The extracellular matrix (ECM) is a deformable dynamic structure that dictates the behavior, function and integrity of blood vessels. The composition, density, chemistry and architecture of major ...globular and fibrillar proteins of the matrisome regulate the mechanical properties of the vasculature (i.e., stiffness/compliance). ECM proteins are linked via integrins to a protein adhesome directly connected to the actin cytoskeleton and various downstream signaling pathways that enable the cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. However, cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, ischemia and aging compromise the mechanical balance of the vascular wall. Stiffening of large blood vessels is associated with well-known qualitative and quantitative changes of fibrillar and fibrous macromolecules of the vascular matrisome. However, the mechanical properties of the thin-walled microvasculature are essentially defined by components of the subendothelial matrix. Cellular communication network (CCN) 1 and 2 proteins (aka Cyr61 and CTGF, respectively) of the CCN protein family localize in and act on the pericellular matrix of microvessels and constitute primary candidate markers and regulators of microvascular compliance. CCN1 and CCN2 bind various integrin and non-integrin receptors and initiate signaling pathways that regulate connective tissue remodeling and response to injury, the associated mechanoresponse of vascular cells, and the subsequent inflammatory response. The CCN1 and CCN2 genes are themselves responsive to mechanical stimuli in vascular cells, wherein mechanotransduction signaling converges into the common Rho GTPase pathway, which promotes actomyosin-based contractility and cellular stiffening. However, CCN1 and CCN2 each exhibit unique functional attributes in these processes. A better understanding of their synergistic or antagonistic effects on the maintenance (or loss) of microvascular compliance in physiological and pathological situations will assist more broadly based studies of their functional properties and translational value.
The retina is a highly specialized tissue composed of a network of neurons, glia, and vascular and epithelial cells; all working together to coordinate and transduce visual signals to the brain. The ...retinal extracellular matrix (ECM) shapes the structural environment in the retina but also supplies resident cells with proper chemical and mechanical signals to regulate cell function and behavior and maintain tissue homeostasis. As such, the ECM affects virtually all aspects of retina development, function and pathology. ECM-derived regulatory cues influence intracellular signaling and cell function. Reversibly, changes in intracellular signaling programs result in alteration of the ECM and downstream ECM-mediated signaling network. Our functional studies in vitro, genetic studies in mice, and multi omics analyses have provided evidence that a subset of ECM proteins referred to as cellular communication network (CCN) affects several aspects of retinal neuronal and vascular development and function. Retinal progenitor, glia and vascular cells are major sources of CCN proteins particularly CCN1 and CCN2. We found that expression of the CCN1 and CCN2 genes is dependent on the activity of YAP, the core component of the hippo-YAP signaling pathway. Central to the Hippo pathway is a conserved cascade of inhibitory kinases that regulate the activity of YAP, the final transducer of this pathway. Reversibly, YAP expression and/or activity is dependent on CCN1 and CCN2 downstream signaling, which creates a positive or negative feedforward loop driving developmental processes (e.g., neurogenesis, gliogenesis, angiogenesis, barriergenesis) and, when dysregulated, disease progression in a range of retinal neurovascular disorders. Here we describe mechanistic hints involving the CCN–Hippo–YAP regulatory axis in retina development and function. This regulatory pathway represents an opportunity for targeted therapies in neurovascular and neurodegenerative diseases.
Graphical abstract
The CCN-YAP regulatory loop in development and pathology
Cellular communication network 2 (CCN2), also known as connective tissue growth factor (CTGF) regulates diverse cellular processes, some at odds with others, including adhesion, proliferation, ...apoptosis, and extracellular matrix (ECM) protein synthesis. Although a cause-and-effect relationship between CCN2/CTGF expression and local fibrotic reactions has initially been established, CCN2/CTGF manifests cell-, tissue-, and context-specific functions and differentially affects developmental and pathological processes ranging from progenitor cell fate decisions and angiogenesis to inflammation and tumorigenesis. CCN2/CTGF multimodular structure, binding to and activation or inhibition of multiple cell surface receptors, growth factors and ECM proteins, and susceptibility for proteolytic cleavage highlight the complexity to CCN2/CTGF biochemical attributes. CCN2/CTGF expression and dosage in the local environment affects a defined community of its interacting partners, and this results in sequestration of growth factors, interference with or potentiation of ligand-receptor binding, cellular internalization of CCN2/CTGF, inhibition or activation of proteases, and generation of CCN2/CTGF degradome products that add molecular diversity and expand the repertoire of functional modules in the cells and their microenvironment. Through these interactions, different intracellular signals and cellular responses are elicited culminating into physiological or pathological reactions. Thus, the CCN2/CTGF interactome is a defining factor of its tissue- and context-specific effects. Mapping of new CCN2/CTGF binding partners might shed light on yet unknown roles of CCN2/CTGF and provide a solid basis for tissue-specific targeting this molecule or its interacting partners in a therapeutic context.
The extracellular matrix (ECM) is critical in all aspects of vascular development and health: supporting cell anchorage, providing structure, organization and mechanical stability, and serving as a ...sink for growth factors and sustained survival signals. Abnormal changes in ECM protein expression, organization, and/or properties, and the ensuing changes in vascular compliance affect vasodilator responses, microvascular pressure transmission, and collateral perfusion. The changes in microvascular compliance are independent factors initiating, driving, and/or exacerbating a plethora of microvascular diseases of the eye including diabetic retinopathy (DR) and vitreoretinopathy, retinopathy of prematurity (ROP), wet age-related macular degeneration (AMD), and neovascular glaucoma. Congruently, one of the major challenges with most vascular regenerative therapies utilizing localized growth factor, endothelial progenitor, or genetically engineered cell delivery, is the regeneration of blood vessels with physiological compliance properties. Interestingly, vascular cells sense physical forces, including the stiffness of their ECM, through mechanosensitive integrins, their associated proteins and the actomyosin cytoskeleton, which generates biochemical signals that culminate in a rapid expression of matricellular proteins such as cellular communication network 1 (CCN1) and CCN2 (aka connective tissue growth factor or CTGF). Loss or gain of function of these proteins alters genetic programs of cell growth, ECM biosynthesis, and intercellular signaling, that culminate in changes in cell behavior, polarization, and barrier function. In particular, the function of the matricellular protein CCN2/CTGF is critical during retinal vessel development and regeneration wherein new blood vessels form and invest a preformed avascular neural retina following putative gradients of matrix stiffness. These observations underscore the need for further in-depth characterization of the ECM-derived cues that dictate structural and functional properties of the microvasculature, along with the development of new therapeutic strategies addressing the ECM-dependent regulation of pathophysiological stiffening of blood vessels in ischemic retinopathies.
The vasculature forms a highly branched network investing every organ of vertebrate organisms. The retinal circulation, in particular, is supported by a central retinal artery branching into ...superficial arteries, which dive into the retina to form a dense network of capillaries in the deeper retinal layers. The function of the retina is highly dependent on the integrity and proper functioning of its vascular network and numerous ocular diseases including diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity are caused by vascular abnormalities culminating in total and sometimes irreversible loss of vision. CCN1 and CCN2 are inducible extracellular matrix (ECM) proteins which play a major role in normal and aberrant formation of blood vessels as their expression is associated with developmental and pathological angiogenesis. Both CCN1 and CCN2 achieve disparate cell-type and context-dependent activities through modulation of the angiogenic and synthetic phenotype of vascular and mesenchymal cells respectively. At the molecular level, CCN1 and CCN2 may control capillary growth and vascular cell differentiation by altering the composition or function of the constitutive ECM proteins, potentiating or interfering with the activity of various ligands and/or their receptors, physically interfering with the ECM-cell surface interconnections, and/or reprogramming gene expression driving cells toward new phenotypes. As such, these proteins emerged as important prognostic markers and potential therapeutic targets in neovascular and fibrovascular diseases of the eye. The purpose of this review is to highlight our current knowledge and understanding of the most recent data linking CCN1 and CCN2 signaling to ocular neovascularization bolstering the potential value of targeting these proteins in a therapeutic context.
CYR61-CTGF-NOV (CCN)1 is a dynamically expressed extracellular matrix (ECM) protein with critical functions in cardiovascular development and tissue repair. Angiogenic endothelial cells (ECs) are a ...major cellular source of CCN1 which, once secreted, associates with the ECM and the cell surface and tightly controls the bidirectional flow of information between cells and the surrounding matrix. Endothelium-specific CCN1 deletion in mice using a cre/lox strategy induces EC hyperplasia and causes blood vessels to coalesce into large flat hyperplastic sinuses with no distinctive hierarchical organization. This is consistent with the role of CCN1 as a negative feedback regulator of vascular endothelial growth factor (VEGF) receptor activation. In the mouse model of oxygen-induced retinopathy (OIR), pericytes become the predominant CCN1 producing cells. Pericyte-specific deletion of CCN1 significantly decreases pathological retinal neovascularization following OIR. CCN1 induces the expression of the non-canonical Wnt5a in pericyte but not in EC cultures. In turn, exogenous Wnt5a inhibits CCN1 gene expression, induces EC proliferation and increases hypersprouting. Concordantly, treatment of mice with TNP470, a non-canonical Wnt5a inhibitor, reestablishes endothelial expression of CCN1 and significantly decreases pathological neovascular growth in OIR. Our data highlight the significance of CCN1-EC and CCN1-pericyte communication signals in driving physiological and pathological angiogenesis.
In celebration of the twentieth anniversary of the inception of the CCN society, and of the first post-Covid-19 live meeting, the executive board of the ICCNS had chosen Nice as the venue for the ...11th International workshop on the CCN family of genes. On this occasion participation in the meeting was extended to colleagues from other cell signaling fields who were invited to present both an overview of their work and the future directions of their laboratory. Also, for the first time, the members of the JCCS Editorial Board were invited to participate in a JCCS special session during which all aspects of the journal « life » were addressed and opened to free critical discussion. The scientific presentations and the discussions that followed showed once more that an expansion of the session topics was beneficial to the quality of the meeting and confirmed that the ARBIOCOM project discussed last April in Nice was now on track to be launched in 2023. The participants unanimously welcomed Professor Attramadal’s proposition to organize the 2024, 12th International CCN workshop in Oslo, Norway.
Connective tissue growth factor (CTGF) or cellular communication network 2 (CCN2) is a matricellular protein essential for normal embryonic development and tissue repair. CTGF exhibits cell- and ...context-dependent activities, but CTGF function in vascular development and barrier function is unknown. We show that endothelial cells (ECs) are one of the major cellular sources of CTGF in the developing and adult retinal vasculature. Mice lacking CTGF expression either globally or specifically in ECs exhibit impaired vascular cell growth and morphogenesis and blood barrier breakdown. The global molecular signature of CTGF includes cytoskeletal and extracellular matrix protein, growth factor, and transcriptional co-regulator genes such as yes-associated protein (YAP). YAP, itself a transcriptional activator of CTGF, mediates several CTGF-controlled angiogenic and barriergenic transcriptional programs. Re-expression of YAP rescues, at least partially, angiogenesis and barriergenesis in CTGF mutant mouse retinas. Thus, the CTGF-YAP regulatory loop is integral to retinal vascular development and barrier function.
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•CTGF has a strong and persistent expression in the retinal vasculature•Mice lacking CTGF exhibit defects in angiogenesis and blood barrier integrity•CTGF-targeted genes include matrix, growth, and transcription co-factors like YAP•YAP re-expression partly rescues angiogenic and barriergenic defects of CTGF loss
Molecular Biology; Cell Biology; Transcriptomics
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.
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.
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