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.     

Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.     

Cloning, expression and purification of the CCN family of proteins in Escherichia coli

The CCN proteins are extracellular matrix associated proteins involved in critical cell activities and several aggressive forms of cancer. The proteins share a modular structure of four discrete domains and 38 conserved cysteine residues. The absence of any structural information of these proteins has resulted in a need for the ability to produce substantial amounts of pure CCN protein. Through bacterial expression and inclusion body based purification, pure recombinant CCN proteins have been produced for use in structural and biochemical experiments.     

Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy

Abstract

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.     

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.     

Domain-and species-specific monoclonal antibodies recognize the Von Willebrand Factor-C domain of CCN5

The CCN family of proteins typically consists of four distinct peptide domains: an insulin-like growth factor binding protein-type (IGFBP) domain, a Von Willebrand Factor C (VWC) domain, a thrombospondin type 1 repeat (TSP1) domain, and a carboxy-terminal (CT) domain. The six family members participate in many processes, including proliferation, motility, cell-matrix signaling, angiogenesis, and wound healing. Accumulating evidence suggests that truncated and alternatively spliced isoforms are responsible for the diverse functions of CCN proteins in both normal and pathophysiologic states. Analysis of the properties and functions of individual CCN domains further corroborates this idea. CCN5 is unique among the CCN family members because it lacks the CT-domain. To dissect the domain functions of CCN5, we are developing domain-specific mouse monoclonal antibodies. Monoclonal antibodies have the advantages of great specificity, reproducibility, and ease of long-term storage and production. In this communication, we injected mixtures of GST-fused rat CCN5 domains into mice to generate monoclonal antibodies. To identify the domains recognized by the antibodies, we constructed serial expression plasmids that express dual-tagged rat CCN5 domains. All of the monoclonal antibodies generated to date recognize the VWC domain, indicating it is the most highly immunogenic of the CCN5 domains. We characterized one particular clone, 22H10, and found that it recognizes mouse and rat CCN5, but not human recombinant CCN5. Purified 22H10 was successfully applied in Western Blot analysis, immunofluorescence of cultured cells and tissues, and immunoprecipitation, indicating that it will be a useful tool for domain analysis and studies of mouse-human tumor models.     

Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis

Connective tissue growth factor (CTGF, CCN2) is a secreted protein with major roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. It is a member of the CCN family of immediate-early gene products which are characterised by four discrete protein modules in which reside growth factor binding domains, functional motifs for integrin recognition, heparin and proteoglycan binding, and dimerization motifs. A primary function of CTGF is to modulate and coordinate signaling responses involving cell surface proteoglycans, key components of the extracellular matrix, and growth factors. Integration of these molecular cues regulates growth factor and receptor interactions, cell motility and mesenchymal cell activation and differentiation in tissue remodelling. Abnormal amplification of CTGF dependent signals results in a failure to terminate tissue repair, leading pathological scarring in conditions such as fibrosis and cancer.     

Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.     

Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes

CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.     

Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice

CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.     

The CCN2/CTGF interactome: an approach to understanding the versatility of CCN2/CTGF molecular activities

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.     

Trial by CCN2: a standardized test for fibroproliferative disease?

A major issue concerning clinical trials is the availability of standardized assays to evaluate drug efficacy. Ideally, such assays should test the effect of a putative drug on the expression of a biomarker in biological fluids. In a recent study by Kuiper et al. (PLOS One, 3(7): e2675). The relative levels of vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor [CTGF]) were examined in proliferative diabetic retinopathy (PDR). This paper is the subject of this commentary.     

IGFBP7 contributes to epithelial-mesenchymal transition of HPAEpiC cells in response to radiation

Radiation-induced lung injury (RILI) frequently occurs in patients with thoracic malignancies. In response to radiation, alveolar epithelial cells (AEC) undergo epithelial-mesenchymal transition (EMT) and contribute to the pathogenesis of RILI. Insulin-like growth factor binding protein 7 (IGFBP7) is reported as a downstream mediator of transforming growth factor-β1 (TGF-β1) pathway, which plays a crucial role in radiation-induced EMT. In the present study, the levels of IGFBP7 and TGF-β1 were simultaneously increased in experimental RILI models and radiation-treated AEC (human pulmonary alveolar epithelial cells [HPAEpic]). The expression of IGFBP7 in radiation-treated HPAEpic cells was obviously inhibited by the specific inhibitor of TGF-β receptor antagonist SB431542 and TGF-β1 neutralizing antibody, and time-dependently enhanced by TGF-β1 treatment. Moreover, IGFBP7 knockdown significantly attenuated the effects of radiation on morphology change, cell migration, expression of EMT-related markers (E-cadherin, α-SMA, and Vimentin), and phosphorylation of extracellular-signal-regulated kinase (ERK). The effects of IGFBP7 overexpression on the expression of EMT-related markers were partially reversed by the ERK inhibitor PD98059. In conclusion, IGFBP7, was enhanced by TGF-β1, may be involved in radiation-induced EMT of AEC via the ERK signaling pathway, thus contributing to the pathogenesis of RILI.     

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.