Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.     

Fluid forces control endothelial sprouting

During angiogenesis, endothelial cells (ECs) from intact blood vessels quickly infiltrate avascular regions via vascular sprouting. This process is fundamental to many normal and pathological processes such as wound healing and tumor growth, but its initiation and control are poorly understood. Vascular endothelial cell growth factor (VEGF) can promote vessel dilation and angiogenic sprouting, but given the complex nature of vascular morphogenesis, additional signals are likely necessary to determine, for example, which vessel segments sprout, which dilate, and which remain quiescent. Fluid forces exerted by blood and plasma are prime candidates that might codirect these processes, but it is not known whether VEGF cooperates with mechanical fluid forces to mediate angiogenesis. Using a microfluidic tissue analog of angiogenic sprouting, we found that fluid shear stress, such as exerted by flowing blood, attenuates EC sprouting in a nitric oxide-dependent manner and that interstitial flow, such as produced by extravasating plasma, directs endothelial morphogenesis and sprout formation. Furthermore, positive VEGF gradients initiated sprouting but negative gradients inhibited sprouting, promoting instead sheet-like migration analogous to vessel dilation. These results suggest that ECs integrate signals from fluid forces and local VEGF gradients to achieve such varied goals as vessel dilation and sprouting.     

A novel role for inducible Fut2 in angiogenesis

Rationale

Angiogenesis plays an important role in wound healing and tumor growth. Fucosyltransferases synthesize fucosylated glycans and may play a major role in vascular biology.

Objective

To examine the role of an alpha(1,2) fucosyltransferase (Fut2) in angiogenesis.

Methods and results

We found that Fut2 mRNA and protein expression is inducible in human dermal microvascular endothelial cells (HMVECs). After finding that Fut2 is inducible in HMVECs, we examined if Fut2 contributes to angiogenesis. We found that Fut2 null endothelial cell (EC) migration and tube formation were significantly less compared to wild type (wt) ECs. Angiogenesis was impaired in Fut2 null compared to wt mice in the mouse Matrigel plug and the sponge granuloma angiogenesis assays. To assess the characteristics of Fut2 null ECs in vivo, we performed Matrigel plug angiogenesis assays in wt mice using Fut2 null and wt mouse ECs. We found a significant decrease in Fut2 null EC incorporation in neoangiogenesis compared to wt ECs. ERK1/2 activation, fibroblast growth factor receptor2, and vascular endothelial growth factor expression were less in Fut2 null ECs, suggesting a possible mechanism of impaired angiogenesis when Fut2 is lacking.

Conclusions

These data suggest a novel role for Fut2 as a regulator of angiogenesis.     

Deficiency of the cysteine protease cathepsin S impairs microvessel growth

During angiogenesis, microvascular endothelial cells (ECs) secrete proteinases that permit penetration of the vascular basement membrane as well as the interstitial extracellular matrix. This study tested the hypothesis that cathepsin S (Cat S) contributes to angiogenesis. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated the expression of Cat S, whereas inhibition of Cat S activity reduced microtubule formation by impairing cell invasion. ECs from Cat S-deficient mice showed reduced collagenolytic activity and impaired invasion of collagens type I and IV. Cat S-deficient mice displayed defective microvessel development during wound repair. This abnormal angiogenesis occurred despite normal vascular endothelial growth factor and basic fibroblast growth factor levels, implying an essential role for extracellular matrix degradation by Cat S during microvessel formation. These results demonstrate a novel function of endothelium-derived Cat S in angiogenesis.     

Loss of bcl-2 during the senescence exacerbates the impaired angiogenic functions in endothelial cells by deteriorating the mitochondrial redox state

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.     

神经生长因子对体外培养的小鼠主动脉环血管新生的影响及TrkA的关键作用

目的 探讨神经生长因子（NGF）对体外培养的小鼠主动脉环血管新生的影响。方法 ①将小鼠主动脉环在三维基质胶中以含不同浓度的NGF的无血清培养基培养不同天数后,对出芽的细胞进行免疫荧光染色细胞学鉴定,或在倒置显微镜下观察、摄像,并以Image Pro图像分析软件计算每高倍视野中平均出芽的面积。②向培养基中预先加入抗NGF的中和抗体或血管内皮生长因子受体2（VEGFR2）拮抗剂SU5416,观察NGF或VEGF对出芽性血管新生的影响。③向培养基中预先加入酪氨酸受体激酶A（TrkA）受体拮抗剂K252a,观察NGF是否可通过TrkA受体促血管新生。结果①外源性NGF呈浓度依赖性促进基质胶中培养的小鼠主动脉环出芽。出芽的细胞上CD31的表达阳性。抗NGF的中和抗体能明显阻断NGF诱导的出芽性血管的新生。②SU5416可显著减少VEGF诱导的血管新生;但对NGF诱导的血管新生无明显影响。③K252a可明显抑制NGF诱导的血管新生。结论 NGF可通过TrkA受体促进小鼠主动脉环血管新生,为促血管新生治疗方案的选择提供了新的思路。     

Migration inhibitory factor mediates angiogenesis via mitogen-activated protein kinase and phosphatidylinositol kinase

In this study, we investigated the effects of migration inhibitory factor (rhMIF) on angiogenesis-related signaling cascades and apoptosis in human endothelial cells (ECs). We show that in vitro rhMIF induces migration and tube formation in Matrigel of human dermal microvascular endothelial cells (HMVECs), with potency comparable to that of basic fibroblast growth factor. In vivo, rhMIF induces angiogenesis in Matrigel plugs and in the corneal bioassay. Using panels of relatively specific kinase inhibitors, antisense oligonucleotides, and dominant-negative mutants, we show that mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) are critical for MIF-dependent HMVEC migration, whereas Src and p38 kinases are nonessential. Moreover, we demonstrate that rhMIF induces time-dependent increases in phosphorylation levels of MEK1/2, Erk1/2, and Elk-1, as well as PI3K, and its effector kinase, Akt, in HMVECs. Studies with dominant-negative mutants and antisense oligonucleotides corroborate these effects in HMVECs. Furthermore, we demonstrate that rhMIF-induced angiogenesis in the rat cornea in vivo and in the ex vivo endothelial cell morphogenesis assay is also MAPK- and PI3K-dependent. Our findings support a role for MIF as an angiogenic factor and provide a rationale for the use of MIF as a therapeutic inducer of neovascularization in the development of collateral circulation in coronary artery disease.     

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.     

Activation of sphingosine kinase-1 mediates induction of endothelial cell proliferation and angiogenesis by epoxyeicosatrienoic acids

AIMS: Recent evidence suggests that the epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess mitogenic and angiogenic effects in vascular endothelial cells. However, the mechanisms underlying these effects are not fully elucidated. Because sphingosine kinase (SK) and its product S1P play essential roles in cell growth, survival and migration, we hypothesized that SK activation by EETs may mediate some of its angiogenic effects. METHODS AND RESULTS: We studied the effects of EETs on SK activity in human umbilical vein endothelial cells (HUVECs). Treatment with EETs, particularly 11,12-EET, markedly augmented SK activity in HUVECs. At the concentration of 1 micromol/L, 11,12-EET increased SK activity by 110% and the maximal effect on SK activation was observed at 20 min after 11,12-EET addition. Furthermore, inhibition of SK by a specific inhibitor, SKI-II, markedly attenuated 11,12-EET-induced EC proliferation. Importantly, 11,12-EET-induced activation of Akt kinase and transactivation of the epidermal growth factor (EGF) receptor was also inhibited by SKI-II. To investigate the isoform-specific role of SK in EET-induced angiogenesis, inhibition of SK1 by expression of dominant-negative SK1(G82D) substantially attenuated 11,12-EET-induced EC proliferation, migration, and tube formation in vitro and Matrigel plug angiogenesis in vivo. Furthermore, knockdown of SK1 expression by specific siRNA also inhibited 11,12-EET-induced EC proliferation and migration, whereas SK2 siRNA knockdown was without effect. CONCLUSION: These results suggest that SK1 is an important mediator of the 11,12-EET-induced angiogenic effects in human ECs. Thus, SK1 may represent a novel therapeutic modality for the treatment of angiogenesis-related diseases such as cancer and ischaemia.     

AMP-activated protein kinase signaling stimulates VEGF expression and angiogenesis in skeletal muscle

AMP-activated protein kinase (AMPK) is regulated by various cellular stresses. Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is also upregulated by several stress-inducible factors such as hypoxia and stimulation by cytokines and growth factors. Here, we investigated whether AMPK signaling in muscle has a role in regulating VEGF-mediated angiogenic processes. AICAR stimulated VEGF mRNA and protein levels in C2C12 myotube cultures. Transduction with dominant-negative AMPK abolished AICAR-induced VEGF expression at both steady state mRNA and protein levels. AICAR increased VEGF mRNA stability without affecting VEGF promoter activity. AICAR also stimulated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Activation of p38 MAPK was suppressed by transduction with dominant-negative AMPK, suggesting that AMPK is upstream of p38 MAPK. The p38 MAPK inhibitor SB203580 blocked AICAR-induced increase in VEGF mRNA and protein levels, indicating that AICAR-mediated VEGF induction is dependent on p38 MAPK signaling. AICAR treatment increased VEGF expression and accelerated angiogenic repair of ischemic hindlimbs in mice in an AMPK-dependent manner. These data indicate that AMPK-p38 MAPK signaling cascade can increase VEGF production in muscle and promote angiogenesis in response to ischemic injury.     

Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay.

OBJECTIVES: An endothelial cell (EC)-specific angiogenesis assay was developed to functionally characterize angiogenic properties of 2 distinct putative endothelial progenitor cells (EPCs): early EPCs and late outgrowth endothelial cells (OECs). BACKGROUND: Endothelial progenitor cells promote revascularization of ischemic tissue. However, the nature of different EPCs and their role in angiogenesis remains debated. METHODS: Tubulogenesis was assessed by immunohistochemistry in co-cultures of differentiated ECs (including human umbilical vein, coronary artery, and microvascular ECs) or non-ECs with monolayers of human fibroblasts (MRC5). Using adaptations of the co-culture assay, early EPCs and OECs, isolated from peripheral blood mononuclear cells, were assessed by 3-dimensional immunofluorescence microscopy for their capacity for: 1) independent tubulogenesis; 2) incorporation into pre-existing vascular networks; and 3) paracrine angiogenic effects using transwell cultures. RESULTS: Branched interconnecting EC-specific tubules formed with all differentiated ECs after 72 h. Proangiogenic and antiangiogenic agents modulated tubulogenesis appropriately (vascular endothelial growth factor 10 ng: +142 +/- 13%, 1 microM anti-vascular endothelial growth factor: -44 +/- 7% vs. control, p < 0.001). In contrast, early EPCs, along with nonendothelial cell types, failed to independently form tubules or incorporate into differentiated EC tubules. Nevertheless, early EPCs indirectly augmented tubulogenesis by differentiated ECs even when physically separated by transwells (+115 +/- 4% vs. control; p < 0.001). By contrast, OECs independently formed tubules and incorporated into differentiated EC tubules but exerted no significant paracrine angiogenic effects. CONCLUSIONS: A novel EC-specific tubulogenesis assay highlights strikingly different angiogenic properties of different EPCs: late OECs directly participate in tubulogenesis, whereas early EPCs augment angiogenesis in a paracrine fashion, with implications for optimizing cell therapies for 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.     

Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ₃

Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)β(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)β(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.     

A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices

Interactions between migratory endothelial cells (ECs) and surrounding extracellular matrix (ECM) are of central importance to vascular growth. Here, we present a new model of EC migration and morphogenesis within three-dimensional ECM termed "radial invasion of matrix by aggregated cells" (RIMAC). In the RIMAC model, single aggregates of defined numbers of bovine aortic ECs were embedded within small, lenticular gels of type I collagen supported by annuli of nylon mesh. Culture of the gels in nutrient media resulted in quantifiable, reproducible, radial migration of ECs into the collagen. The angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) each stimulated migration of ECs in a concentration-dependent manner. In combination, bFGF and VEGF stimulated migration synergistically. In contrast, transforming growth factor-beta1 inhibited migration of ECs. Low concentrations (0.1-0.5 ng/ml) of VEGF induced ECs to form multicellular sprouts, some of which possessed lumen-like spaces. Mitomycin C, an inhibitor of cell proliferation, did not affect the migration of ECs into collagen induced by 0.5 ng/ml VEGF but moderately inhibited migration induced by 5 ng/ml VEGF. Increasing the density (concentration) of the collagen gel inhibited the migration of single ECs and increased the branching and anastomosis of multicellular sprouts. We conclude that the RIMAC model is a highly efficacious assay for the screening of potentially angiogenic and angiostatic compounds and, moreover, is advantageous for mechanistic studies of vascular morphogenesis.     

Lysophosphatidic acid mediates interleukin-8 expression in human endometrial stromal cells through its receptor and nuclear factor-kappaB-dependent pathway: a possible role in angiogenesis of endometrium and placenta

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid molecule involved in inflammation, angiogenesis, would healing, and cancer invasion. Whereas serum lysophospholipase D activity increases in women with pregnancy, the role of LPA in pregnancy remains unclear. We investigated the expression of LPA receptors and function of LPA in endometrial stromal cells. Histologically normal endometrium was obtained from surgical specimens of women undergoing hysterectomy for leiomyoma. First-trimester decidua was obtained from women receiving elective termination of pregnancy. We examined the expressions of LPA1, LPA2, and LPA3 receptors in endometrial stromal cells. The effects of LPA on the expression of vascular endothelial growth factor, IL-6, and IL-8 were examined. Signal pathways of LPA were delineated. Functions of secretory angiogenic factors were tested using human endometrial microvascular endothelial cells. Immunoreactivity and mRNA of LPA1 receptors were identified in endometrial stromal cells. LPA enhanced IL-8 expression in a dose- and time-dependent manner, whereas vascular endothelial growth factor or IL-6 expression was not affected by LPA treatment. Mechanistic dissection disclosed that LPA functioned via the Gi protein, MAPK/p38 and nuclear factor-kappaB pathway. LPA-induced IL-8 enhanced migration, permeability, capillary tube formation, and proliferation of human endometrial microvascular endothelial cells. Endometrial stromal cells express LPA1 receptors. Through the LPA1 receptor, LPA induces IL-8 expression via a nuclear factor-kappaB-dependent signal pathway. These results could suggest that LPA may play a role in angiogenesis of endometrium and placenta through induction of IL-8 in endometrial stromal cells during pregnancy.     

Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice

Angiogenesis is the main mechanism of vascular remodeling during late development and, after birth, in wound healing. Perturbations of angiogenesis occur in cancer, diabetes, ischemia, and inflammation. While much progress has been made in identifying factors that control angiogenesis, the understanding of the precise molecular mechanisms involved is incomplete. Here we identify a small GTPase, Rap1b, as a positive regulator of angiogenesis. Rap1b-deficient mice had a decreased level of Matrigel plug and neonatal retinal neovascularization, and aortas isolated from Rap1b-deficient animals had a reduced microvessel sprouting response to 2 major physiological regulators of angiogenesis: vascular endothelial growth factor (VEGF) and basic fibroblasts growth factor (bFGF), indicating an intrinsic defect in endothelial cells. Proliferation of retinal endothelial cells in situ and in vitro migration of lung endothelial cells isolated from Rap1b-deficient mice were inhibited. At the molecular level, activation of 2 MAP kinases, p38 MAPK and p42/44 ERK, important regulators of endothelial migration and proliferation, was decreased in Rap1b-deficient endothelial cells in response to VEGF stimulation. These studies provide evidence that Rap1b is required for normal angiogenesis and reveal a novel role of Rap1 in regulation of proangiogenic signaling in endothelial cells.