Connective tissue growth factor (CTGF) and cancer progression

Connective tissue growth factor (CTGF) is a member of the CCN family of secreted, matrix-associated proteins encoded by immediate early genes that play various roles in angiogenesis and tumor growth. CCN family proteins share uniform modular structure which mediates various cellular functions such as regulation of cell division, chemotaxis, apoptosis, adhesion, motility, angiogenesis, neoplastic transformation, and ion transport. Recently, CTGF expression has been shown to be associated with tumor development and progression. There is growing body of evidence that CTGF may regulate cancer cell migration, invasion, angiogenesis, and anoikis. In this review, we will highlight the influence of CTGF expression on the biological behavior and progression of various cancer cells, as well as its regulation on various types of protein signals and their mechanisms.     

内皮细胞生长状态对血管平滑肌细胞增生迁移的影响

实验通过建立细胞共培养体系,探讨内皮细胞生长状态对血管平滑肌细胞增生迁移的影响及机制。检测指标包括~3H-TdR掺入、细胞周期、细胞迁移计数和α-SM-actin mRNA表达。结果显示,融合生长内皮使平滑肌细胞~3H-TdR掺入量明显降低,增加平滑肌细胞停留在G_0/G_1期的比例,上调平滑肌细胞α-SM-actin mRNA表达;而对数生长内皮细胞使平滑肌细胞~3H-TdR掺入量明显升高,促进平滑肌细胞由 G_0/G_1期进入G_2/M和S期,下调平滑肌细胞α-SM-actin mRNA表达。对照组平滑肌细胞在基础状态下存在少量迁移,对数增殖内皮细胞组平滑肌迁移数比对照组增高约4倍(P<0.01),而融合生长内皮细胞组平滑肌迁移数仅为对照组的0.5倍(P<0.05)。结果提示内皮细胞生长状态不同,对平滑肌细胞生物学特性的影响也不同,增殖期内皮明显促进平滑肌细胞增生迁移、下调平滑肌细胞α-SM-actin mRNA表达。     

Connective tissue growth factor (CTGF) in the human dermis through ontogenesis

Connective tissue growth factor (CTGF) was examined in the structures of dermis of humans with different ages, from 20 weeks of pregnancy to 85 years. By immunohistochemistry, the fibroblasts and blood vessels positively stained for CTGF were observed in the dermis of all examined ages. An age-dependent increase in the percent of the fibroblasts and blood vessels positively stained for CTGF in the dermis was observed. A statistically significant negative correlation was found between the age-dependent changes in the total number of fibroblasts, percent of the fibroblasts with positive staining for proliferating cell nuclear antigen, and portion of the fibroblasts with positive staining for CTGF. Another statistically significant negative correlation was found between the age-dependent changes in the number of blood vessels and portion of the blood vessels with a positive staining for CTGF. The results suggest that CTGF has an inhibitory influence on the angiogenesis and fibroblast renewal in the human dermis through ontogenesis.     

Connective tissue growth factor and vascular endothelial growth factor from airway smooth muscle interact with the extracellular matrix

Airway remodeling describes the structural changes that occur in the asthmatic airway that include airway smooth muscle hyperplasia, increases in vascularity due to angiogenesis, and thickening of the basement membrane. Our aim in this study was to examine the effect of transforming growth factor-beta on the release of connective tissue growth factor and vascular endothelial growth factor from human airway smooth muscle cells derived from asthmatic and nonasthmatic patients. In addition we studied the immunohistochemical localization of these cytokines in the extracellular matrix after stimulating bronchial rings with transforming growth factor-beta. Connective tissue growth factor and vascular endothelial growth factor were released from both cell types and colocalized in the surrounding extracellular matrix. Prostaglandin E2 inhibited the increase in connective tissue growth factor mRNA but augmented the release of vascular endothelial growth factor. Matrix metalloproteinase-2 decreased the amount of connective tissue growth factor and vascular endothelial growth factor, but not fibronectin deposited in the extracellular matrix. This report provides the first evidence that connective tissue growth factor may anchor vascular endothelial growth factor to the extracellular matrix and that this deposition is decreased by matrix metalloproteinase-2 and prostaglandin E2. This relationship has the potential to contribute to the changes that constitute airway remodeling, therefore providing a novel focus for therapeutic intervention in asthma.     

Hepatocyte growth factor triggers signaling cascades mediating vascular smooth muscle cell migration

A key event in neointima formation and atherogenesis is the migration of vascular smooth muscle cells (VSMCs) into the intima. This is controlled by cytokines and extracellular matix (ECM) components within the microenvironment of the diseased vessel wall. At present, these signals have only been partially identified. In this study, we demonstrate that Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), is expressed on VSMCs isolated from the intima of atherosclerotic plaques of carotid arteries. Stimulation with HGF led to activation of Met as well as to activation of PI3-K, PKB/Akt, MEK, and the MAP kinases Erk1 and -2. Moreover, HGF induced lamellipodia formation, a characteristic feature of motile cells, and promoted VSMC migration across fibronectin-coated filters. The HGF-induced cell migration was mediated by beta1 integrins and required PI3-K activation. Our results suggest a role for the HGF-Met signaling pathway in the pathogenesis of atherosclerosis and restenosis.     

Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) growth. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived growth factor (PDGF) AB or 20 ng/ml fibroblast growth factor. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for fibroblasts or renal epithelial cells. The time course for uric acid stimulation of VSMC growth was slower than serum, suggesting induction of an autocrine growth mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC growth via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.     

Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation

Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) are both involved in integrin-mediated cell migration. However, the molecular mechanism, and the relationship between FAK and ILK activity in signaling transduction for the osteopontin (OPN)-induced migration of vascular smooth muscle cells (VSMCs) remain unclear. Here, we show that treating VSMCs with OPN could result in the dissociation of FAK with ILK by inducing phosphorylation of the former and dephosphorylation of the latter. Furthermore, we demonstrate that FAK phosphorylation induced by OPN is coupled with ILK dephosphorylation. We also provide evidence that ILK acts downstream of FAK in the signaling pathways that mediate OPN-induced VSMC migration. These findings suggest that FAK phosphorylation and ILK dephosphorylation play important roles in VSMC migration induced by OPN.     

Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration

Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.     

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.     

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.     

20-Hydroxyeicosatetraenoic acid (20-HETE) stimulates migration of vascular smooth muscle cells.

AIM: We tested the hypothesis that 20-HETE production contributes to platelet derived growth factor (PDGF)-BB stimulated migration of VSMC in a cell culture model. METHODS: Studies were performed with A10 cells which are a rat vascular smooth muscle derived cell line. Migration was determined using a Boyden chamber chemotactic assay. RESULTS: Pre-treatment of cells with two doses of 20-HETE (100 and 500 nM) significantly increased PDGF-BB stimulated VSMC migration by 34-58% of control; whereas, prior incubation of cells with inhibitors of 20-HETE production, 17-ODYA (1-25 M) or HET0016 (100 nM), significantly decreased PDGF-BB stimulated migration by 40-90%. 20-HETE mediated increase in PDGF-BB migration was completely prevented by the 20-HETE antagonist, WIT-002. In order to determine what second messenger pathways are involved in the 20-HETE mediated stimulation of VSM migration, experiments were performed with specific inhibitors of tyrosine kinase (tyrphostin 25, 10 microM), mitogen-activated extracellular signal-regulated kinase (MEK, PD98059, 20 microM and U0126, 10 microM), protein kinase C (Myr-PKC, 50 microM), and phosphoinositide 3-kinases (PI3Ks) (wortmannin, 50 nM). Blockade of MEK and PI3K all abolished the increase in 20-HETE mediated migration. CONCLUSION: 20-HETE stimulates PDGF-mediated VSM migration acting through pathways that involve MEK and PI3K.     

Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth

We have previously hypothesized that the development of severe angioproliferative pulmonary hypertension is associated with not only initial endothelial cell (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC but also with proliferation of vascular smooth muscle cells (VSMC). We have demonstrated that EC death results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype. We postulate here that the initial apoptosis of EC induces the release of mediators that cause VSMC proliferation. We cultured EC in an artificial capillary CellMax system designed to simulate the highly efficient functions of the human capillary system. We induced apoptosis of microvascular EC using shear stress and the combined VEGF receptor (VEGFR-1 and -2) inhibitor SU-5416. Flow cytometry for the proliferation marker bromodeoxyuridine showed that serum-free medium conditioned by apoptosed EC induced proliferation of VSMC, whereas serum-free medium conditioned by nonapoptosed EC did not. We also show that medium conditioned by apoptosed EC is characterized by increased concentrations of transforming growth factor (TGF)-beta1 and VEGF compared with medium conditioned by nonapoptosed EC and that TGF-beta1 blockade prevented the proliferation of cultured VSMC. In conclusion, EC death induced by high shear stress and VEGFR blockade leads to the production of factors, in particular TGF-beta1, that activate VSMC proliferation.     

Connective tissue growth factor (CCN2, CTGF) and organ fibrosis: lessons from transgenic animals

In recent months, four different systems have been reported in the literature in which CCN2 transgenes were individually expressed in podocytes, hepatocytes, cardiomyocytes or respiratory epithelial cells to achieve overexpression in, respectively, the kidney, liver, heart, or lung. These transgenic systems have provided valuable information about the contribution of CCN2 to fibrosis in vivo and have begun to reveal the complexities of the underlying mechanisms involved. On the one hand, studies of these animals have revealed that CCN2 overexpression does not necessarily lead directly to fibrotic pathology but may cause severe non-fibrotic tissue damage due to its other effects on cell function (e.g. heart). On the other hand, overexpression of CCN2 in concert with signaling pathways associated with development (e.g. lung) or fibrosing injuries (e.g. kidney, liver) can lead to the initiation or exacerbation of fibrosis. The significance of these studies is discussed in the context of the requirement for interactions between CCN2 and co-stimulatory factors in the microenvironment for the manifestation of CCN2-dependent fibrosis.     

Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix

Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD.     

Connective tissue growth factor (CTGF) promotes activated mesangial cell survival via up-regulation of mitogen-activated protein kinase phosphatase-1 (MKP-1)

Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.     

Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis

Summary The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]