Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.     

Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that limits vessel density in normal tissues and curtails tumor growth. Here, we show that the inhibition of angiogenesis in vitro and in vivo and the induction of apoptosis by thrombospondin-1 all required the sequential activation of CD36, p59fyn, caspase-3 like proteases and p38 mitogen-activated protein kinases. We also detected increased endothelial cell apoptosis in situ at the margins of tumors in mice treated with thrombospondin-1. These results indicate that thrombospondin-1, and possibly other broad-spectrum natural inhibitors of angiogenesis, act in vivo by inducing receptor-mediated apoptosis in activated microvascular endothelial cells.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration

We have observed that the vasoactive peptide endothelin-1 is a potent inducer of migration of primary human brain-derived microvascular endothelial cells. By blocking signal transduction pathways with specific inhibitors, and using dominant negative mutant infections, we have demonstrated that multiple pathways are involved in endothelin-1-induced migration. Absolutely required for migration are protein tyrosine kinase Src, Ras, protein kinase C (PKC), phosphatidylinositol 3-kinase, ERK, and JNK; partial requirements were exhibited by cAMP-activated protein kinase and p38 kinase. Partial elucidation of the signal transduction sequences showed that the MAPKs ERK, JNK, and p38 are positioned downstream of both PKC and cAMP-activated protein kinase in the signal transduction scheme. The results show that human brain endothelial cell migration has distinct characteristics, different from cells derived from other vascular beds, or from other species, often used as model systems. Furthermore, the results indicate that endothelin-1, secreted by many tumors, is an important contributor to tumor-produced proangiogenic microenvironment. This growth factor has been associated with increased microvessel density in tumors and is responsible for endothelial cell proliferation, migration, invasion, and tubule formation. Because many signal transduction pathways investigated in this study are potential or current targets for anti-angiogenesis therapy, these results are of critical importance for designing physiological antiangiogenic protocols. signal transduction; angiogenesis; microvessels; vasoactive peptides     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration: potential roles for JNK and ERK

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGFbeta receptor family that is implicated in angiogenesis and in the pathogenesis of the vascular disease, hereditary hemorrhagic telangiectasia (HHT). In the absence of a specific ligand, ALK1 cellular functions have been mainly studied through the use of a constitutively active form of this receptor (ALK1ca) and are still debated. We previously reported that ALK1ca inhibits proliferation and migration of human endothelial cells suggesting that ALK1 plays an important role in the maturation phase of angiogenesis (Lamouille et al., 2002, Blood 100: 4495-4501). In the present work, we further analyzed the role of ALK1 in the migration of human dermal microvascular endothelial cell (HMVEC-d) and observed that silencing endogenous ALK1 expression with siRNAs accelerates endothelial cell migration in the wound assay. Further, we demonstrate that ALK1-induced inhibition of migration is Smad-independent. Using a panel of kinase inhibitors, we found that HMVEC-d wound closure was completely inhibited by a JNK inhibitor and to a lower degree by an ERK kinase inhibitor. Further, HMVEC-d wounding induced activation of both JNK and ERK, and these were inhibited by ALK1ca expression. Taken together, these results support a significant role for ALK1 as a negative regulator of endothelial cell migration and suggest the implication of JNK and ERK as mediators of this effect.     

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.     

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.     

Endostatin: a promising drug for antiangiogenic therapy

Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size. Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor. Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors. Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not. Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis. The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown. Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclinical and clinical trials.     

Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.     

Regulation of vascular endothelial cell polarization and migration by Hsp70/Hsp90-organizing protein

Hsp70/Hsp90-organizing protein (HOP) is a member of the co-chaperone family, which directly binds to chaperones to regulate their activities. The participation of HOP in cell motility and endothelial cell functions remains largely unknown. In this study, we demonstrate that HOP is critically involved in endothelial cell migration and angiogenesis. Tube formation and capillary sprouting experiments reveal that depletion of HOP expression significantly inhibits vessel formation from endothelial cells. Wound healing and transwell migration assays show that HOP is important for endothelial cell migration. By examination of centrosome reorientation and membrane ruffle dynamics, we find that HOP plays a crucial role in the establishment of cell polarity in response to migratory stimulus. Furthermore, our data show that HOP interacts with tubulin and colocalizes with microtubules in endothelial cells. These findings indicate HOP as a novel regulator of angiogenesis that functions through promoting vascular endothelial cell polarization and migration.     

Sphingosine 1-phosphate and cell migration: resistance to angiogenesis inhibitors

Naturally occurring angiogenesis inhibitors can inhibit different steps of the angiogenic process, such as endothelial cell migration. However, the mechanisms underlying this inhibition have not been elucidated. We demonstrate that migration of human umbilical vein endothelial cells induced by the potent endothelial cell chemoattractant sphingosine 1-phosphate is refractory to inhibition by well-characterized angiogenesis inhibitors such as endostatin and plasminogen-related protein-B. Our data support the contention that for effective blockage of tumor-induced angiogenesis, antagonists of both G protein-coupled receptor signaling and receptor tyrosine kinase signaling must be combined.     

Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase

Ischemia induces angiogenesis as a compensatory response. Although ischemia is known to causes synthesis and release of calcitonin gene-related peptide (CGRP), it is not clear whether CGRP regulates angiogenesis under ischemia and how does it function. Thus we investigated the role of CGRP in angiogenesis and the involved mechanisms. We found that CGRP level was increased in the rat hindlimb ischemic tissue. The expression of exogenous CGRP by adenovirus vectors enhanced blood flow recovery and increased capillary density in ischemic hindlimbs. In vitro, CGRP promoted human umbilical vein endothelial cell (HUVEC) tube formation and migration. Further more, CGRP activated AMP-activated protein kinase (AMPK) both in vivo and in vitro, and pharmacological inhibition of CGRP and cAMP attenuated the CGRP-activated AMPK in vitro. CGRP also induced endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs at Ser1177 and Ser633 in a time-dependent manner, and such effects were abolished by AMPK inhibitor Compound C. As well, Compound C blocked CGRP-enhanced HUVEC tube formation and migration. These findings indicate that CGRP promotes angiogenesis by activating the AMPK-eNOS pathway in endothelial cells.     

Green tea catechin inhibits ephrin-A1-mediated cell migration and angiogenesis of human umbilical vein endothelial cells

Angiogenesis, the formation of new blood vessels from preexisting capillaries, is essential for tumor progression and metastasis. During tumor neovascularization, vascular endothelial growth factor and ephrin (Eph) families emerge as critical mediators of angiogenesis. The green tea catechin epigallocatechin gallate (EGCG), a tyrosine kinase inhibitor, has been demonstrated in previous studies to be an effective antiangiogenesis agent. However, the inhibitory effect of green tea catechins on ephrin-A1-mediated tumor angiogenesis has not been demonstrated yet. Thus, in this study, we investigated the molecular mechanism of ephrin-A1-mediated cell migration and angiogenesis, as well as the inhibitory effects of EGCG. Here we show that ephrin-A1 mediates endothelial cell migration and regulates vascular remodeling in tumor neovascularization in vitro. We also demonstrated that ephrin-A1-mediated cell migration required the activation of extracellular-regulated kinase (ERK-1/2) but not of phosphatidylinositol-3-kinase. The green tea catechin EGCG inhibited ephrin-A1-mediated endothelial cell migration, as well as tumor angiogenesis, in a dose-dependent manner. Furthermore, EGCG inhibited the ephrin-A1-mediated phosphorylation of EphA2 and ERK-1/2. Taken together, these data indicated that activation of ERK-1/2 plays an essential role in ephrin-A1-mediated cell migration. EGCG inhibited ephrin-A1-mediated endothelial migration and angiogenesis. It suggests a novel antiangiogenesis application of EGCG in cancer chemoprevention.     

N-terminal parathyroid hormone-related peptide hyperpolarizes endothelial cells and causes a reduction of the coronary resistance of the rat heart via endothelial hyperpolarization

Parathyroid hormone-related peptide (PTHrP) is known to be a strong vasorelaxant peptide. The mechanisms by which PTHrP reduces the coronary resistance of the rat heart have not been worked out but seem to be independent of the classical PTH/PTHrP receptor-mediated, cAMP-dependent effect. In this study we hypothesized that PTHrP reduces the coronary resistance of the rat heart via endothelial cell hyperpolarization. Isolated microvascular endothelial cells from rat heart were incubated with PTHrP(1-36), and changes in the membrane potential were recorded via DiBAC fluorescence. Cells exposed to PTHrP showed a hyperpolarization of approximately 7mV. In the isolated Langendorff preparation, PTHrP-dependent vasodilatation of l-nitro-arginine-exposed hearts was abolished under depolarizing conditions (high potassium). Denudation of the endothelial cell layer significantly impaired the vasodilatory effect of PTHrP. In the presence of H89 (a cAMP/protein kinase A pathway antagonist) and indomethacin (a cyclooxygenase inhibitor), PTHrP dilated the vessels. In conclusion, PTHrP exerted a nitric oxide-independent vasodilatory effect that depends on endothelial cell hyperpolarization.     

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

VEGF-E activates endothelial nitric oxide synthase to induce angiogenesis via cGMP and PKG-independent pathways

Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cgamma (PLCgamma) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCgamma but independent of cGMP and PKG.