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.     

Ceramide inhibits connective tissue growth factor expression by human retinal pigment epithelial cells

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-β. Real-time PCR analysis showed downregulation of CTGF mRNA by C₂ ceramide and upregulation by TGF-β. C₂ ceramide also inhibited constitutive and TGF-β-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

Regulation of pancreatic function by connective tissue growth factor (CTGF,CCN2)

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular secreted protein that regulates diverse cell functions including adhesion, migration, proliferation, differentiation, survival, senescence and apoptosis. In the pancreas, CTGF/CCN2 regulates critical functions including β cell replication during embryogenesis, stimulation of fibrogenic pathways in pancreatic stellate cells during pancreatitis, and regulation of the epithelial and stromal components in pancreatic ductal adenocarcinoma. This article reviews the evidence establishing CTGF/CCN2 as an important player in pancreatic physiology and pathology, highlighting the specific cell types that are involved in each process and the importance of CTGF/CCN2 as a component of autocrine or paracrine signaling within or between these various cells. Translational applications, including the potential for CTGF/CCN2-based therapies in diabetes, fibrosis, or cancer, are discussed.     

Dendrobium candidum protects against diabetic kidney lesions through regulating vascular endothelial growth factor,Glucose Transporter 1, and connective tissue growth factor expression in rats

Diabetes mellitus (DM) remains a great health problem with approximate 30% of patients with DM eventually suffering from diabetic nephropathy. The search for exogenous protective factors has recently received wide attention. The current study aimed to investigate the protective effects of Dendrobium candidum (DC) on kidneys in diabetic rats. Initially, streptozotocin-induced diabetic rats were established and randomly divided into the model group, DC group (0.2, 0.4, and 0.8 g/kg) and irbesartan group (17.5 mg/kg). The biochemical indexes, pathological changes, and the expressions of vascular endothelial growth factor (VEGF), GLUT-1, and CTGF were examined. It was found that as compared with the model group, the kidney index, serum creatine, blood urea nitrogen, 24-hour urine protein, and VEGF of DC treatment groups were significantly decreased, and pathological changes in kidney were improved in the DC groups and irbesartan group ( P < 0.05 for each parameter). The protein and messenger RNA levels of GLUT-1 and CTGF in treatment groups were significantly lower than those in rats' renal cortex without treatment. Our data suggest that DC may protect the kidneys of diabetic rats via regulating expression of VEGF, GLUT-1, and CTGF.     

Adenoviral-mediated inhibition of connective tissue growth factor in Rat-2 cells

Connective tissue growth factor (CTGF) is a profibrotic factor shown to induce extracellular matrix production and angiogenesis, two processes involved in the development of diabetic retinopathy (DR). In this study we tested the effect of a recombinant adenovirus encoding for a CTGF antisense oligonucleotide (rAdASO) on the levels of transforming growth factor-beta (TGF-beta) induced expression of CTGF in Rat-2 fibroblasts. Using semi-quantitative RT-PCR, there was a 2-fold increase in CTGF message induced by TGF-beta. Western blot and immunocytochemical analyses revealed a significant increase in CTGF protein level. This upregulation of CTGF by TGF-beta was inhibited by infection with rAdASO. These findings indicate that infection of the Rat-2 cells with rAdASO was effective in decreasing TGF-beta-induced CTGF expression. These results indicate that this viral vector might have therapeutic potential to control elevated CTGF levels that occur in DR.     

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.     

Taurine promotes connective tissue growth factor (CTGF) expression in osteoblasts through the ERK signal pathway

Summary. Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.     

Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte-fibroblast coculture contributes to keloid pathogenesis

Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial-mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial-mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast-single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial-mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions.     

Abundant retention and release of connective tissue growth factor (CTGF/CCN2) by platelets

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.     

Ratio of the vitreous vascular endothelial growth factor and pigment epithelial-derived factor in Eales disease

Eales disease (ED) is an idiopathic inflammatory venous occlusion of the peripheral retina. As neovascularization is prominent in ED, this study attempts to look at the ratio of VEGF, the angiogenic factor, and PEDF, an anti-angiogenic factor in the vitreous of ED patients in comparison with the macular hole (MH) and Proliferative Diabetic Retinopathy (PDR). Vitreous levels of VEGF and PEDF were determined in the undiluted vitreous specimen obtained from 26 ED cases, 17 PDR, and seven patients with MH. The vitreous levels of VEGF and PEDF were estimated by ELISA. The immunohistochemistry (IHC) for VEGF and PEDF were done in the epiretinal membrane of ED and PDR case. The VEGF/PEDF ratio was found to be significantly increased in ED (p = 0.014) and PDR (p = 0.000) compared to MH. However the ratio was 3.5-fold higher in PDR than ED (p = 0.009). The IHC data on the ERM specimen from ED showed the presence of VEGF and PEDF similar to PDR. The high angiogenic potential seen as the ratio of VEGF/PEDF correlates with the peak clinical onset of the disease in the age group 21–30 years and the diseases usually self-resolves above the age of 40, which is reflected by the low ratio of VEGF/PEDF. The study shows that the VEGF/PEDF ratio is significantly increased in ED though the angiogenic potential is higher in PDR than in ED. Clinically Eales Disease is known as a self-limiting disease, while PDR is a progressive disease.     

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.     

Gene expression and distribution of connective tissue growth factor (CCN2/CTGF) during secondary ossification center formation.

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.     

Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non-malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro-survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy.     

Connective tissue growth factor is induced in bleomycin-induced skin scleroderma

The origin of fibrotic cells within connective tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofibroblasts present in dermal fibrosis in uncertain. Connective tissue growth factor (CTGF/CCN2) is a marker and mediator of fibrosis. In this report, we use an antibody recognizing CCN2 to assess the cell types in mouse dermis which express CCN2 in the bleomycin model of skin scleroderma. Control (PBS injected) and fibrotic (bleomycin-injected) dermis was examined for CCN2, α-smooth muscle actin (α-SMA) (to detect myofibroblasts), and NG2 (to detect pericytes) expression. Consistent with previously published data, CCN2 expression was largely absent in the dermis of control mice. However, upon exposure to bleomycin, CCN2 was observed in the dermis. Cells that expressed CCN2 were α−SMA-expressing myofibroblasts. Approximately 85% of myofibroblasts were NG2-positive, CCN2-expressing pericytes, indicating that pericytes significantly contributed to the presence of myofibroblasts in sclerotic dermis. Thus CCN2 is induced in fibrotic skin, correlating with the induction of myofibroblast induction. Moreover, CCN2-expressing pericytes significantly contribute to the appearance of myofibroblasts in bleomycin-induced skin scleroderma.