Objective-Angiotensin II is recognized as one of the major mediators of cardiovascular pathology. Because connective tissue growth factor (CTGF) is involved in the pathophysiologic processes underlying fibrotic diseases, its regulation by angiotensin II was investigated. Methods and Results-In the 2-kidney, 1-clip model of renovascular hypertension, increased expression of CTGF was detectable in the hypertrophic left ventricle. ngiotensin II is the primary effector molecule of the renin-angiotensin system, and as such, it plays a central role in the regulation of arterial blood pressure and the etiology of hypertension. Apart from its pressure effects, angiotensin II exerts a variety of nonhemodynamic effects that are linked to cardiovascular and renal pathology. 1 By binding to and activation of angiotensin II type 1 (AT 1 ) receptors, angiotensin II mediates renal or cardiac fibrosis. 2 The fibrogenic effects of angiotensin II are often related to the cytokine transforming growth factor-␤ (TGF-␤). In vivo and in vitro data show the high capacity of TGF-␤ to induce the synthesis of extracellular matrix proteins and to prevent their degradation, thus leading to excess deposition of extracellular matrix and fibrosis (summarized in Eddy 3 ). It was shown in various cellular systems and animal studies that interference with TGF-␤ signaling reduced angiotensin II-mediated synthesis of matrix molecules (reviewed in Kim and Iwao 1 and Williams 4 ).Connective tissue growth factor (CTGF) is a member of the CYR61, CTGF, and NOV protein family, structurally characterized by their cysteine-rich sequence. 5 Functionally, CTGF was characterized as a downstream mediator of TGF-␤, mediating many but not all of the profibrotic actions of this cytokine. 6 TGF-␤ was shown to be the strongest inducer of CTGF in most cells, but other factors such as bioactive lipids have also been implicated in CTGF induction. 7,8 In injury-induced animal models of cardiac hypertrophy, increased levels of CTGF were observed. 9,10 In cyclosporin A-induced myocardial lesions, CTGF was reduced when the renin-angiotensin II system was blocked. 11 CTGF was similarly reduced in diabetic nephropathy when the animals were treated with an angiotensin II receptor blocker, 12 suggesting a link between angiotensin II and CTGF expression. These animal models, however, did not allow elucidation of the molecular mechanisms of this relation.Angiotensin II exerts its diverse biologic effects by binding to 2 types of receptors, AT 1 and AT 2 , both of which belong to the group of heptahelical transmembrane receptors. Depending on the cell type and stimulus, coupling to various different intracellular pathways mediates the physiologic and pathophysiologic actions of angiotensin II (summarized in Touyz and Schiffrin 13 ). Given the profibrotic effects of angiotensin II, we hypothesized that

Statins were identified as potent inhibitors of ccn2 (ctgf) mRNA expression in mesangial cells, and therefore might be of potential use to modulate the excessive ccn2 (ctgf) expression in mesangial cells related to glomerular fibrosis.

BackgroundHypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model.ResultsMicrovascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells.ConclusionsOur data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo. (Mol Cancer Res 2009;7(2):180–8)

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