High-density lipoprotein (HDL) promotes angiogenesis via S1P3-dependent VEGFR2 activation

High-density lipoprotein (HDL) has previously been shown to promote angiogenesis. However, the mechanisms by which HDL enhances the formation of blood vessels remain to be defined. To address this, the effects of HDL on the proliferation, transwell migration and tube formation of human umbilical vein endothelial cells were investigated. By examining the abundance and phosphorylation (i.e., activation) of the vascular endothelial growth factor receptor VEGFR2 and modulating the activity of the sphingosine-1 phosphate receptors S1P1–3 and VEGFR2, we characterized mechanisms controlling angiogenic responses in response to HDL exposure. Here, we report that HDL dose-dependently increased endothelial proliferation, migration and tube formation. These events were in association with increased VEGFR2 abundance and rapid VEGFR2 phosphorylation at Tyr1054/Tyr1059 and Tyr1175 residues in response to HDL. Blockade of VEGFR2 activation by the VEGFR2 inhibitor SU1498 markedly abrogated the pro-angiogenic capacity of HDL. Moreover, the S1P3 inhibitor suramin prevented VEGFR2 expression and abolished endothelial migration and tube formation, while the S1P1 agonist CYM-5442 and the S1P2 inhibitor JTE-013 had no effect. Last, the role of S1P3 was further confirmed in regulation of S1P-induced endothelial proliferation, migration and tube formation via up-regulation and activation of VEGFR2. Together, these findings argue that HDL promotes angiogenesis via S1P3-dependent up-regulation and activation of VEGFR2 and also suggest that the S1P–S1P3–VEGFR2 signaling cascades as a novel target for HDL-modulating therapy implicated in vascular remodeling and functional recovery in atherosclerotic diseases such as myocardial infarction and ischemic stroke.     

Hypoxia paradoxically inhibits the angiogenic response of isolated vessel explants while inducing overexpression of vascular endothelial growth factor

This study was designed to investigate how changes in O₂ levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1–4 % O₂. Additionally, aortic neovessels preformed in atmospheric O₂ lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O₂. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O₂ levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.     

RETRACTED ARTICLE: Gold nanoparticles inhibit vascular endothelial growth factor-induced angiogenesis and vascular permeability via Src dependent pathway in retinal endothelial cells

The purpose of this study was to investigate the effect of gold nanoparticles on the signaling cascade related to angiogenesis and vascular permeability induced by Vascular Endothelial Growth Factor (VEGF) in Bovine retinal endothelial cells (BRECs). The effect of VEGF and gold nanoparticles on cell viability, migration and tubule formation was assessed. PP2 (Src Tyrosine Kinase inhibitor) was used as the positive control and the inhibitor assay was performed to compare the effect of AuNPs on VEGF induced angiogenesis. The transient transfection assay was performed to study the VEGFR2/Src activity during experimental conditions and was confirmed using western blot analysis. Treatment of BRECs with VEGF significantly increased the cell proliferation, migration and tube formation. Furthermore, gold nanoparticles (500 nM) significantly inhibited the proliferation, migration and tube formation, in the presence of VEGF in BRECs. The gold nanoparticles also inhibited VEGF induced Src phosphorylation through which their mode of action in inhibiting angiogenic pathways is revealed. The fate of the gold nanoparticles within the cells is being analyzed using the TEM images obtained. The potential of AuNPs to inhibit the VEGF165-induced VEGFR-2 phosphorylation is also being confirmed through the receptor assay which elucidates one of the possible mechanism by which AuNPs inhibit VEGF induced angiogenesis. These results indicate that gold nanoparticles can block VEGF activation of important signaling pathways, specifically Src in BRECs and hence modulation of these pathways may contribute to gold nanoparticles ability to block VEGF-induced retinal neovascularization.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

IQGAP1, a novel vascular endothelial growth factor receptor binding protein, is involved in reactive oxygen species--dependent endothelial migration and proliferation

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.     

Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.     

Effects of vascular endothelial growth factors and their receptors on megakaryocytes and platelets and related diseases

It is well known that vascular endothelial growth factors (VEGFs) and their receptors (vascular endothelial growth factor receptors, VEGFRs) are expressed in different tissues, and VEGF‐VEGFR loops regulate a wide range of responses, including metabolic homeostasis, cell proliferation, migration and tubuleogenesis. As ligands, VEGFs act on three structurally related VEGFRs (VEGFR1, VEGFR2 and VEGFR3 [also termed FLT1, KDR and FLT4, respectively]) that deliver downstream signals. Haematopoietic stem cells (HSCs), megakaryocytic cell lines, cultured megakaryocytes (MKs), primary MKs and abnormal MKs express and secrete VEGFs. During the development from HSCs to MKs, VEGFR1, VEGFR2 and VEGFR3 are expressed at different developmental stages, respectively, and re‐expressed, e.g., VEGFR2, and play different roles in commitment, differentiation, proliferation, survival and polyplodization of HSCs/MKs via autocrine, paracrine and/or even intracrine loops. Moreover, VEGFs and their receptors are abnormally expressed in MK‐related diseases, including myeloproliferative neoplasms, myelodysplastic syndromes and acute megakaryocytic leukaemia (a rare subtype of acute myeloid leukaemia), and they lead to the disordered proliferation/differentiation of bone marrow cells and angiogenesis, indicating that they are closely related to these diseases. Thus, targeting VEGF‐VEGFR loops may be of potential therapeutic value.     

Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts- and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.     

Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia

OBJECTIVE: Electronegative low density lipoprotein (LDL) subfractions are cytotoxic to endothelial cells. To continue our study of homozygotic familial hypercholesterolemic (FH)-LDL, we report the effects of FH-LDL subfractions (FH-L1 to FH-L5) on the angiogenic processes in cultured endothelial cells. METHODS AND RESULTS: Subconfluent bovine aortic endothelial cells (BAEC) were treated with LDL subfractions (20 microg/ml), and the effects on angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, secretion of matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) were determined. The electronegative FH-L4 and FH-L5 inhibited cell proliferation while the other FH-LDL subfractions and LDL from normocholesterolemic subjects (N-LDL) had negligible effects. Like Cu2+ ox-LDL, FH-L5 strongly inhibited endothelial cell viability and FH-L4 had a milder effects. Similarly, FH-L4 and FH-L5 but not the other subfractions retarded cell migration, induced cell apoptosis, and perturbed tube formation by BAEC in matrigel. FH-L5 inhibited secretion of MMP-2 and MMP-9 by BAEC without affecting their endogenous levels. In contrast, FH-L5 increased the VEGF expression in endothelial cells. CONCLUSIONS: Our results show for the first time that FH-L5, a circulating LDL subfraction from hypercholesterolemic patients, modulates various angiogenic processes, thereby dysregulating endothelial function in a way that may be atherogenic.     

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.     

Integrating the VEGF signals leading to actin-based motility in vascular endothelial cells

Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor that stimulates endothelial cell proliferation and migration, two key events of the angiogenic process. The intracellular signals leading to these events have recently been investigated and a better understanding on how VEGF induces its angiogenic functions is emerging. Herein, we summarize recent findings on how VEGF stimulates endothelial cell migration and contributes to angiogenesis. In particular, the role of the VEGF receptors involved in initiating the coordinated signals that leads to actin-based motility is discussed.     

Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family

The central role of vascular endothelial growth factor (VEGF) in angiogenesis in health and disease makes it attractive both as a therapeutic target for anti-angiogenic drugs and as a pro-angiogenic cytokine for the treatment of ischaemic heart disease. While VEGF binds to two receptor protein tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (KDR), most biological functions of VEGF are mediated via VEGFR2, and the role of VEGFR1 is currently unknown. Neuropilin-1, a non-tyrosine kinase transmembrane molecule, may function as a co-receptor for VEGFR2. Considerable progress has recently been made towards delineating the signal transduction pathways distal to activation of VEGFR2. Activation of the mitogen-activated protein kinase, protein kinase C and Akt pathways are all strongly implicated in mediating diverse cellular biological functions of VEGF, including cell survival, proliferation, the generation of nitric oxide and prostacyclin and angiogenesis. Upregulation of metalloproteinases, activation of focal adhesion kinase and interactions between VEGF receptors and integrins are strongly implicated in VEGF-induced endothelial cell migration. Recent findings suggest important roles for the vasodilators nitric oxide and prostacyclin, in linking post-receptor signaling networks to downstream biological effects and in mediating some in vivo endothelial functions of VEGF.     

Insulin stimulates the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor.

Insulin treatment is known epidemiologically as an independent risk factor for the progression of diabetic retinopathy. However, how insulin exacerbates the retinopathy is not yet fully understood. In this study, we investigate the effects of insulin on the growth and tube formation of microvascular endothelial cells (EC). When human skin microvascular EC were grown under various concentrations of insulin, DNA synthesis as well as tube formation of EC was found to be significantly stimulated. We obtained evidence that it is mainly vascular endothelial growth factor (VEGF) that mediates the angiogenic activity of insulin as follows. (1) Insulin upregulates the level of mRNA coding for secretory forms of VEGF, while the expression of the two VEGF receptor genes, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase1 (flt1), was essentially unchanged by exposure to insulin. (2) A monoclonal antibody against human VEGF can completely neutralize both the proliferation and the tube formation of EC induced by insulin. The angiogenic effects of insulin were additive with those of hypoxia, a principal factor that causes angiogenesis. Further, insulin significantly stimulated plasminogen activator inhibitor-1 activity in EC. The results thus suggest that insulin not only elicits angiogenesis through the induction of autocrine VEGF but also is a predisposing factor for thrombogenesis, which may give rise to focal ischemia that could superdrive angiogenesis, thereby leading to the exacerbation of diabetic retinopathy.     

RETRACTED ARTICLE: PEDF inhibits VEGF- and EPO- induced angiogenesis in retinal endothelial cells through interruption of PI3K/Akt phosphorylation

Retinal angiogenesis in diabetes may lead to visual impairment and even irreversible blindness in people of working age group worldwide. The main pathological feature of proliferative diabetic retinopathy (PDR) is hypoxia, and overproduction of growth factors like vascular endothelial growth factor (VEGF) and erythropoietin (Epo). This results in pathological proliferation of retinal endothelial cells (RECs), leading to new vessel formation (angiogenesis). Inhibition of angiogenesis is a promising strategy for treatment of PDR and other retinal neovascular disorders. Pigment epithelium-derived factor (PEDF), a 50-kDa protein secreted by retinal pigment epithelium, inhibits the growth of new blood vessel induced in the eye in a variety of ways with a yet elusive mechanism. Here, we investigated the possible mechanism by which PEDF inhibits VEGF- and Epo-induced angiogenic effects in RECs is mediated through PI3K/Akt pathway. PEDF treatment induced the apoptosis in RECs by activating caspase-3 and DNA fragmentation. We found a dose-dependent increase in cell survival with VEGF or Epo, which was attenuated in the presence of PEDF. In addition, PEDF significantly (P < 0.05) inhibited migration and in vitro tube formation in RECs in the presence of VEGF as like PI3K/Akt inhibitor. Of interest, PEDF effectively abrogated VEGF-mediated phosphorylation of PI3K/Akt. Further studies using RECs transfected with constitutively active and dominant-negative forms of Akt suggest that PEDF could inhibit VEGF- and also Epo-induced angiogenesis by disruption of PI3K/Akt signaling.     

晚期糖基化终产物对缺氧条件下内皮细胞血管生成能力的影响

目的明确晚期糖基化终产物(AGE)对缺氧条件下内皮细胞血管生成能力的影响,并探讨其可能机制。方法将酶消化法分离、培养的大鼠心肌微血管内皮细胞随机分为5组,对照组、AGE组、缺氧组、AGE+缺氧组、抑制剂组。MTT法测定细胞增殖能力;Transwell法检测细胞迁移;管样结构形成实验观察血管生成能力;Westernblot检测细胞外信号调节激酶(ERK)5磷酸化水平;ELISA检测血管内皮细胞生长因子(VEGF)分泌。结果与对照组比较,缺氧组内皮细胞的增殖、迁移、管样结构形成能力和VEGF分泌水平显著提高。与AGE+缺氧组比较,抑制剂组内皮细胞的增殖、迁移、管样结构形成能力增强,ERK5磷酸化水平明显降低,VEGF分泌水平增加。结论 AGE可抑制缺氧条件下内皮细胞的血管生成能力,其机制可能与ERK5的磷酸化抑制VEGF的表达有关。     

Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells

Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. Recent data indicate that angiotensin type 2 (AT2) receptor stimulation is antiangiogenic. Therefore, we studied the effect of angiotensin II (Ang II) on VEGF-induced migration and in vitro tube formation of human endothelial cells. Ang II inhibited VEGF-induced migration of EA.hy926 cells, human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) as well as tube formation by HDMECs. The AT2 receptor antagonist PD123,319 but not the AT1 receptor antagonist losartan blocked the inhibitory effect of Ang II. The inhibitory effect of Ang II on VEGF-induced migration of endothelial cells was mimicked by the specific AT2 receptor agonist CGP-42112A. The phosphorylation of Akt and its downstream effector endothelial NO synthase (eNOS) is pivotal to VEGF-induced angiogenesis. We therefore investigated the effect of Ang II on VEGF-induced Akt and eNOS phosphorylation. Ang II diminished the VEGF-induced phosphorylation of Akt and eNOS in endothelial cells, whereas the autophosphorylation of VEGF receptors was unaffected. CGP-42112A again mimicked and PD123,319 but not losartan blocked the inhibitory effect of Ang II. Treatment of endothelial cells with pertussis toxin (PTX) totally abolished the AT2 receptor-mediated inhibition of VEGF-induced endothelial cell migration and blocked the inhibition of Akt and eNOS phosphorylation. In conclusion, this study indicates that AT2 receptor stimulation inhibits VEGF-induced endothelial cell migration and tube formation via activation of a PTX-sensitive G protein. These findings may explain the reported antiangiogenic properties of the AT2 receptor.     

Mapping genetic determinants of coronary microvascular remodeling in the spontaneously hypertensive rat

This study aimed to analyze the role of endothelial progenitor cell (EPC)-derived angiogenic factors and chemokines in the multistep process driving angiogenesis with a focus on the recently discovered macrophage migration inhibitory factor (MIF)/chemokine receptor axis. Primary murine and murine embryonic EPCs (eEPCs) were analyzed for the expression of angiogenic/chemokines and components of the MIF/CXC chemokine receptor axis, focusing on the influence of hypoxic versus normoxic stimulation. Hypoxia induced an upregulation of CXCR2 and CXCR4 but not CD74 on EPCs and triggered the secretion of CXCL12, CXCL1, MIF, and vascular endothelial growth factor (VEGF). These factors stimulated the transmigration activity and adhesive capacity of EPCs, with MIF and VEGF exhibiting the strongest effects under hypoxia. MIF-, VEGF-, CXCL12-, and CXCL1-stimulated EPCs enhanced tube formation, with MIF and VEGF exhibiting again the strongest effect following hypoxia. Tube formation following in vivo implantation utilizing angiogenic factor-loaded Matrigel plugs was only promoted by VEGF. Coloading of plugs with eEPCs led to enhanced tube formation only by CXCL12, whereas MIF was the only factor which induced differentiation towards an endothelial and smooth muscle cell (SMC) phenotype, indicating an angiogenic and differentiation capacity in vivo. Surprisingly, CXCL12, a chemoattractant for smooth muscle progenitor cells, inhibited SMC differentiation. We have identified a role for EPC-derived proangiogenic MIF, VEGF and MIF receptors in EPC recruitment following hypoxia, EPC differentiation and subsequent tube and vessel formation, whereas CXCL12, a mediator of early EPC recruitment, does not contribute to the remodeling process. By discerning the contributions of key angiogenic chemokines and EPCs, these findings offer valuable mechanistic insight into mouse models of angiogenesis and help to define the intricate interplay between EPC-derived angiogenic cargo factors, EPCs, and the angiogenic target tissue.     

Inhibition of mini-TyrRS-induced angiogenesis response in endothelial cells by VE-cadherin-dependent mini-TrpRS

To clarify whether a VE-cadherin-dependent pathway allows mini-TrpRS to inhibit mini-TrpRS-induced new blood vessel formation in endothelial cells (ECs), the inhibitory effects of mutant mini-TrpRS and VE-cadherin on mini-TrpRS-induced angiogenesis were investigated. The effects of mini-TyrRS and mini-TrpRS on EC proliferation were evaluated using an MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The angiogenic activity in vitro was evaluated by transwell migration assay and matrigel-induced capillary tube formation. It was found that mini-TrpRS does not inhibit the mini-TyrRS-induced proliferation and migration of EC under the condition of VE-cadherin knockout. While wild-type mini-TrpRS inhibited mini-TyrRS-induced angiogenesis, this activity vanished for the mutant protein. Also, the promotion of angiogenesis by mini-TyrRS and the inhibition of angiogenesis by mini-TrpRS were VEGFR2 dependent but not VEGF dependent. Mini-TyrRS was able to increase the protein expression of VEGFR-2 in the presence of VE-cadherin, while no stimulatory effect of mini-TyrRS was detected when VE-cadherin was not present. Angiogenesis is therefore stimulated by mini-TyrRS and inhibited by mini-TrpRS, raising the possibility that mini-TyrRS and mini-TrpRS stimulate a common downstream signaling event: VE-cadherin. Thus, naturally occurring fragments of the two proteins involved in translation, TyrRS and TrpRS, have opposing activities on angiogenesis. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and antiangiogenic stimuli.