induced expression of chemokines. In addition, MXRA5 down‐regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFβ1. Furthermore, in clear cell renal cancer, von Hippel–Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFβ1‐ and VHL‐regulated protein and, for the first time, we identify MXRA5 functions as an anti‐inflammatory and anti‐fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.

Astragaloside IV inhibits renal tubulointerstitial fibrosis by blocking TGF-β/Smad signaling pathway in vivo and in vitro.

Astragaloside IV (AS-IV) is a major active ingredient from Radix astragali, which has been considered as a renoprotective agent; however, its molecular mechanisms are unclear. Thus, we designed to investigate the renoprotective effects and mechanisms of AS-IV in rat model of renal fibrosis induced by unilateral ureteral obstruction (UUO) in vivo and TGF-β1-stimulated rat renal fibroblasts (NRK-49F) in vitro. Sprague-Dawley rats were randomly divided into six groups: sham operation, UUO, UUO/AS-IV (3.3, 10, 33 mg·kg(-1)·d(-1)), and UUO/enalapril (4 mg·kg(-1)·d(-1)). Renal function, tubulointerstitial damage index score, extracellular matrix (ECM) deposition, and the expressions of TGF-β1, connective tissue growth factor (CTGF), α-SMA, fibronectin, collagen I, III, Smad2/3, phosphorylated-Smad2/3, and Smad7 were measured. In addition, the expressions of CTGF, α-SMA, fibronectin, collagen I, III, Smad2/3, phosphorylated-Smad2/3, and Smad7 were measured in TGF-β1-stiumlated NRK-49F cell line. AS-IV significantly decreased UUO-induced renal fibrosis and functional impairment, which are associated with inhibition of TGF-β1, CTGF, α-SMA, and collagen matrix expression, and a decrease in serum creatinine and urea nitrogen. The renoprotective effects of AS-IV on fibrosis were associated with up-regulation of Smad7, thereby blocking up-regulations of TGF-β1, CTGF, and α-SMA, and activation of phosphorylated-Smad2/3. These effects were further conformed in NRK-49F cell line stimulated by TGF-β1. Moreover, knockdown of Smad7 gene in NRK-49F cells was able to prevent AS-IV-induced inhibition to Smad2/3 signaling activation, expression of CTGF, α-SMA, and ECM proteins in response to TGF-β1. Renal tubulointerstitial fibrosis was attenuated by treatment with AS-IV, which was closely related to induction of Smad7, thereby inhibiting TGF-β/Smad signaling.

Extracellular Matrix and Liver Disease

The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF' apoptosis, senescence, and reversal to quiescence. We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new "omics" tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs.

Competing Roles of TGFβ and Nma/BAMBI in Odontoblasts

Nma/BAMBI is a novel pseudoreceptor with homology to a TGFβ type I receptor that lacks a serine/threonine kinase domain. Nma/BAMBI functions as a dominant-negative protein that regulates reciprocal epithelial-mesenchymal interactions during organogenesis. Therefore, we hypothesized that Nma/BAMBI regulates TGFβ signaling and downstream gene expression during dentinogenesis. To test this hypothesis, we examined the downstream gene expression profiles of major dentin extracellular matrix proteins in response to Nma/BAMBI, and we examined the roles of Nma/BAMBI and TGFβ-1 during dentinogenesis. Overexpression of Nma/BAMBI in the mouse odontoblast-like cell line MD10-A2 down-regulated expression of DSPP by 66% and up-regulated expression of DMP1 four-fold. TGFβ treatment reversed Nma/BAMBI’s negative effect on DSPP expression. Furthermore, we demonstrated that TGFβ negatively regulates Nma/BAMBI’s expression levels in MD10-A2 odontoblast-like cells. Analysis of these data, together, indicates that TGFβ and Nma/BAMBI are inversely regulated and that the sequence of expression determines the net effect on downstream gene expression.

The extracellular matrix composition and responsiveness to breast carcinoma therapy

It is established that stroma surrounding breast carcinoma can be altered in comparison with its normal counterpart, and histological observations recognize lesions with loose stroma rich in hyaluronic acid (HA) and recognize lesions with dense stroma rich in fibulin-1, collagens, laminins, fibronectin and fibrillins. Previous studies have shown that adhesion of tumor cells to different extracellular matrix (ECM) components interferes with drug responses. Thus, to address the functional and biological behavior of the breast cancer-related ECM proteins in response to cytotoxic treatments, the breast carcinoma cell line MDAMB361 was injected into athymic mice in the presence of a matrix containing high levels of fibulin-1, laminin-1 and collagens. The grown tumors displayed significantly (P = 0.01) reduced sensitivity to DXR compared with the same cells injected without the matrix, strongly indicating that the ECM milieu of tumor impacts the responsiveness of tumor cells to drugs. The analysis of changes in the ECM components in response to DXR treatment revealed that the human breast carcinoma cell lines SKBR-3, MCF-7, MDAMB157 and MDAMB361 upmodulated fibulin-1 transcript [1] and protein levels, particularly in a form exhibiting a molecular weight (about 50 kDa) lower than expected (about 74 kDa), thus suggesting that tumor cells may actively reorganize their matrix environment. In parallel with the increase of fibulin-1 the same stress conditions determined decreased expression of hyaluronan synthetases, the enzymes involved in the synthesis of HA. These preliminary findings provide support for a role of ECM in response to drugs and suggest that reorganization of the ECM following chemotherapy is the basis of drug-induced resistance.

Osteopontin in Melanocytic Lesions—A First Step Towards Invasion?

Determination of the earliest molecular changes that occur during the process of cellular transformation is of intense interest to the field of tumor biology but represents a considerable technical challenge in that it is not normally amenable to pathological examination. As the transition from melanocyte hyperplasia to primary cutaneous melanoma often occurs within dysplastic nevi at the dermal–epidermal junction on sun exposed sites of the skin, it presents a unique opportunity to examine these initial stages of tumor progression. Aberrant changes associated with this transition can be identified via gene expression profiling using high-density DNA microarrays probed with samples prepared from melanocytic lesions or cell lines (Carr et al, 2003) or conversely using immunohistochemical staining to determine expression in tissue section microarrays from defined stages (Pacifico et al, 2004). Genes identified as being differentially expressed in this way provide prognostic markers as well as insight into the process of cellular transformation. The combination of both of these techniques were used in the report ofZhou et al (2005), in order to discover genes highly upregulated in melanocytic lesions. Comparison of metastatic tumor nodules with benign nevi has found over 190 genes potentially implicated in this process of early stage transformation, with the osteopontin gene already recognized for its involvement in transformation of a number of diverse tumor types (Wai and Kuo, 2004) showing the highest abundance of differential expression.

MCP-1-stimulated monocytes preferentially utilize beta 2-integrins to migrate on laminin and fibronectin

Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl-phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins.

Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4.

Interleukin 4 (also known as "B cell stimulatory factor-1"), a cytokine product of T lymphocytes and mast cells, stimulates synthesis of the extracellular matrix proteins, types I and III collagen and fibronectin, by human dermal fibroblasts in vitro. Stimulation of collagen by human recombinant (hr)IL-4 was also demonstrated in several fibroblastic synovial cell lines obtained from patients with rheumatoid arthritis and osteoarthritis. The stimulatory effect of hrIL-4 on fibroblast collagen synthesis was specifically neutralized by rabbit anti-hrIL-4 Ig. IL-4 specifically increased the steady-state levels of types I and III procollagen and fibronectin mRNAs, with no effect on cytoplasmic beta-actin mRNA. Quantitative analysis of the levels of Pro alpha 1(I) collagen transcripts in IL-4-treated fibroblast cultures was also corroborated by antisense RNA-mRNA hybridization and RNAse resistant hybrids which showed that IL-4-treated fibroblasts expressed higher levels of Pro alpha 1(I) collagen transcripts. Nuclear run-off transcription experiments indicated that IL-4 stimulated the rates of mRNA biogenesis. Based on these observations we conclude that IL-4 exerts its effect on collagen and fibronectin synthesis at the pretranslational level, resulting in synthesis of these extracellular matrix proteins. These and other data suggest that IL-4 may be a "fibrogenic cytokine" that could be important in promoting biogenesis of extracellular matrix proteins in normal wound healing and in pathological fibrosis in which mast cells and T lymphocytes play a central role.