Brahim Chaqour

Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins

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.

Mechanical Regulation of the Proangiogenic Factor CCN1/CYR61 Gene Requires the Combined Activities of MRTF-A and CREB-binding Protein Histone Acetyltransferase

Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A−/− cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF·MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.

Forkhead Transcription Factor FOXO3a Is a Negative Regulator of Angiogenic Immediate Early Gene CYR61, Leading to Inhibition of Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia

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 (0.06±0.02 versus 0.20±0.07 mm2, P<0.01). 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.

Mechanical Stretch Modulates the Promoter Activity of the Profibrotic Factor CCN2 through Increased Actin Polymerization and NF-κB Activation

The connective tissue growth factor known as CCN2 is an inducible, profibrotic molecule that becomes aberrantly expressed in mechanical overload-bearing tissues. In this study, we found that CCN2 gene expression is rapidly induced in cyclically stretched bladder smooth muscle cells (SMCs) in vitro and in the detrusor muscle of a mechanically overloaded bladder in a rat model of experimental urethral obstruction. The activity of CCN2 promoter constructs, transiently transfected into cultured SMCs, was increased (up to 6-fold) by continuous cyclic stretching. Molecular analyses of the CCN2 promoter by serial construct deletions, cis-element mutagenesis, and electrophoretic mobility shift assays revealed that a highly conserved NF-κB binding site located within the CCN2 proximal promoter region is responsible for the activation of the promoter by stretch. Chromatin immunoprecipitation assays showed that NF-κB binds to the endogenous CCN2 promoter in both stretched cells and mechanically overloaded bladder tissues. Furthermore, stretch-dependent CCN2 promoter activity was significantly reduced upon inhibition of either phosphatidylinositol 3-kinase, p38 stress-activated kinase, or RhoA GTPase and was completely abolished upon inhibition of actin polymerization. Concordantly, actin polymerization was increased in either mechanically stretched cells or overloaded bladder tissues. Incubation of cultured SMCs with a cell-penetrating peptide containing the N-terminal sequence, Ac-EEED, of smooth muscle α-actin, altered both actin cytoskeleton organization and stretch-mediated nuclear relocation of NF-κB, and subsequently, it reduced CCN2 promoter activity. Thus, mechanical stretch-induced changes in actin dynamics mediate NF-κB activation and induce CCN2 gene expression, which probably initiates the fibrotic reactions observed in mechanical overloadassociated pathologies.

Mechanical strain activates a program of genes functionally involved in paracrine signaling of angiogenesis

Studies were performed to examine the extent to which mechanical stimuli mediate control of angiogenesis in bladder cells both in vitro and in vivo. Differential gene expression between control nonstretched and cyclically stretched bladder smooth muscle cells was assessed using oligonucleotide microarrays and pathway analysis by the web tool Fast Assignment and Transference of Information (FatiGO). Data showed that a substantial proportion (33 of 86) of mechanically responsive genes were angiogenesis-related and include cytokines, growth-related factors, adhesion proteins, and matricellular, signal transduction, extracellular matrix (ECM), and inflammatory molecules. Integrative knowledge of protein-protein interactions revealed that 12 mechano-sensitive gene-encoded proteins have interacting partner(s) in the vascular system confirming their potential role in paracrine regulation of angiogenesis. Angiogenic genes include matricellular proteins such as Cyr61/CCN1, CTGF/CCN2 and tenascin C, components of the VEGF and IGF systems, ECM proteins such as type I collagen and proteoglycans, and matrix metalloproteinases. In an in vivo model of bladder overdistension, 5 of 11 mechano-responsive angiogenic genes, independently tested by real-time PCR, were upregulated as a result of pressure overload including Cyr61/CCN1, CTGF/CCN2, MCP-1, VEGF-A, MMP-1, and midkine. Meanwhile, the molecular anatomy of angiogenic gene promoters reveals the presence of GA box-binding for the myc-associated zinc finger protein, MAZ, often found adjacent to binding sites for mechano-responsive transcription factors (e.g., NF-kappaB), suggesting that the coordinated activity of these factors may induce selective angiogenic gene transcription. These data suggest that mechanical control of angiogenic genes is an integral part of the adaptive and plasticity responses to mechanical overload.

Cysteine-Rich Protein 61 and Connective Tissue Growth Factor Induce Deadhesion and Anoikis of Retinal Pericytes

Loss of retinal pericytes is one of the distinctive features of diabetic retinopathy (DR), which is characterized by retinal capillary obliteration. The matricellular proteins, cysteine-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF), are aberrantly expressed in the retinal vasculature from the early stages of DR, but their effects on retinal pericytes are unknown. We show herein that rat retinal pericytes (RRPs) exposed to advanced glycosylation-end products, an important injurious stimulus of diabetes, express increased levels of both Cyr61 and CTGF, and concomitantly undergo anoikis, a form of apoptosis by loss of cell-matrix interactions. Adenovirus-mediated expression of Cyr61 and/or CTGF conferred an anoikis-prone phenotype to rat retinal pericytes, including decreased phosphotyrosine protein levels at focal adhesion points and formation of cortical actin rings. When used as substrates for pericyte attachment and compared with other matrix proteins (e.g. type IV collagen), recombinant Cyr61 and CTGF proteins exhibited antiadhesive and apoptogenic activities. Phosphatase inhibitors reversed these effects, suggesting that Cyr61 and CTGF promote dephosphorylation events. Furthermore, Cyr61- and CTGF-induced apoptosis was mediated through the intrinsic pathway and involved the expression of genes that have been functionally grouped as p53 target genes. Expression of the matrix metalloproteinase-2 gene, a known target of p53, was increased in pericytes overexpressing either Cyr61 or CTGF. Inhibition of matrix metalloproteinase-2 had, at least in part, a protective effect against Cyr61- and CTGF-induced apoptosis. Taken together, these findings support the involvement of Cyr61 and CTGF in pericyte detachment and anoikis, implicating these proteins in the pathogenesis of DR.

Connective tissue growth factor regulates retinal neovascularization through p53 protein-dependent transactivation of the matrix metalloproteinase (MMP)-2 gene.

Background: The role of connective tissue growth factor (CTGF/CCN2) in pathological angiogenesis in the retina is unknown. Results: CTGF/CCN2 stimulates retinal neovascularization through transactivation of p53 target genes such as matrix metalloproteinase (MMP)-2. Conclusion: CTGF/CCN2 effects on abnormal vessel formation in the retina are mediated by p53 and MMP-2. Significance: CTGF/CCN2 and its downstream effectors are potential targets in the development of new antiangiogenic treatments. Pathological angiogenesis in the retina is driven by dysregulation of hypoxia-driven stimuli that coordinate physiological vessel growth. How the various components of the neovascularization signaling network are integrated to yield pathological changes has not been defined. Connective tissue growth factor (CTGF/CCN2) is an inducible matricellular protein that plays a major role in fibroproliferative disorders. Here, we show that CTGF/CCN2 was dynamically expressed in the developing murine retinal vasculature and was abnormally increased and localized within neovascular tufts in the mouse eye with oxygen-induced retinopathy. Consistent with its propitious vascular localization, ectopic expression of the CTGF/CCN2 gene further accelerated neovascularization, whereas lentivirus-mediated loss-of-function or -expression of CTGF/CCN2 harnessed ischemia-induced neovessel outgrowth in oxygen-induced retinopathy mice. The neovascular effects of CTGF/CCN2 were mediated, at least in part, through increased expression and activity of matrix metalloproteinase (MMP)-2, which drives vascular remodeling through degradation of matrix and non matrix proteins, migration and invasion of endothelial cells, and formation of new vascular patterns. In cultured cells, CTGF/CCN2 activated the MMP-2 promoter through increased expression and tethering of the p53 transcription factor to a highly conserved p53-binding sequence within the MMP-2 promoter. Concordantly, the neovascular effects of CTGF/CCN2 were suppressed by p53 inhibition that culminated in reduced enrichment of the MMP-2 promoter with p53 and decreased MMP-2 gene expression. Our data identified new gene targets and downstream effectors of CTGF/CCN2 and provided the rational basis for targeting the p53 pathway to curtail the effects of CTGF/CCN2 on neovessel formation associated with ischemic retinopathy.

The Matricellular Protein Cysteine-rich Protein 61 (CCN1/Cyr61) Enhances Physiological Adaptation of Retinal Vessels and Reduces Pathological Neovascularization Associated with Ischemic Retinopathy

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.

Positive Transcription Elongation Factor b Activity in Compensatory Myocardial Hypertrophy is Regulated by Cardiac Lineage Protein-1

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb complex, composed of cyclin T and cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl-terminal domain of RNA polymerase (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We also generated bigenic mice (MHC–cyclin T1/CLP-1+/−) by crossing MHC–cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.

The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses. Summary: Mice lacking endothelial CCN1 show severe defects in retinal vessel network formation and barrier integrity, associated with endothelial cell hyperproliferation.