David R. Brigstock

Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61)

Connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61) are prototypical members of the CCN family which also contains nephroblastoma overexpressed (NOV) and Wnt-induced secreted proteins-1, -2 and -3 (WISP-1, -2, -3). These proteins function as extracellular matrix (ECM)-associated signaling molecules that contain structural modules allowing them to bind directly with other moieties in the pericellular environment. Although multiple target cell types have been identified for CCN proteins, there is strong evidence supporting a role for CTGF and CYR61 in the regulation of endothelial cell function and angiogenesis. Both CTGF and CYR61 can promote endothelial cell growth, migration, adhesion and survival in vitro and at least some of these effects are mediated through cell surface integrins. Both proteins are transcriptionally activated in endothelial cells in response to basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), and endothelial cell proliferation and migration in vitro is reduced by antagonists of CTGF production or action. The expression pattern of CTGF and CYR61 in endothelial cells of vessels in situ supports a role for these molecules in normal endothelial homeostasis, as well as participating in the angiogenic process during embryonic development, placentation, tumor formation, fibrosis, and wound healing. CTGF or CYR61 knockout mice exhibit vascular defects during embryogenesis and fetal development. Both CTGF and CYR61 are intrinsically active in in vivo asssays for angiogenic activity. However, they can also regulate the production and/or activity of other angiogenic molecules (e.g. bFGF, VEGF) as well molecules that affect the integrity or stability of the ECM (e.g. collagen, matrix metalloproteases (MMPs), tissue inhibitors of MMPs (TIMPs)). Therefore, through their paracrine action as products of cells such as fibroblasts or smooth muscle cells or through their autocrine action as products of endothelial cells, CTGF and CYR61 participate in a variety of direct and indirect mechanisms by which angiogenesis is regulated at multiple control points.

Connective Tissue Growth Factor (CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin αvβ3 and Heparan Sulfate Proteoglycan

Connective tissue growth factor (CCN2, also known as CTGF) is a matricellular protein that appears to play an important role in hepatic stellate cell (HSC)-mediated fibrogenesis. After signal peptide cleavage, the full-length CCN2 molecule comprises four structural modules (CCN21–4) and is susceptible to proteolysis by HSC yielding isoforms comprising essentially modules 3 and 4 (CCN23–4) or module 4 alone (CCN24). In this study we show that rat activated HSC are capable of adhesion to all three CCN2 isoforms via the binding of module 4 to integrin αvβ3, a process that is dependent on interactions between module 4 and cell surface heparan sulfate proteoglycans (HSPGs). These findings are based on several lines of evidence. First, integrin αvβ3 was detected in HSC lysates by immunoprecipitation and Western blot, and CCN24-mediated HSC adhesion was blocked by anti-integrin αvβ3 antibody. Second, as assessed by immunoprecipitation and solid phase binding assay, CCN24 bound directly to integrin αvβ3 in cell-free systems. Third, destruction or inhibition of synthesis of cell surface HSPGs with, respectively, heparinase or sodium chlorate abrogated HSC adhesion to CCN24. Fourth, prior occupancy of heparin-binding sites on CCN24 with soluble heparin completely blocked HSC adhesion. These findings indicate that integrin αvβ3 functions as a co-receptor with HSPGs for CCN24-mediated HSC adhesion. Furthermore, by peptide mapping and site-directed mutagenesis we demonstrated that the sequence IRTPKISKPIKFELSG within CCN24 is a unique binding domain for integrin αvβ3 that is sufficient to mediate integrin αvβ3- and HSPG-dependent HSC adhesion. These findings offer the possibility of developing novel antifibrotic therapies that target the integrin-binding domain.

Connective tissue growth factor (CTGF/CCN2) in hepatic fibrosis.

Connective tissue growth factor (CTGF/CCN2) is a highly profibrogenic molecule which is overexpressed in many fibrotic lesions, including those of the liver. CTGF/CCN2 is transcriptionally activated by transforming growth factor-beta (TGF-beta) and appears to mediate some of the extracellular matrix (ECM)-inducing properties that have been previously attributed to TGF-beta. CTGF/CCN2 and TGF-beta stimulate connective tissue cell proliferation and ECM synthesis in vitro and exhibit shared fibrogenic and angiogenic properties in vivo. In fibrotic liver, CTGF/CCN2 mRNA and protein are produced by fibroblasts, myofibroblasts, hepatic stellate cells (HSCs), endothelial cells, and bile duct epithelial cells. CTGF/CCN2 is also produced at high levels in hepatocytes during cytochrome P-4502E1-mediated ethanol oxidation. CTGF/CCN2 expression in cultured HSCs is enhanced following their activation or stimulation by TGF-beta while exogenous CTGF/CCN2 is able to promote HSC adhesion, proliferation, locomotion, and collagen production. Collectively, these data suggest that during initiating or downstream fibrogenic events in the liver, production of CTGF/CCN2 is regulated primarily by TGF-beta in one or more cell types and that CTGF/CCN2 plays important roles in HSC activation and progression of fibrosis. This article reviews the data that support the importance of CTGF/CCN2 in hepatic fibrosis and highlights the concept that CTGF/CCN2 may represent a new therapeutic target in this disease.

Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids. Identification as heparin-regulated Mr 10,000 forms of connective tissue growth factor.

Uterine growth factors are potential effector molecules in embryo growth signaling pathways. Pig uterine luminal flushings contained a heparin-binding growth factor (HBGF) that required 0.8 m NaCl for elution from heparin columns and was termed HBGF-0.8. This factor, which was heat- and acid-labile and of M r 10,000 as assessed by gel filtration, stimulated DNA synthesis in fibroblasts and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent M r of 10,000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EENIKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKXIRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247–262 and 248–259 of the 349-residue predicted primary translation product of porcine connective tissue growth factor (pCTGF). 10-kDa CTGF-mediated fibroblast DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and 20 kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel growth factor in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.

Structural and Functional Properties of CCN Proteins

The CCN family currently comprises six members (CCN1-6) that regulate diverse cell functions, including mitogenesis, adhesion, apoptosis, extracellular matrix (ECM) production, growth arrest, and migration. These properties can result in a multiplicity of effects during development, differentiation, wound healing, and disease states, such as tumorigenesis and fibrosis. CCN proteins have emerged as major regulators of chondrogenesis, angiogenesis, and fibrogenesis. CCN proteins are mosaic in nature and consist of up to four structurally conserved modules, at least two of which are involved in binding to cell surfaces via molecules that include integrins, heparan sulfate proteoglycans, and low-density lipoprotein receptor-related protein. CCN proteins use integrins as signal transducing receptors to regulate context-dependent responses in individual cell types. The involvement of integrins in mediating CCN signaling allows for considerable plasticity in response because some effects are specific for certain integrin subtypes and integrin signaling is coordinated with other signaling pathways in the cell. In addition to their own biological properties, CCN proteins regulate the functions of other bioactive molecules (e.g., growth factors) via direct binding interactions. CCN molecules demonstrate complex multifaceted modes of action and regulation and have emerged as important matricellular regulators of cell function.

Characterization of Cell-Associated and Soluble Forms of Connective Tissue Growth Factor (CTGF) Produced by Fibroblast Cells In Vitro

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate M(r) 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4 kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non- or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells

BACKGROUND/AIMS Connective tissue growth factor is a recently described mitogenic protein implicated in a variety of fibrotic disorders. Connective tissue growth factor may be a downstream mediator of the pro-fibrotic and mitogenic actions of transforming growth factor-beta, promoting extracellular matrix deposition and fibrogenesis. As transforming growth factor-beta is considered important to the pathogenesis of hepatic fibrosis, we examined the possible contribution of connective tissue growth factor to this process. METHODS Connective tissue growth factor expression was examined in normal and fibrotic human and rat livers using RT-PCR and ribonuclease protection assays, and in primary cultures of rat hepatic stellate cells by Northern and Western blotting. RESULTS Ribonuclease protection assays demonstrated connective tissue growth factor mRNA was increased 3-5-fold in human fibrotic liver compared with normal. RT-PCR showed this mRNA was increased in carbon-tetrachloride-treated rat liver. Northern analysis showed connective tissue growth factor mRNA was increasingly expressed during progressive activation of cultured rat hepatic stellate cells. Western analysis confirmed that freshly isolated hepatic stellate cells secreted relatively little connective tissue growth factor compared with hepatic stellate cells activated in culture. Hepatic stellate cells stimulated with transforming growth factor-beta showed increased expression of connective tissue growth factor mRNA and protein. CONCLUSIONS Connective tissue growth factor mRNA is consistently upregulated in human liver cirrhosis of various aetiologies, supporting a role for this growth factor in hepatic fibrogenesis. Our studies suggest that hepatic stellate cells may be an important source of hepatic connective tissue growth factor in vivo, particularly following stimulation with transforming growth factor-beta.

Kupffer Cells Mediate Leptin-Induced Liver Fibrosis

BACKGROUND & AIMS Leptin has profibrogenic effects in liver, although the mechanisms of this process are unclear. We sought to elucidate the direct and indirect effects of leptin on hepatic stellate cells (HSCs). METHODS HSCs from Sprague-Dawley rats were exposed to leptin and expression of collagen-I, tissue inhibitor of matrix metalloproteinases-1 (TIMP1), transforming growth factor beta1 (TGF-beta1), and connective tissue growth factor (CTGF/CCN2) was assessed. The effects of medium from Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) following leptin were evaluated in HSCs; alpha-smooth muscle actin (alphaSMA) production and KC signaling were analyzed. RESULTS HSCs were not activated by incubation with leptin. However, HSCs cultured with medium taken from KCs that were incubated with leptin had increased expression of collagen I, TIMP1, TGF-beta1, and CTGF/CCN2, as well as alphaSMA protein levels and proliferation. These effects were leptin receptor dependent because conditioned medium from KCs isolated from leptin receptor-deficient Zucker (fa/fa) rats did not activate HSCs. In KCs incubated with leptin, messenger RNA and protein expression of TGF-beta1 and CTGF/CCN2 increased. Leptin potentiated signal transducer and activator of transcription 3, AKT, and extracellular signal-related kinase 1/2 phosphorylation in KCs and increased AP-1 and nuclear factor-kappaB DNA binding. Finally, addition of anti-TGF-beta to KC-conditioned medium inhibited HSC expression of collagen I, TIMP1, and CTGF/CCN2, whereas signal transducer and activator of transcription 3 inhibitor attenuated TGF-beta1 production by KC. CONCLUSIONS Leptin mediates HSC activation and liver fibrosis through indirect effects on KC; these effects are partly mediated by TGF-beta1.

Low density lipoprotein receptor-related protein (LRP) is a heparin-dependent adhesion receptor for connective tissue growth factor (CTGF) in rat activated hepatic stellate cells

Connective tissue growth factor (CTGF) is a cysteine-rich, extracellular matrix-associated heparin-binding protein implicated in a variety of fibrotic disorders. CTGF is initially synthesized as a mosaic protein containing four discrete structural modules (CTGF(1-4)) but this is susceptible to proteolytic cleavage yielding isoforms comprising modules 3 and 4 (CTGF(3-4)) or module 4 alone (CTGF(4)). In this study, we show that cultured rat hepatic stellate cells (HSCs) produce CTGF(1-4) and CTGF(3-4) following treatment with transforming growth factor-beta and that CTGF is a cell adhesion factor for activated HSCs. Low density lipoprotein receptor-associated protein (LRP) is a receptor for CTGF(1-4) or CTGF(3-4), but not CTGF(4), whereas cell surface heparan sulfate proteoglycans (HSPGs) are binding sites for all CTGF isoforms. Prior occupancy of LRP with other LRP ligands, receptor associated protein, anti-LRP, or a thrombospondin type I peptide (TEWSACSKTCG) resulted in a 50% decrease in the adhesion of activated HSCs to CTGF(1-4) or CTGF(3-4) whereas there was no effect on CTGF(4)-mediated adhesion. Co-incubation of CTGF with heparin or perturbation of cell surface HSPGs with heparinase or sodium chlorate completely blocked adhesion of activated HSCs to all CTGF isoforms. Freshly isolated HSCs demonstrated only weak binding to CTGF but strong binding to fibronectin. Thus HSC adhesion is at least partially promoted by CTGF through its binding to LRP, a process that is heparin-dependent. CTGF-LRP interactions are likely mediated by module 3 and CTGF-heparin interactions occur principally in module 4, although additional motifs may account for the heparin-dependency of LRP binding. These data show that LRP and HSPGs are utilized by HSCs for binding to CTGF and suggest that these cell surface molecules may be involved in mediating CTGF activity or adhesive signaling during the activation process.

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.