Overexpression Of Fibronectin Research Articles

Fibronectin Regulates Wnt7a Signaling and Satellite Cell Expansion

The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a coreceptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche via transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, invivo overexpression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 coreceptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

BackgroundPirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.MethodsThe expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.ResultsTGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.ConclusionWe concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway

BackgroundInflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified.ResultsU937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway.ConclusionThe present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.

Inhibition of Connective Tissue Growth Factor Overexpression in Diabetic Retinopathy by SERPINA3K via Blocking the WNT/β-Catenin Pathway

OBJECTIVEConnective tissue growth factor (CTGF) is a major fibrogenic factor. Increased retinal CTGF levels have been implicated to play a role in diabetic retinopathy. SERPINA3K is a serine proteinase inhibitor, and its levels were decreased in retinas with diabetic retinopathy. The purpose of this study was to investigate the role of SERPINA3K in the regulation of CTGF and fibrogenesis and its mechanism of action.RESEARCH DESIGN AND METHODSAdenovirus expressing SERPINA3K was injected intravitreally into streptozotocin-induced diabetic rats. CTGF expression was measured using Western blot analysis and real-time RT-PCR. Fibrosis was evaluated by quantifying retinal fibronectin using enzyme-linked immunosorbent assay. Wnt pathway activation was determined by phosphorylation of LDL receptor–related protein 6, a coreceptor of Wnt ligands, and stabilization of β-catenin, an essential effector of the canonical Wnt pathway.RESULTSAd-SERPINA3K attenuated the CTGF and fibronectin overexpression in retinas of diabetic rats. In cultured retinal cells, SERPINA3K blocked the overproduction of CTGF induced by high glucose. Dickkopf-1, a specific Wnt antagonist, also attenuated the high-glucose–induced CTGF overexpression, indicating a role of Wnt signaling in CTGF overexpression in diabetes. Similarly, increased SERPINA3K blocked Wnt pathway activation in diabetic retinas and in cells treated with high glucose. Further, SERPINA3K also attenuated the Wnt3a-induced activation of the canonical Wnt pathway and the overexpression of CTGF.CONCLUSIONSERPINA3K is an antifibrogenic factor, and its antifibrogenic activity is through blocking the Wnt pathway. Decreased SERPINA3K levels may contribute to the fibrosis in diabetic retinopathy.

Cannabinoid Receptor 1 Blockade Ameliorates Albuminuria in Experimental Diabetic Nephropathy

OBJECTIVECannabinoid receptor 1 (CB1) is localized in the central nervous system and in peripheral tissues involved in energy metabolism control. However, CB1 receptors are also expressed at low level within the glomeruli, and the aim of this study was to investigate their potential relevance in the pathogenesis of proteinuria in experimental type 1 diabetes.RESEARCH DESIGN AND METHODSStreptozotocin-induced diabetic mice were treated with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,3-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), a selective CB1-receptor antagonist, at the dosage of 1 mg · kg−1 · day−1 via intraperitoneal injection for 14 weeks. Urinary albumin excretion was measured by enzyme-linked immunosorbent assay. CB1 receptor expression was studied by immunohistochemistry, immunoblotting, and real-time PCR. Expression of nephrin, podocin, synaptopodin, and zonula occludens-1 (ZO-1) was assessed by immunofluorescence and real-time PCR. Fibronectin, transforming growth factor-β1 (TGF-β1), and connective tissue growth factor (CTGF) mRNA levels were quantitated by real-time PCR.RESULTSIn diabetic mice, the CB1 receptor was overexpressed within the glomeruli, predominantly by glomerular podocytes. Blockade of the CB1 receptor did not affect body weight, blood glucose, and blood pressure levels in either diabetic or control mice. Albuminuria was increased in diabetic mice compared with control animals and was significantly ameliorated by treatment with AM251. Furthermore, CB1 blockade completely prevented diabetes-induced downregulation of nephrin, podocin, and ZO-1. By contrast overexpression of fibronectin, TGF-β1, and CTGF in renal cortex of diabetic mice was unaltered by AM251 administration.CONCLUSIONSIn experimental type 1 diabetes, the CB1 receptor is overexpressed by glomerular podocytes, and blockade of the CB1 receptor ameliorates albuminuria possibly via prevention of nephrin, podocin, and ZO-1 loss.

Glucose-induced fibronectin expression in endothelial cells is mediated by protein kinase C.

One of the crucial pathophysiological changes in diabetic angiopathy might be the glucose-induced synthesis of components of the extracellular matrix-like fibronectin. This effect has been described for endothelial and mesangial cells. In order to gain further insight into the mechanisms how glucose-induced fibronectin expression is regulated, confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated with varying concentrations of D-glucose (5-30 mM) in a time-dependent manner. Using Westernblotting techniques with a monoclonal antibody, a 3 +/- 0.2-fold increase of the 220 kDa-signal corresponding to human fibronectin was visible after an incubation time of 6 h indicating de-novo synthesis. Using incubation times of 6 or 10 days, glucose-mediated fibronectin overexpression was still visible, reaching a maximum at 15 mM D-glucose. The corresponding maximum values were 2.4 +/- 0.3 (6 d) and 2.3 +/- 0.2 (10 d). There was a concomitant glucose-dependent onset of expression of Protein Kinase C (PKC)-isoforms PKC-delta (2.5 +/- 0.3-fold), PKC-epsilon (2 +/- 0.2-fold) and PKC-zeta (1.8 +/- 0.2-fold), which reached a maximum after 12 h and was still visible during long-term culture. Induction of fibronectin expression was also obtained using the PKC-activating phorbolester Phorbol-12-myristat-13-acetat (PMA) which mimicks glucose-induced PKC activation. Glucose-induced fibronectin expression was decreased by the PKC-inhibitor H-7 (1- [5-isoquinolinylsulfonyl]-2-methylpiperazine). These experiments suggest that: 1. Short-term induction of fibronectin expression mediated by glucose is probably PKC-triggered since concomitant induction of PKC-isoforms PKC-delta, PKC-epsilon and PKC-zeta was observed. 2. Long-term incubation with D-glucose leads to an ongoing concentration-dependent coexpression of fibronection and various PKC-isoforms. If glucose-induced, PKC-mediated de-novo synthesis of components of the connective tissue or the extracellular matrix may be important in the pathomechanism of diabetic angiopathy, has to be proven.

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β1 signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β1 in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β1. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.

Tight Glycemic Control Regulates Fibronectin Expression and Basement Membrane Thickening in Retinal and Glomerular Capillaries of Diabetic Rats

To determine whether tight glycemic control prevents development of basement membrane (BM) thickening in retinal and glomerular capillaries of diabetic rats and whether the extent to which BM thickening develops is linked to fibronectin (FN) overexpression and the degree of hyperglycemia. Retinal and renal cortical tissues obtained from the tightly controlled diabetic (TC), poorly controlled diabetic (D), and nondiabetic (N) control rats were subjected to morphometric and biochemical analyses. In both tissues, capillary BM thickening was determined by electron microscopy, and FN protein level was assessed by Western blot analysis. Routine measurements of blood glucose level and glycohemoglobin level were performed throughout the study. The HbA1c level was significantly increased in D rats, but not in TC rats, compared with those of the N rats with a concomitant increase in capillary BM thickness and FN protein expression in retinal and renal tissues. A strong correlation was observed between retinal and glomerular capillary BM thickness (r=0.79, P=0.0001), between retinal and kidney FN protein levels (r=0.7, P=0.005), between HbA1c and FN protein levels in the retina (r=0.66, P=0.006) and kidney (r= .84, P=0.0003), and between HbA1c level and BM thickness in retinal (r=0.76, P=0.0002) and renal tissues (r=0.64, P=0.004). In diabetes BM thickening develops in retinal and glomerular capillaries in a correlated manner. Tight glycemic control may be beneficial in preventing the pathologic development of capillary BM thickening and FN overexpression in retinal and renal tissues, two target tissues of diabetic microangiopathy.

Molecular Classification of Thyroid Nodules by Cytology

Fine needle aspiration (FNA) biopsy of thyroid nodules provides cytologic specimens whose interpretation can direct patients toward either thyroidectomy or observation. Approximately 20% of FNA specimens yield an indeterminate result. Recent studies have characterized differences in gene expression between benign and malignant conditions, most often using whole tissue. Our goal was to determine the feasibility of quantitative polymerase chain reaction (qPCR)-based gene expression analysis in cytologic samples. For five genes shown to be over-expressed in thyroid carcinomas (fibronectin, galectin-3, Met/HGFR, MUC1, and GA733-precursor), we compared expression among pathologic states. Prospective laboratory analysis of 20 thyroidectomy specimens. Routine microscopy was performed. Cytologic samples were obtained from the dominant nodules, and RNA was extracted. Preliminary analysis using fluorometry and reverse-transcriptase (RT)-PCR was performed. Expression levels of the test genes in nodules and from control samples were measured by real-time qPCR. Fold changes in gene expression were compared. Only one specimen did not yield sufficient intact RNA for gene expression analysis. RT-PCR revealed satisfactory RNA recovery in all other specimens. qPCR showed significant over-expression of fibronectin in the papillary carcinomas compared with the goiters (P = .0013), follicular adenomas (P = .0014), and follicular carcinomas (P = .0001). Differences in both fibronectin and MUC1 expression between the follicular carcinomas and the follicular adenomas were also significant (P = .025 and .045, respectively). Cytologic specimens were a satisfactory source of tissue for qPCR-based gene expression analysis. Both fibronectin and MUC1 were differentially expressed in follicular adenomas and follicular carcinomas, and fibronectin expression differed in papillary carcinomas compared with the other lesions. These results may form the basis of a clinical predictor for lesions with indeterminate or suspicious cytology.

Epithelial-Mesenchymal Transition (EMT) and Activated Extracellular Signal-regulated Kinase (p-Erk) in Surgically Resected Pancreatic Cancer

EMT or transformation to the mesenchymal phenotype plays an important role in tumor invasion and metastasis. In vitro data suggest that mesenchymal transformation may correlate with the activation of PI3 kinase and Ras/Erk pathways. We investigated the expression of EMT markers (low E-cadherin, high fibronectin, and vimentin) and their association with p-Erk in resected pancreatic cancer. Clinical data/surgical specimens from 34 consecutive pancreatic cancer patients (pts) who underwent pancreatectomy were included. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues using monoclonal antibodies against vimentin, fibronectin, E-cadherin, and p-Erk. The results were correlated with clinicopathological parameters and survival. Survival analysis (log-rank test, Cox proportional hazard model), categorical data analysis (Pearson's chi-square, Fisher's exact test) and Kendall's tau were performed at a significance level of 0.05. The patient population was formed from 13 males and 21 females, with a median age of 66 years (range 38-84 years); American Joint Committee on Cancer (AJCC) stage 1 (n = 2), 2 (n = 27), 3 (n = 5); histological grade 1 (n = 4), 2 (n = 13), 3 (n = 16), 4 (n = 1). Median survival was 15 months (95% CI: 11-24 months). Fibronectin overexpression correlated with the presence of vimentin (p = 0.0048) and activated Erk (p = 0.0264). There was a borderline association of fibronectin with worsening grade (p = 0.06). A negative association between vimentin and E-cadherin was noted (p = 0.0024). Increased fibronectin or vimentin and decreased E-cadherin correlated with poor survival. EMT is associated with poor survival in surgically resected pancreatic adenocarcinoma. A correlation between activated Erk and fibronectin was identified that may open avenues for targeted therapy for this subgroup.

Rosiglitazone inhibits angiotensin II-induced CTGF expression in vascular smooth muscle cells––Role of PPAR-γ in vascular fibrosis

Angiotensin (Ang) II plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. Connective tissue growth factor (CTGF) is a potent profibrotic factor implicated in the Ang II-induced pathologic fibrosis process. PPAR-γ activators thiazolidinediones have been recently reported to have beneficial vascular effects. However, their effects and related molecular mechanisms on extracellular matrix (ECM) turnover in vascular smooth muscle cells (VSMCs) are unknown. The present study evaluated the regulation of Ang II-induced CTGF, ECM production and cell growth by rosiglitazone in VSMCs. In aorta of Ang II-infused rats, CTGF expression was markedly increased, and type III collagen and fibronectin overexpression was observed. Cotreatment with rosiglitazone diminished these changes, whereas increased nuclear PPAR-γ expression in VSMCs. In growth-arrested VSMCs, rosiglitazone attenuated the proliferation and apoptosis, increased PPAR-γ production and activation, and reduced CTGF and ECM production in response to Ang II in a dose-dependent fashion. These inhibitory effects were attenuated by the pretreatment of cells with PPAR-γ antagonist GW9662 or bisphenol A diglycidyl ether (BADGE). Furthermore, rosiglitazone inhibited Ang II-induced Smad2 production and phosphorylation but had no effect on transforming growth factor-β1 (TGF-β1) expression. These results suggest that in Ang II-stimulated VSMCs, rosiglitazone caused an antiproliferative, antiapototic effect and reduces ECM production through mechanisms that include reducing CTGF expression, and a crosstalk between PPAR-γ and Smad may be involved in the inhibitory effects of rosiglitazone. This novel finding suggests a role of PPAR-γ activators in preventing Ang II-induced vascular fibrosis.

Salutary Effect of Pigment Epithelium–Derived Factor in Diabetic Nephropathy

Diabetic nephropathy is a major complication of diabetes and a leading cause of end-stage renal diseases in the U.S. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor that has been extensively studied in diabetic retinopathy. Recently, we reported that PEDF is expressed at high levels in normal kidneys and that PEDF levels are decreased in kidneys of streptozotocin (STZ)-induced diabetic rats. In the present study, we injected STZ-diabetic rats with an adenovirus expressing PEDF (Ad-PEDF) to evaluate its effects in diabetes. The results showed that increased expression of PEDF in the kidney in response to Ad-PEDF delivery significantly alleviated microalbuminuria in early stages of diabetes. Administration of Ad-PEDF was found to prevent the overexpression of two major fibrogenic factors, transforming growth factor-beta (TGF-beta)1 and connective tissue growth factor (CTGF), and to significantly reduce the production of an extracellular matrix (ECM) protein in the diabetic kidney. Moreover, PEDF upregulated metalloproteinase-2 expression in diabetic kidney, which is responsible for ECM degradation. In cultured human mesangial cells, PEDF significantly inhibited the overexpression of TGF-beta1 and fibronectin induced by angiotensin II. PEDF also blocked the fibronectin production induced by TGF-beta1 through inhibition of Smad3 activation. These findings suggest that PEDF functions as an endogenous anti-TGF-beta and antifibrogenic factor in the kidney. A therapeutic potential of PEDF in diabetic nephropathy is supported by its downregulation in diabetes; its prevention of the overexpression of TGF-beta, CTGF, and ECM proteins in diabetic kidney; and its amelioration of proteinuria in diabetic rats following Ad-PEDF injection.

Notoginsenoside R1 inhibits TNF-α-induced fibronectin production in smooth muscle cells via the ROS/ERK pathway

The matrix fibronectin protein plays an important role in vascular remodeling. Notoginsenoside R1 is the main ingredient with cardiovascular activity in Panax notoginseng; however, its molecular mechanisms are poorly understood. We report that notoginsenoside R1 significantly decreased TNF-α-induced activation of fibronectin mRNA, protein levels, and secretion in human arterial smooth muscle cells (HASMCs) in a dose-dependent manner. Notoginsenoside R1 scavenged hydrogen peroxide (H 2O 2) in a dose-dependent manner in the test tube. TNF-α significantly increased intracellular ROS generation and then ERK activation, which was blocked by notoginsenoside R1 or DPI and apocynin, inhibitors of NADPH oxidase, or the antioxidant NAC. Our data demonstrated that TNF-α-induced upregulation of fibronectin mRNA and protein levels occurs via activation of ROS/ERK, which was prevented by treatment with notoginsenoside R1, DPI, apocynin, NAC, or MAPK/ERK inhibitors PD098059 and U0126. Notoginsenoside R1 significantly inhibited H 2O 2-induced upregulation of fibronectin mRNA and protein levels and secretion; it also significantly inhibited TNF-α and H 2O 2-induced migration. These results suggest that notoginsenoside R1 inhibits TNF-α-induced ERK activation and subsequent fibronectin overexpression and migration in HASMCs by suppressing NADPH oxidase-mediated ROS generation and directly scavenging ROS.

Effect of Combined Antisense Oligonucleotides Against High-Glucose-and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability

The effect of combined antisense oligonucleotides (AS-oligos) against overexpression of extracellular matrix (ECM) components, fibronectin, laminin, and collagen IV and on cell monolayer permeability was examined in rat microvascular endothelial cells (RMECs) grown in high glucose medium and on retinal vascular permeability in diabetic rats. RMECs grown in high glucose for 10 days and transfected with combined AS-oligos showed a significantly reduced fibronectin, laminin, and collagen IV protein level. In parallel studies, high-glucose–induced excess monolayer permeability was also reduced in RMECs transfected with the combined AS-oligos. Similarly, diabetic rats intravitreally injected with the combined AS-oligos and examined after 2 months of diabetes showed significant reduction in retinal fibronectin, laminin, and collagen IV expression. In addition, vascular permeability, as determined from extravasation of fluorescein isothiocyanate–BSA in the surrounding areas of the retinal capillaries, was partially reduced in the combined AS-oligos–treated diabetic retinas. Our results indicate that the combined AS-oligos strategy is effective in simultaneously reducing fibronectin, collagen IV, and laminin overexpression and reducing vascular leakage in the retinal capillaries of diabetic rat retinas. The findings suggest that abnormal synthesis of ECM components may contribute to vascular leakage in the diabetic retina.

Effect of Combined Antisense Oligonucleotides Against High-Glucose–and Diabetes-Induced Overexpression of Extracellular Matrix Components and Increased Vascular Permeability

The effect of combined antisense oligonucleotides (AS-oligos) against overexpression of extracellular matrix (ECM) components, fibronectin, laminin, and collagen IV and on cell monolayer permeability was examined in rat microvascular endothelial cells (RMECs) grown in high glucose medium and on retinal vascular permeability in diabetic rats. RMECs grown in high glucose for 10 days and transfected with combined AS-oligos showed a significantly reduced fibronectin, laminin, and collagen IV protein level. In parallel studies, high-glucose-induced excess monolayer permeability was also reduced in RMECs transfected with the combined AS-oligos. Similarly, diabetic rats intravitreally injected with the combined AS-oligos and examined after 2 months of diabetes showed significant reduction in retinal fibronectin, laminin, and collagen IV expression. In addition, vascular permeability, as determined from extravasation of fluorescein isothiocyanate-BSA in the surrounding areas of the retinal capillaries, was partially reduced in the combined AS-oligos-treated diabetic retinas. Our results indicate that the combined AS-oligos strategy is effective in simultaneously reducing fibronectin, collagen IV, and laminin overexpression and reducing vascular leakage in the retinal capillaries of diabetic rat retinas. The findings suggest that abnormal synthesis of ECM components may contribute to vascular leakage in the diabetic retina.

Ascorbic acid and uric acid suppress glucose-induced fibronectin and vascular endothelial growth factor production in human endothelial cells

Human umbilical vein endothelial cells (HUVECs) were used to investigate the individual effect of ascorbic (0.01 and 0.05 mM) and uric acid (3, 5, and 7 mg/dl), as well as the combined effect of these two agents on glucose-induced overexpression of fibronectin (Fn), glutathione peroxidase (GPx) activity, H 2O 2 level and vascular endothelial growth factor (VEGF) protein levels. Under 30 mM glucose stress, GPx activity significantly decreased, and lactate dehydrogenase (LDH) release, H 2O 2 level, and Fn and VEGF production significantly increased in HUVECs ( P<.05). The addition of ascorbic acid at 0.05 mM or uric acid at 5 and 7 mg/dl significantly reduced LDH release, recovered GPx activity, suppressed H 2O 2 production, and decreased Fn and VEGF production ( P<.05). The combination of 0.05 mM ascorbic acid and 3, 5, or 7 mg/dl provided significantly greater effect in enhancing GPx activity and lowering H 2O 2 level than ascorbic acid or uric acid treatment alone did ( P<.05). These data suggest that ascorbic acid alone or combined with uric acid in these concentrations may be beneficial to ameliorate glucose-induced diabetic deterioration.

HGF induces fibronectin matrix synthesis in melanoma cells through MAP kinase-dependent signaling pathway and induction of Egr-1

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Cultured tubule cells from TGF-β1 null mice exhibit impaired hypertrophy and fibronectin expression in high glucose

To firmly establish the role of the transforming growth factor-beta1 (TGF-beta1) isoform in the pathophysiology of diabetic tubulointerstitial hypertrophy and fibrosis, we examined how the total absence of TGF-beta1 would alter the effect of high glucose on cellular hypertrophy and matrix expression in tubuloepithelial cells cultured from TGF-beta1 null mice. Primary tubule cell cultures, obtained from kidneys of TGF-beta1 knockout mice and their wild-type littermates, were treated with exogenous TGF-beta1 or high glucose. The TGF-beta system was characterized at the ligand and receptor levels using Northern and Western blotting. Cellular hypertrophy and growth were assessed by thymidine incorporation, cell counting, leucine incorporation, and protein content. Fibronectin expression was assessed by Northern analysis and enzyme-linked immunosorbent assay (ELISA). Knockout cells did not express TGF-beta1 but did express TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors. Exogenous TGF-beta1 down-regulated the ligand-binding type II receptor but up-regulated type I receptor expression. Knockout cells proliferated more rapidly than wild-type cells, but restoring TGF-beta1 to knockout cells slowed their proliferation. In wild-type cells, high glucose caused cellular hypertrophy, evidenced by greater leucine incorporation and protein content along with decreased thymidine incorporation. High glucose also increased fibronectin message and protein. However, in knockout cells, high glucose failed to induce hypertrophy and was severely limited in its capacity to stimulate fibronectin. In tubular epithelial cells, TGF-beta1 mediates the hypertrophic and fibronectin-stimulatory effects of high glucose, confirming the role of the TGF-beta1 isoform in the pathogenesis of diabetic tubular hypertrophy and fibronectin overexpression.

1067-135 Over-expression of fibronectin involves activation of mitogen-activated protein-kinase-kinase of the stress activated protein kinase/C-jun N-terminal kinase in aortic regurgitation

1067-135 Over-expression of fibronectin involves activation of mitogen-activated protein-kinase-kinase of the stress activated protein kinase/C-jun N-terminal kinase in aortic regurgitation

Effective suppression of fibronectin synthesis by retrovirus delivered shRNA in rat cardiac fibroblasts

Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling.