CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.     

Overexpressing STAMP2 attenuates adipose tissue angiogenesis and insulin resistance in diabetic ApoE−/−/LDLR−/− mouse via a PPARγ/CD36 pathway

The aim of this study was to investigate whether overexpression of STAMP2 improves insulin resistance by regulating angiogenesis in adipose tissues. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Histological and morphological analysis demonstrated that STAMP2 gene overexpression reduced adipocyte size, angiogenesis in epididymal and brown adipose tissues. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited after STAMP2 gene overexpression. The cellular effect of STAMP2 on angiogenesis was explored in human umbilical vein endothelial cells (HUVECs) model. Correlation of STAMP2 and angiogenesis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. In vitro, overexpression of STAMP2 significantly inhibited endothelial cell migration, tube formation. The effects of Ad‐STAMP2 transfection on HUVECs were abolished by treatment with PPARγ antagonist GW9662 (2.5 μM), and the roles of STAMP2 siRNA on HUVECs were also reversed by treatment with PPARγ agonist rosiglitazone (RSG) (0.1 mM). RT‐PCR indicated that STAMP2 could regulate levels of adhesion molecules, vascular endothelial growth factor A and CD36. The expression of PPARγ and CD36 was decreased when STAMP2 was inhibited by siRNA, while PPARγ and CD36 were highly expressed after overexpression of STAMP2. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via attenuating angiogenesis in epididymal and brown adipose tissues through the PPARγ/CD36 signalling pathway.     

Impaired tumor angiogenesis and VEG-Finduced pathway in endothelial CD146 knockout mice

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146^EC-KO) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146^EC-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatmentwas impairedinendothelial cells (ECs) of CD146^EC-KO mice. Mechanistic studies further confirmed that VEGFinduced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-κB activation were inhibited in these CD146-null ECs, whichmight present theunderlyingcause for theobserved inhibition of tumor angiogenesis in CD146^EC-KO mice. These results suggest thatCD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.     

Endothelial CD146 is required for in vitro tumor-induced angiogenesis: The role of a disulfide bond in signaling and dimerization

Tumor angiogenesis, induced by tumor-secreted pro-angiogenic factors, is an essential process for cancer development and metastasis. CD146 is identified as an endothelial cell adhesion molecule and implicated in blood vessel formation, however, its exact role in angiogenesis, particularly tumor angiogenesis, and its potential function of mediating downstream signaling are still unclear. In present study, we evidenced that silencing endogenous endothelial CD146 by RNAi significantly impaired hepatocarcinoma cell secretions-promoted tubular morphogenesis and -enhanced motility of endothelial cells. Biochemical studies revealed that CD146 was required for the activation of p38/IKK/NFκB signaling cascade and up-regulation of NFκB downstream pro-angiogenic genes, notably IL-8, ICAM-1 and MMP9, in response to tumor secretions. Interestingly, specific anti-CD146 mAb AA98, which bound a conformational epitope depending on C452–C499 disulfide bond, could abrogate NFκB activation and tumor angiogenesis, whereas another anti-CD146 mAb AA1 recognizing a linear epitope containing aa50–54 did not have such effects. Further structure–function analysis identified that C452–C499 disulfide bond within the fifth extracellular Ig domain was indispensible for CD146-mediated signaling and tube formation. Moreover, dimerization of CD146, which was enhanced by tumor secretions and suppressed by AA98 but not AA1, also relied on C452 and C499. Together, this study for the first time uncovered the pro-angiogenic role of CD146 and also pinpointed the key structural basis responsible for its signaling function and dimerization. These findings also suggested that CD146 might serve as not just a cell adhesion molecule but also a membrane signal receptor in tumor-induced angiogenesis.     

Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146

CD146, also known as melanoma cell adhesion molecule or MCAM, is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis. CD146 promotes tumor progression of many cancers including melanoma and prostate. Strikingly, its expression is frequently lost in breast carcinoma cells, and it may act as a suppressor of breast cancer progression. While upstream mechanisms regulating CD146 are well documented, our understanding of the downstream molecular events underlying its mode of action remains to be elucidated. This review aims to focus on the progress in understanding the signaling mechanisms and the functional relevance of CD146, a multifaceted molecule, in cancer with particular emphasis on its role in inhibiting breast cancer progression.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.     

CD146: the unveiling of a pro-angiogenic netrin receptor

Netrin-1, a classic neuronal guidance cue, can promote angiogenesis under certain developmental and pathological conditions, but key receptors on vascular endothelium have remained elusive. A recent study published in Cell Research by Tu et al. reveals that CD146, an endothelial receptor of the immunoglobulin superfamily, binds netrin-1 with high affinity and may play an important role in regulating angiogenesis.The anatomical similarity of the nervous and vascular systems has long been recognized, and it is now known that many of the neuronal guidance cues, such as netrins, slits, semaphorins and ephrins, also regulate angiogenesis — the sprouting of new blood vessels. During neurogenesis axonal growth cones play a key navigational role, sensing local cues in the environment and relaying signals that culminate in axon extension or retraction. Similarly, endothelial tip cells direct new blood vessel sprouts during angiogenesis. Netrins, derived from the Sanskrit word ''netr'' meaning ''one who guides'', were first shown to promote the outgrowth of commissural axons in 1994 and are comprised of a family that includes three secreted members (netrins 1, 3, and 4) and two GPI-linked members (netrins G1 and G2). Netrin-1, the prototypical netrin, attracts or repulses growth cones depending on receptor status. Beginning in 2004, several published studies have revealed that netrin-1 also has pro-angiogenic or anti-angiogenic activity, the balance depending on ligand concentration and presumably the receptors expressed on the endothelium lining the vessels, although the identity of the pro-angiogenic netrin receptor has remained elusive^1,2,3,4.To stimulate neuronal outgrowth, netrin-1 binds Deleted in Colorectal Cancer (DCC), a type I cell surface receptor of the immunoglobulin superfamily critical for neurogenesis (Figure 1). A related DCC family member, neogenin, also promotes axon guidance, whereas members of the uncoordinated 5 family (UNC5A-D) predominantly inhibit neurogenesis. Recent structural studies demonstrate that netrin-1 is able to crosslink its receptors, with DCC/DCC homodimers promoting chemoattraction and DCC/UNC5 heterodimers promoting chemorepulsion⁵. In the vascular system UNC5B is highly expressed in endothelial cells during embryonic development and pathological angiogenesis and, like the role of UNC5s in repression of neurogenesis, may mediate netrin-1 antiangiogenic activity³. However, it has been unclear how the pro-angiogenic activity of netrin-1 is mediated because DCC and neogenin are not detected in most endothelium. The previous lack of a clear angiogenesis-promoting netrin receptor on proliferating vascular endothelium led Mehlen''s group to propose that UNC5B could act as a dual purpose “dependence receptor” either stimulating or blocking endothelial apoptosis depending on ligand availability⁶.Open in a separate windowFigure 1Receptors of the immunoglobulin superfamily that bind Netrin-1. Schematic representation of netrin-1 and its receptors. Netrin-1 contains an N-terminal laminin type VI domain, a V region containing three laminin-like EGF domains, followed by a C-terminal netrin-like domain (NTR). The VI and V domains play an important role in receptor binding. The receptor binding sites for netrin-1 have been mapped to the regions shown in yellow. DSCAM, Down''s syndrome cell adhesion molecule; DCC, Deleted in Colorectal Cancer; DD, death domain; FNIII, fibronectin type III domain; Ig, immunoglobulin domain; TSP, thrombospondin type-1 domain; P1-3, conserved regions of the DCC cytoplasmic domain; UPA, UNC5, PIDD and ankyrin domain; ZU5, zona occludens 5 domain.In an elegant study recently published in Cell Research, Tao Tu and coworkers from Xiyun Yan''s laboratory provide compelling evidence that CD146, an endothelial cell surface receptor of the immunoglobulin superfamily (Figure 1) can stimulate angiogenesis in response to netrin-1⁷. CD146 was first described in 1987 based on its elevated expression in malignant versus normal melanocytes, and is also called Melanoma Cell Adhesion Molecule (MCAM or Mel-CAM). Soon after its discovery, CD146 was also found to be widely expressed on vascular endothelium and smooth muscle. CD146 is elevated in some tumor types, including melanoma, prostate and breast cancers, and recent studies demonstrate that it can promote epithelial to mesenchymal transition. In cultured endothelial cells CD146 has been shown to promote migration, proliferation and tube formation⁷. Although CD146-null mice are viable, impaired tumor angiogenesis and tumor growth in these mice suggests that this receptor may play a critical role in pathological angiogenesis⁸.Tu et al.⁷ show that netrin-1 directly binds CD146 with high affinity (Kd = 1.3 nM), somewhat higher than its affinity for the repulsive netrin receptor UNC5B (Kd = 5.1 nM). Through deletion analysis they show that the V domain of netrin-1, which contains three laminin-like EGF domains, plays an important role in binding. By analogy with data from the crystal structure of netrin-1 in complex with DCC⁵, regions in both the V domain and the N-terminal VI domain of netrin-1 may bind CD146, potentially explaining the residual binding the authors observe with their V domain mutant. Netrin-1 treatment of endothelial cells resulted in a biphasic response with low doses (50-200 ng/mL) inducing proliferation, migration and tube formation and high doses (1 000-2 000 ng/mL) inhibiting these effects. Knockdown of CD146 inhibited the pro-angiogenic phenotypes observed at low doses, whereas knockdown of UNC5B prevented the anti-angiogenic effects at high doses. Importantly, the authors demonstrate the importance of CD146 for angiogenesis in vivo using a CD146 neutralizing antibody and conditional knockout mice that lack CD146 in endothelium. Furthermore, the authors demonstrate that knockdown of either CD146 or netrin-1 leads to a similar defect in parachodal vessel formation during zebrafish development.Tu et al. find that addition of netrin-1 to endothelial cells is able to induce the formation of an unusually stable CD146 dimer that remains intact following exposure to the anionic detergent SDS. The authors also find that VEGFR2, the main VEGF receptor involved in VEGF-mediated angiogenesis, is rapidly phosphorylated upon addition of netrin-1 to serum-starved endothelial cells, which in turn activates signaling through ERK and p38 kinase, while CD146 knockdown prevents these effects. Netrin-1 was unable to bind VEGFR2 directly, and further studies are needed to determine exactly how these pathways intersect and the biological significance of these interactions in vivo. Like any landmark study, the work raises many new questions. For example, is the pro-angiogenic role of netrin-1 through CD146 completely dependent on the increased VEGFR2 signaling, or can netrin-1 also stimulate angiogenesis independent of VEGFR2? Is VEGF required for the rapid VEGFR2 phosphorylation in response to netrin-1 — for example, can netrin-1 mobilize matrix-bound VEGF or promote intracrine signaling, or does the purified recombinant netrin-1 contain trace amounts of VEGF? Can CD146 form heterodimers with other netrin receptors, like UNC5B? Can netrin signal through CD146 in non-endothelial cells, such as pericytes and smooth muscle cells that are also CD146 positive?Another important area concerns the role of netrin/CD146 signaling in pathological angiogenesis. While CD146 has been found to be widely expressed in adult normal vascular endothelium through gene expression profiling and using various anti-CD146 antibodies, for example the s-Endo-1 or the P1H12 antibodies, the AA98 mouse anti-human CD146 antibody developed in Yan''s laboratory appears to preferentially label CD146 on tumor-associated endothelium compared to normal endothelium^9,10. AA98, which binds the same region of CD146 as netrin-1, prevents dimerization of the receptor^7,11. If particular regions of CD146 are uniquely exposed during pathological angiogenesis, for example surfaces on the newly produced monomeric receptor that are normally masked upon ligand binding and dimerization, then this could potentially be exploited for specific targeting of tumor angiogenesis.Although the knockdown studies in zebrafish and cultured endothelial cells provide important evidence for the role of this receptor in angiogenesis, so far no clear developmental angiogenesis defect in CD146 and netrin-1 knockout mice has been reported. One possible explanation is ligand or receptor redundancy. In that regard, netrin-3 and netrin-4, which have not yet been tested for CD146 binding, have also been found to have angiogenic activity in vivo. Integrins, which are widely expressed on endothelium, have also been implicated as netrin-binding receptors¹². Another possibility is that the netrin/CD146 interactions are more important for angiogenesis associated with wound repair, ischemia and other pathological conditions than for development. If so, then targeting the netrin/CD146 axis could have important therapeutic implications for various angiogenesis-dependent diseases.     

Role of autophagy in angiogenesis in aortic endothelial cells

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.     

Visualization of CD146 dimerization and its regulation in living cells

Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescence-tagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.     

Proangiogenic function of CD40 ligand-CD40 interactions

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.     

Vascular endothelial growth factor receptor 2-based DNA immunization delays development of herpetic stromal keratitis by antiangiogenic effects

Stromal keratitis (SK) is an immunoinflammatory eye lesion caused by HSV-1 infection. One essential step in the pathogenesis is neovascularization of the normally avascular cornea, a process that involves the vascular endothelial growth factor (VEGF) family of proteins. In this report, we targeted the proliferating vascular endothelial cells expressing VEGFR-2 in the SK cornea by immunization with recombinant Salmonella typhimurium containing a plasmid encoding murine VEGFR-2. This form of DNA immunization resulted in diminished angiogenesis and delayed development of SK caused by HSV-1 infection and also reduced angiogenesis resulting from corneal implantation with rVEGF. CTL responses against endothelial cells expressing VEGFR-2 were evident in the VEGFR-2-immunized group and in vivo CD8+ T cell depletion resulted in the marked reduction of the antiangiogenic immune response. These results indicate a role for CD8+ T cells in the antiangiogenic effects. Our results may also imply that the anti-VEGFR-2 vaccination approach might prove useful to control pathological ocular angiogenesis and its consequences.     

Modulation of functional responses of endothelial cells linked to angiogenesis and inflammation by shear stress: differential effects of the mechanotransducer CD31

We investigated the roles of the "mechanotransducer" CD31 in the effects of shear stress on endothelial gene expression and functional responses relevant to angiogenesis and inflammation. Human or murine endothelial cells (hEC or mEC) were exposed to different levels of shear stress, while expression of CD31 was modified using siRNA in the hEC, or mEC from CD31(-/-) mice. Quantitation of expression of genes linked to inflammation or angiogenesis showed several were sensitive to shear. In a "wound" assay, exposure of endothelial cells (EC) to shear stress tended to align migration with the direction of flow and decrease the rate of closure compared to static cultures. When EC were cultured on filters, shear stress promoted migration away from the luminal surface. EC conditioned by shear stress recruited fewer flowing neutrophils, and showed reduced up-regulation of E-selectin after stimulation with tumor necrosis factor-α (TNF). Use of siRNA against CD31 in the hEC, or testing of mEC from mice lacking CD31, indicated that expression of CD31 was not required for the shear-induced modification of wound closure. However, shear modulation of response to TNF was less effective in the absence of CD31, while reduction of CD31 reduced shear-sensitivity in some genes (e.g., eNOS), but not others (e.g., KLF-2). Thus, CD31 played a role in shear-sensitivity of some genes and of neutrophil recruitment, but not in modulation of endothelial migration. Different mechanotransducers may mediate different functional effects of shear stress. Hence, identification of the specific pathways may provide targets for therapeutic manipulation of angiogenesis or inflammation.     

CD105 antagonizes the inhibitory signaling of transforming growth factor beta1 on human vascular endothelial cells.

CD105 (endoglin), a receptor for transforming growth factor beta (TGFbeta), is highly expressed in tissue-cultured, activated endothelial cells in vitro and in tissues undergoing angiogenesis in vivo. The absence of CD105 in knockout mice leads to their death from defective vascular development, but the role of CD105 in the modulation of angiogenesis has not been elucidated. TGFbeta1 is a well-recognized regulator of angiogenesis. Using an antisense approach, we have shown that inhibition of CD105 protein translation in cultured human endothelial cells enhances the ability of TGFbeta1 to suppress growth and migration in these cells. The ability of endothelial cells to form capillary tubes was evaluated by the use of a 3-dimensional collagen matrix system where TGFbeta1 not only reduced the length of capillary-like structures, but also caused massive mortality in CD105-deficient cells compared to control cultures. These results provide direct evidence that CD105 antagonizes the inhibitory effects of TGFbeta1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

The PPARδ ligand L-165041 inhibits VEGF-induced angiogenesis, but the antiangiogenic effect is not related to PPARδ

Peroxisome proliferator-activated receptor (PPAR)δ is known to be expressed ubiquitously and involved in lipid and glucose metabolism. Recent studies have demonstrated that PPARδ is expressed in endothelial cells (ECs) and plays a potential role in endothelial survival and proliferation. Although PPARα and PPARγ are well recognized to play anti-inflammatory, antiproliferative, and antiangiogenic roles in ECs, the general effect of PPARδ on angiogenesis in ECs remains unclear. Thus, we investigated the effect of the PPARδ ligand L-165041 on vascular EC proliferation and angiogenesis in vitro as well as in vivo. Our data show that L-165041 inhibited VEGF-induced cell proliferation and migration in human umbilical vein ECs (HUVECs). L-165041 also inhibited angiogenesis in the Matrigel plug assay and aortic ring assay. Flow cytometric analysis indicated that L-165041 reduced the number of ECs in the S phase and the expression levels of cell cycle regulatory proteins such as cyclin A, cyclin E, CDK2, and CDK4; phosphorylation of the retinoblastoma protein was suppressed by pretreatment with L-165041. We confirmed whether these antiangiogenic effects of L-165041 were PPARδ-dependent using GW501516 and PPARδ siRNA. GW501516 treatment did not inhibit VEGF-induced angiogenesis, and transfection of PPARδ siRNA did not reverse this antiangiogenic effect of L-165041, suggesting that the antiangiogenic effect of L-165041 on ECs is PPARδ-independent. Together, these data indicate that the PPARδ ligand L-165041 inhibits VEGF-stimulated angiogenesis by suppressing the cell cycle progression independently of PPARδ. This study highlights the therapeutic potential of L-165041 in the treatment of many disorders related to pathological angiogenesis.     

Outside-in signaling pathway linked to CD146 engagement in human endothelial cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.     

Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation

Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.