Determination of a Potential Role of the CCN Family of Growth Regulators in Connexin43 Transfected C6 Glioma Cells

Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.     

CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression

Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.     

Determination of a potential role of the CCN family of growth regulators in connexin43 transfected C6 glioma cells

Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.     

More insights into the CCN3/Connexin43 interaction complex and its role for signaling

Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc.     

The CCN family of genes: a perspective on CCN biology and therapeutic potential

The CCN family of genes currently comprises six secreted proteins (designated CCN1–6 after Cyr61/CCN1; ctgf/CCN2; Nov/CCN3; WISP1/CCN4; WISP2/CCN5, WISP3/CCN6) with a similar mosaic primary structure. It is now well accepted that CCN proteins are not growth factors but matricellular proteins that modify signaling of other molecules, in particular those associated with the extracellular matrix. CCN proteins are involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Since their first identification as matricellular factors, the CCN proteins now figure prominently in a variety of major diseases and are now considered valid candidates for therapeutic targeting. Dissection of the molecular mechanisms governing the biological properties of these proteins is being actively pursued by an expanding network of scientists around the globe who will meet this year at the 5th International Workshop on the CCN family of Genes, organized by the International CCN Society (http://ccnsociety.com), home for an international cadre of collaborators working in the CCN field.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_j and γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_j in Cx40/Cx40 pairs, but decreased g_j in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_j suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_j involved a decrease in both γ_j and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

A single bout of exercise with high mechanical loading induces the expression of Cyr61/CCN1 and CTGF/CCN2 in human skeletal muscle.

High mechanical loading was hypothesized to induce the expression of angiogenic and/or lymphangiogenic extracellular matrix (ECM) proteins in skeletal muscle. Eight men performed a strenuous exercise protocol, which consisted of 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion. Muscle biopsies were taken 30 min and 48 h postexercise from the vastus lateralis muscle and analyzed for the following parameters: mRNA and protein expression of ECM-associated CCN proteins [cysteine-rich angiogenic protein 61 (Cyr61)/CCN1, connective tissue growth factor (CTGF)/CCN2], and mRNA expression of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1alpha. The mRNA expression of Cyr61 and CTGF increased 30 min after the exercise (14- and 2.5-fold, respectively; P < 0.001). Cyr61 remained elevated 48 h postexercise (threefold; P < 0.05). The mRNA levels of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or hypoxia-inducible factor-1alpha did not change significantly at either 30 min or 48 h postexercise; however, the variation between subjects increased markedly in VEGF-A and VEGF-B mRNA. Cyr61 protein levels were higher at both 30 min and 48 h after the exercise compared with the control (P < 0.05). Cyr61 and CTGF proteins were localized to muscle fibers and the surrounding ECM by immunohistochemistry. Fast fibers stained more intensively than slow fibers. In conclusion, mechanical loading induces rapid expression of CCN proteins in human skeletal muscle. This may be one of the early mechanisms involved in skeletal muscle remodeling after exercise, since Cyr61 and CTGF regulate the expression of genes involved in angiogenesis and ECM remodeling.     

Connexin Over-Expression Differentially Suppresses Glioma Growth and Contributes to the Bystander Effect Following HSV-Thymidine Kinase Gene Therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

Connexin over-expression differentially suppresses glioma growth and contributes to the bystander effect following HSV-thymidine kinase gene therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.     

Selective effect of PDGF on connexin43 versus connexin40 comprised gap junction channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g(j) and gamma(j), respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g(j) in Cx40/Cx40 pairs, but decreased g(j) in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g(j) suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g(j) involved a decrease in both gamma(j) and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

CYR61 (CCN1) is essential for placental development and vascular integrity

CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.     

CCN1/Cyr61 is regulated by the canonical Wnt signal and plays an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_jand γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_jin Cx40/Cx40 pairs, but decreased g_jin the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.The CCN (CYR61/Connective Tissue Growth Factor/Nephroblastoma Overexpressed) family of multimodular proteins mediates diverse cellular functions, including cell adhesion, migration, and proliferation (1–3). Overexpression of CCN3, one of the founding members of the family, inhibits proliferation in most types of tumors such as glioblastoma and Ewing sarcoma (4, 5). Similarly, down-regulation of CCN3 has been suggested to promote melanoma progression (6). On the other hand, CCN3 can also promote migration in sarcoma and glioblastoma (4, 7), although a separate study shows that it decreases the invasion of melanoma (6). Therefore, in contrast to its role in growth suppression, the role of CCN3 signaling in cell motility is less clear.Most evidence suggests CCN3 mediates its effects by binding to the integrin proteins, such as the αVβ3 receptors (8, 9), and that CCN3 alters cell adhesion in an integrin-dependent fashion (4, 10). In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43³ (15, 16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.Gap junction proteins are best known for forming channels between cells, contributing to intercellular communication by allowing the exchange of small ions and molecules (17, 18). Consequently, attenuated intercellular communication has been implicated in promoting carcinogenesis (19, 20). Recent evidence has indicated that connexins can mediate channel-independent growth control through interaction of their C-terminal cytoplasmic tail with various intracellular signaling molecules (21–23). In addition, many Cx43-interacting proteins, including ZO-1 (zonula occludens-1) (24), Drebrin (25), and N-cadherin (26) associate with F-actin, thus placing Cx43 in close proximity to the actin cytoskeleton.In this study, we show for the first time that CCN3 reorganizes the actin cytoskeleton of the breast cancer cells MDA-MB-231 with the formation of multiple cell protrusions, possibly by activating the small GTPase Rac1. Our results also suggest an alternative route by which Cx43 may be functionally linked to actin cytoskeletal signaling via CCN3.     

Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.