Angiogenic properties of the chemokine RANTES/CCL5

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.     

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.     

Ezrin regulates synovial angiogenesis in rheumatoid arthritis through YAP and Akt signalling

This study aimed to investigate the role and regulatory mechanisms of Ezrin in synovial vessels in rheumatoid arthritis (RA). Synovial tissues were obtained from people with osteoarthritis people and patients with RA patients. We also used an antigen-induced arthritis (AIA) mice model by using Freund's adjuvant injections. Ezrin expression was analysed by immunofluorescence and immunohistochemical staining in synovial vessels of patients with RA and AIA mice. We investigated the role of Ezrin on vascular endothelial cells and its regulatory mechanism in vivo and in vitro by adenoviral transfection technology. Our results suggest a role for the Ezrin protein in proliferation, migration and angiogenesis of vascular endothelial cells in RA. We also demonstrate that Ezrin plays an important role in vascular endothelial cell migration and tube formation through regulation of the Hippo-yes-associated protein 1 (YAP) pathway. YAP, as a key protein, can further regulate the activity of PI3K/Akt signalling pathway in vascular endothelial cells. In AIA mice experiments, we observed that the inhibition of Ezrin or of its downstream YAP pathway can affect synovial angiogenesis and may lead to progression of RA. In conclusion, Ezrin plays an important role in angiogenesis in the RA synovium by regulating YAP nuclear translocation and interacting with the PI3K/Akt signalling pathway.     

Neural driven angiogenesis by overexpression of nerve growth factor

Mechanisms regulating angiogenesis are crucial in adjusting tissue perfusion on metabolic demands. We demonstrate that overexpression of nerve growth factor (NGF) in brown adipose tissue (BAT) of NGF-transgenic mice elevates both mRNA and protein levels of vascular endothelial growth factor (VEGF) and VEGF-receptors. Increased vascular permeability, leukocyte–endothelial interactions (LEI), and tissue perfusion were measured using intravital microscopy. NGF-stimulation of adipocytes and endothelial cells elevates mRNA expression of VEGF and its receptors, an effect blocked by NGF neutralizing antibodies. These data suggest an activation of angiogenesis as a result of both: stimulation of adipozytes and direct mitogenic effects on endothelial cells. The increased nerve density associated with vessels strengthened our hypothesis that tissue perfusion is regulated by neural control of vessels and that the interaction between the NGF and VEGF systems is the critical driver for the activated angiogenic process. The interaction of VEGF- and NGF-systems gives new insights into neural control of organ vascularization and perfusion.     

YAP accelerates vascular senescence via blocking autophagic flux and activating mTOR

Yes-associated protein (YAP), a major effector of the Hippo signalling pathway, is widely implicated in vascular pathophysiology processes. Here, we identify a new role of YAP in the regulation of vascular senescence. The inhibition or deficiency and overexpression of YAP were performed in human umbilical vein endothelial cells (HUVECs) and isolated vascular tissues. Cellular and vascular senescence was assessed by analysis of the senescence-associated β-galactosidase (SA-β-gal) and expression of senescence markers P16, P21, P53, TERT and TRF1. We found that YAP was highly expressed in old vascular tissues, inhibition and knockdown of YAP decreased senescence, while overexpression of YAP increased the senescence in both HUVECs and vascular tissues. In addition, autophagic flux blockage and mTOR pathway activation were observed during YAP-induced HUVECs and vascular senescence, which could be relieved by the inhibition and knockdown of YAP. Moreover, YAP-promoted cellular and vascular senescence could be relieved by mTOR inhibition. Collectively, our findings indicate that YAP may serve as a potential therapeutic target for ageing-associated cardiovascular disease.     

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2

Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.     

Role of caveolin-1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell activation

Flavonoids can protect against inflammatory diseases such as atherosclerosis by decreasing vascular endothelial cell activation. Plasma microdomains called caveolae may be critical in regulating endothelial activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that flavonoids can down-regulate endothelial inflammatory parameters by modulating caveolae-regulated cell signaling. We focused on the role of caveolae and its major protein, caveolin-1, in mechanisms of linoleic-acid-induced endothelial cell activation and protection by the catechin epigallocatechin-3-gallate (EGCG). Exposure to linoleic acid for 6 h induced expression of both caveolin-1 and cyclooxygenase (COX)-2. Pretreatment with EGCG blocked fatty-acid-induced caveolin-1 and COX-2 expression in a time- and concentration-dependent manner. Similar results were observed with nuclear factor-kappa B DNA binding activity, which was also reduced by caveolin-1 silencing. Exposure to linoleic acid rapidly increased phosphorylation of several kinases, including p38 MAPK, extracellular signal regulated kinase 1/2 (ERK1/2) and amino kinase terminal (Akt), with maximal induction at about 10 min. Inhibitors of ERK1/2 and Akt down-regulated the linoleic-acid-induced increase in COX-2 protein, which also occurred after pretreatment with EGCG. Caveolin-1 silencing blocked linoleic-acid-induced phosphorylation of ERK1/2 and protein expression of COX-2, suggesting that specific MAPK signaling is caveolae dependent. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and protection by flavonoids such as EGCG.     

CD44 Regulation of Endothelial Cell Proliferation and Apoptosis via Modulation of CD31 and VE-cadherin Expression

CD44 has been implicated in a diverse array of cell behaviors and in a diverse range of signaling pathway activations under physiological and pathophysiological conditions. We have documented a role for CD44 in mediating vascular barrier integrity via regulation of PECAM-1 (CD31) expression. We now report our findings on the roles of CD44 in modulating proliferation and apoptosis of microvascular endothelial cells via its modulation of CD31 and VE-cadherin expression and the Hippo pathway. In this report, we demonstrate persistent increased proliferation and reduced activations of both effector and initiator caspases in high cell density, postconfluent CD44 knock-out (CD44KO), and CD31KO cultures. We found that reconstitution with murine CD44 or CD31 restored the proliferative and caspase activation rates to WT levels. Moreover, we have confirmed that the CD31 ecto-domain plays a key role in specific caspase cascades as well as cell adhesion-mediated cell growth and found that CD31 deficiency results in a reduction in VE-cadherin expression. Last, we have shown that both CD44KO and CD31KO endothelial cells exhibit a reduced VE-cadherin expression correlating with increased survivin expression and YAP nuclear localization, consistent with inactivation of the Hippo pathway, resulting in increased proliferation and decreased apoptosis. These findings support the concept that CD44 mediates several of its effects on endothelia through modulation of adhesion protein expression, which, in addition to its known modulation of junctional integrity, matrix metalloproteinase levels and activation, interactions with cortical membrane proteins, and selected signaling pathways, plays a key role as a critical regulator of vascular function.     

Focal adhesion kinase controls cellular levels of p27/Kip1 and p21/Cip1 through Skp2-dependent and -independent mechanisms

Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to soluble growth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from both adhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation. Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylation of FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided with elevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaired degradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibiting mitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels of p21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a consequence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completely restored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in the regulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They also provide important insights into the requirement of focal adhesion kinase for normal vascular development and reveal novel regulatory control points for angiogenesis.     

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.     

Geranylgeranyl pyrophosphate-mediated protein geranylgeranylation regulates endothelial cell proliferation and apoptosis during vasculogenesis in mouse embryo

Vascular development is essential for the establishment of the circulatory system during embryonic development and requires the proliferation of endothelial cells. However, the underpinning regulatory mechanisms are not well understood. Here, we report that geranylgeranyl pyrophosphate(GGPP), a metabolite involved in protein geranylgeranylation, plays an indispensable role in embryonic vascular development. GGPP is synthesized by geranylgeranyl pyrophosphate synthase(GGPPS) in the mevalonate pathway. The selective knockout of Ggpps in endothelial cells led to aberrant vascular development and embryonic lethality, resulting from the decreased proliferation and enhanced apoptosis of endothelial cells during vasculogenesis. The defect in protein geranylgeranylation induced by GGPP depletion inhibited the membrane localization of Rho A and enhanced yes-associated protein(YAP) phosphorylation, thereby prohibiting the entry of YAP into the nucleus and the expression of YAP target genes related to cell proliferation and the antiapoptosis process. Moreover, inhibition of the mevalonate pathway by simvastatin induced endothelial cell proliferation defects and apoptosis, which were ameliorated by GGPP. Geranylgeraniol(GGOH), a precursor of GGPP, ameliorated the harmful effects of simvastatin on vascular development of developing fetuses in pregnant mice. These results indicate that GGPP-mediated protein geranylgeranylation is essential for endothelial cell proliferation and the antiapoptosis process during embryonic vascular development.     

Controlling angiogenesis by two unique TGF-β type I receptor signaling pathways

Genetic studies in mice and humans have revealed a pivotal function for transforming growth factor-beta (TGF-β) in vascular development and maintenance of vascular homeostasis. Mice deficient for various TGF-β signaling components develop an embryonic lethality due to vascular defects. In patients, mutations in TGF-β receptors have been linked to vascular dysplasia like Hereditary Hemorrhagic Telangiectasia (HHT) and pulmonary arterial hypertension (PAH). Besides indirect effects by regulating the expression of angiogenic regulators, TGF-β also has potent direct effects on endothelial cell growth and migration, and we have proposed that TGF-β regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways, activin receptor-like kinase (ALK)1 and ALK5. TGF-β is also critical for the differentiation of mural precursors into pericytes and smooth muscle cells. Furthermore, defective paracrine TGF-β signaling between endothelial and neighboring mural cells may be responsible for a leaky vessel phenotype that is characteristic of HHT. In this review, we discuss our current understanding of the TGF-β signaling pathway and its regulation of endothelial and vascular smooth muscle cell function.     

Bi-directional cell contact-dependent regulation of gene expression between endothelial cells and osteoblasts in a three-dimensional spheroidal coculture model

Multiple cell-cell interactions control bone morphogenesis and vascularization. We have employed a spheroidal coculture system of endothelial cells (EC) and osteoblasts (OB) to study cell contact-dependent gene regulation between these two cell types that may play a role in regulating OB differentiation and EC angiogenic properties. Coculture spheroids differentiate spontaneously to organize into a core of OB and a surface layer of endothelial cells. Individual spheroid culture of EC or OB leads to significant alterations in gene expression compared to standard monolayer culture (upregulation of Tie-2 in EC; upregulation of angiopoietin-2 in osteoblasts). More importantly, spheroidal coculture of endothelial cells and osteoblasts leads to significant changes of gene expression in both cell populations (upregulation of VEGFR-2 in EC; downregulation of VEGF, and upregulation of alkaline phosphatase in osteoblasts). These changes are dependent on cell-cell contact and are not seen in stimulation experiments with conditioned supernatants. Collectively, the data demonstrate complex bi-directional gene regulation mechanisms between EC and OB that are likely to play a critical role during OB differentiation and in controlling the properties of angiogenic EC.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

Temporal relationship between MMP production and angiogenic process in HUVECs

Alterations in both cell-cell and cell-matrix interactions are associated with the activation of endothelial cells that initiate angiogenesis. Cell-matrix interactions are affected by changes in both cell surface receptors for matrix proteins and the composition of ECM. One of the molecular mechanisms involved in changes in these components is the action of neutral proteinases, particularly matrix metalloproteinases. To understand the involvement of MMPs in angiogenic processes, the in vitro model of human umbilical vein endothelial cells in culture was used. Zymography and ELISA showed that, as cell-cell contact and network-like structures were formed, there was down regulation of MMP-2 and MMP-9 associated with high levels of their endogenous inhibitors TIMP-1 and TIMP-2. On treatment with aspirin, which inhibited the cell-cell contact and network-like structure formation, there was no down regulation of MMPs and cells continued to produce MMP-2 and MMP-9. These results indicate a temporal relationship between MMP-2 and MMP-9 production by endothelial cells and the onset of angiogenic event.     

Human peripheral blood eosinophils induce angiogenesis

Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.