Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces. These forces are significantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic overload. Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems. Two immediate early gene‐encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine‐rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2), are highly expressed in several mechanical stress‐related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton. Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events. In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibrotic diseases. At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated by various components of the signaling machinery, i.e., small Rho GTPases, mitogen‐activated protein kinases, and actin binding proteins. This review discusses the mechanical regulation of the Cyr61 and CTGF in various tissues and cell culture models with a special attention to the cytoskeletally based mechanisms involved in such regulation.

The cellular components of the cardiovascular, digestive, and urinary systems elicit adaptive responses to mechanical/pressure overload acutely by retooling their cytoskeletal structures (i.e. increasing actin polymerization rate and contractile protein levels) and chronically by remodeling their extracellular environment. These compensatory responses are associated with the activation of angiogenesis to meet increased metabolic demands and improve tissue perfusion (1, 2). In the absence of angiogenesis, hypertrophic growth increases diffusion distance around microvessels resulting in reduced oxygen supply and hypoxia, altered muscle contractility, and organ failure. Clearly, an unsatisfactory/ insufficient vascularization is an important restraint on the adaptive capabilities of mechanically overloaded tissues.Neovascularization in mechanically challenged smooth muscle-rich organs in particular is promoted by various mechanoresponsive angiogenic factors including CCN1, formerly known as cysteine-rich protein 61 (Cyr61), a functionally multifaceted matricellular protein that appears in the extracellular environment particularly during development and pathological states (3, 4). The CCN1 protein acts either independently or in concert with vascular endothelial growth factor to drive sprouting and branching of new blood vessels and provides protection against oxidative stress (5, 6). Essentially, the CCN1 protein enhances angiogenesis by providing structural integrity to blood vessels, supplying necessary growth factors for endothelial and perivascular cells, and modulating extracellular matrix synthesis and degradation. Targeted disruption of the CCN1 gene leads to early or perinatal lethality in mice due to impaired vessel formation and/or branching (7). The CCN1 proangiogenic properties have further been demonstrated in different models of angiogenesis including rabbit ischemic hind limb and rat cornea models in which CCN1 improves angiogenesis and collateral blood flow to an even larger extent than vascular endothelial growth factor (8, 9).Mechanical strain typified by tension, stretch, shear, and pressure largely controls the expression of the CCN1 gene, although little is known about the molecular mechanisms involved (10). CCN1 was induced in mechanically stimulated myocardial fibroblasts and in smooth muscle cells (SMCs) 2 2 h

Cysteine-rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/ CCN1 gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1-phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-1, were responsible for the promoter activity in S1P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.

Abstract-Cysteine-rich angiogenic protein 61 (CYR61, CCN1) is an immediate early gene expressed in vascular smooth muscle cells (VSMCs) on growth factor stimulation, and its expression has been suggested to be associated with postangioplasty restenosis. The forkhead transcription factors are reported to play various roles in cellular proliferation, apoptosis, and even adaptation to cellular stress. We hypothesized that the forkhead transcription factor FOXO3a may regulate CYR61 expression in VSMCs and investigated the CYR61-modulating effect of FOXO3a in the process of vascular response to vasoactive signals and vascular injury. To evaluate the effect of FOXO3a on CYR61 expression, rat VSMCs were infected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). Constitutively active FOXO3a gene transduction suppressed CYR61 expression. Luciferase assay with the deletion constructs of the forkhead factor binding motif in CYR61 promoter region, which resulted in a significant decrease in luciferase expression compared with the intact construct, and chromatin immunoprecipitation analysis confirmed transcriptional regulation of CYR61 by FOXO3a. Serum and angiotensin II rapidly induced CYR61 expression, which was significantly reduced by Ad-TM-FOXO3a. Reduction of VSMC proliferation and migration associated with FOXO3a activation was significantly reversed by cotransfection of adenoviral vector expressing CYR61, whereas apoptosis induction by FOXO3a was not influenced. In a rat balloon carotid arterial injury model, CYR61 was rapidly induced in VSMCs in the early stage of injury and remained elevated until 14 days, which was suppressed by Ad-TM-FOXO3a transfection. After 14 days, there was a significant reduction in neointima by FOXO3a transduction compared with the control group (0.06Ϯ0.02 versus 0.20Ϯ0.07 mm 2 , PϽ0.01). Such reduction of neointimal hyperplasia by Ad-TM-FOXO3a was reversed by CYR61 replenishment. These data suggest that FOXO3a is a negative transcription factor of CYR61 and that suppression of CYR61 is among several mechanisms by which FOXO3a inhibits VSMC proliferation and neointimal hyperplasia. (Circ Res. 2007;100:372-380.)