Connective tissue growth factor (CTGF) is a 36-to 38-kDa peptide that is selectively induced by transforming growth factor-beta (TGF-beta) in fibroblastic cell types. We compared the biologic activities of CTGF with TGF-beta on fibroblasts in culture and in animal models of fibroplasia. CTGF was active as a mitogen in monolayer cultures of normal rat kidney fibroblasts. CTGF did not stimulate anchorage-independent growth of NRK fibroblasts, however, or inhibit the growth of mink lung epithelial cells, distinguishing CTGF's growth-regulatory activities from those of TGF-beta. In NRK fibroblasts, both TGF-beta and CTGF significantly increased the transcripts encoding alpha 1 type I collagen, alpha 5 integrin, and fibronectin. Stimulation of type I collagen and fibronectin protein synthesis by TGF-beta and CTGF was confirmed by pulse labeling of cells with [35S]methionine. Subcutaneous injection of TGF-beta and CTGF into neonatal NIH Swiss mice resulted in a large stimulation of granulation tissue and fibrosis at the site of injection. In situ hybridization studies revealed that TGF-beta injection induced high levels of CTGF mRNA in the dermal fibroblasts at the injection site, demonstrating that TGF-beta can induce the expression of CTGF in connective tissue cells in vivo. No CTGF transcripts were detected in the epidermal cells in either control or TGF-beta-injected skin or in fibroblasts in control (saline-injected) skin. These results demonstrate that, like TGF-beta, CTGF can induce connective tissue cell proliferation and extracellular matrix synthesis.

Summary Background A number of adhesion-mediated signaling pathways and cell cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we have used hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. Results We find that physiological tissue stiffness acts as a cell cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryo fibroblasts, and osteoblasts shows that cell cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as ECM/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediately-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in the mouse shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. Conclusion Matrix remodeling associated with pathogenesis is, in itself, a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.

Summary Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing HDL maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop which increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE-null mice, this stiffening can be reduced by administration of the lysyl oxidase inhibitor, BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics, and this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.

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Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50-70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the "synthetic" state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms.

CTGF is a 38 kDa cysteine-rich peptide whose synthesis and secretion are selectively induced by transforming growth factor beta (TGF-beta) in connective tissue cells. We have investigated the signaling pathways controlling the TGF-beta induction of connective tissue growth factor (CTGF) gene expression. Our studies indicate that inhibitors of tyrosine kinases and protein kinase C do not block the signaling pathway used by TGF-beta to induce CTGF gene expression. In contrast, elevation of cAMP levels within the target cells by a variety of methods blocked the induction of CTGF by TGF-beta. Furthermore, agents that elevate cAMP blocked the induction of anchorage-independent growth (AIG) by TGF-beta. Inhibition of AIG could be overcome by the addition of CTGF, indicating that it was not a general inhibition of growth but a selective inhibition of CTGF synthesis that is responsible for the inhibition of TGF-beta-induced AIG by cAMP. Kinetic studies of the induction of DNA synthesis by CTGF in cells arrested by cAMP indicate that the block occurs in very late G1. These and other studies in monolayer cultures suggest that the CTGF restriction point in the cell cycle is distinct from the adhesion-dependent arrest point.

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Background Microsomal (m) prostaglandin (PG) E2 synthase (S)-1 catalyzes the formation of PGE2 from PGH2, a cyclooxygenase (COX) product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than COX-2 selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation. The role of mPGES-1 in the response to vascular injury is unknown. Methods and Results Mice were subjected to wire injury of the femoral artery. Both neointimal area and vascular stenosis were reduced significantly four weeks after injury in mPGES-1 knock out (KO) mice compared to wild type (WT) controls (65.6±5.7 vs 37.7±5.1×103 pixel area and 70.5±13.4% vs 47.7±17.4%, respectively; p < 0.01). Induction of tenascin C (TN-C) after injury, a pro-proliferative and promigratory extracellular matrix protein, was attenuated in the KOs. Consistent with in vivo rediversion of PG biosynthesis, mPGES-1 deleted vascular smooth muscle cells (VSMC) generated less PGE2, but more PGI2 and expressed reduced TN-C when compared with WT cells. Both suppression of PGE2 and augmentation of PGI2 attenuate TN-C expression, VSMC proliferation and migration in vitro. Conclusions Deletion of mPGES-1 in mice attenuates neointimal hyperplasia after vascular injury, in part by regulating TN-C expression. This raises for consideration the therapeutic potential of mPGES-1 inhibitors as adjuvant therapy for percutaneous coronary intervention.