Inhibition of angiogenesis by vitamin D-binding protein: Characterization of anti-endothelial activity of DBP-maf

Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis ( μg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.     

Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells

VEGF(165), the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro.     

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.     

Osteoprotegerin and RANKL differentially regulate angiogenesis and endothelial cell function

Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.     

Inhibition of vascular endothelial growth factor-associated tyrosine kinase activity with SU5416 blocks sprouting in the microvascular endothelial cell spheroid model of angiogenesis

The angiogenic vascular endothelial growth factor (VEGF) is believed to play a critical role in endothelial cell proliferation, differentiation, and sprouting. Small molecules that selectively inhibit the VEGF receptor-associated tyrosine kinase activities of Flk-1 (KDR) and Flt-1 have been developed. These agents, a prototype being SU5416, have effects on the proliferation of cultured endothelial cells, constrain angiogenesis in vivo, and have been proposed as antitumor drugs. Although SU5416 inhibits in vivo angiogenesis, it is not clear which of the complex processes leading to angiogenesis are impacted by VEGF receptor-associated tyrosine kinase inhibition. We utilized SU5416 and a microvascular endothelial cell line derived from mouse heart (SMHEC4) to specifically examine the role of VEGF receptor-associated tyrosine kinase activity on in vitro models of angiogenesis. We characterized spheroid formation and sprouting, a new model of angiogenesis, in this stable cell line. SU5416 inhibits (approximately 50%) VEGF (50 ng/ml) stimulated and basal DNA synthesis of SMHEC4 cultured in monolayer. SU5416 does not prevent the aggregation and organization of SMHEC4 into tri-dimensional spheroids. CD31, a marker of differentiated endothelial cells, is negligibly expressed in monolayer cultures but highly expressed in SMHEC4 spheroids. The content and biochemical characteristics of spheroidal CD31 are unaltered by SU5416. SU5416 also does not prevent the spontaneous and rapid (approximately 3-h) alignment into cords by SMHEC4 on Matrigel. These two models suggest that the organization and differentiation of endothelial cells is independent of VEGF receptor-associated tyrosine kinase signaling. SMHEC4 spheroids embedded in collagen gels spontaneously and rapidly (approximately 6 h) sprout capillary-like projections and subsequently (1-2 days) form complex self-anastomosing networks. In addition, VEGF (50 ng/ml) markedly stimulates sprouting of capillary-like projections from SMHEC4 spheroids. Both the spontaneous and the VEGF-stimulated sprouting are nearly eliminated by SU5416. This demonstrates that VEGF receptor-associated tyrosine kinase activity is essential to the formation of capillary-like structures from SMHEC4 spheroids. Overall, these observations demonstrate that (a) the spheroid sprouting model is appropriate for the study of angiogenesis since it appears to recapitulate many of its steps and (b) SU5416 can inhibit endothelial cell proliferation and sprouting without impacting the organization and differentiation of endothelial cells.     

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.     

VEGF-mediated elevated intracellular calcium and angiogenesis in human microvascular endothelial cells in vitro are inhibited by dominant negative TRPC6

Objective: Vascular endothelial growth factor (VEGF)‐induced vascular permeability has been shown to be dependent on calcium influx, possibly through a transient receptor potential cation channel (TRPC)‐mediated cation channel with properties of the TRPC3/6/7 subfamily. To investigate further the involvement of this subfamily, we determined the effects of dominant negative TRPC6 overexpression on VEGF‐mediated changes of human microvascular endothelial cell (HMVEC) calcium, proliferation, migration, and sprouting. Methods: Cytoplasmic calcium concentration was estimated by fura‐2 fluorescence spectrophotometry, migration by Boyden chamber assay, sprouting by immunofluorescence imaging of stimulated endothelial cells, and proliferation by flow cytometry. Results: Overexpression of a dominant negative TRPC6 construct in HMVECs inhibited the VEGF‐mediated increases in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild‐type TRPC6 construct increased the proliferation and migration of HMVECs. Conclusions: TRPC6 is an obligatory component of cation channels required for the VEGF‐mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis.     

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.     

Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo

Modulation of Tie2 receptor activity by its angiopoietin ligands is crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity. It has been proposed that angiopoietins 1 (Ang1) and 2 (Ang2) are pro- and anti-angiogenic owing to their respective agonist and antagonist signaling action through the Tie2 receptor. The function of Ang2 has remained controversial, however, with recent reports suggesting that in some circumstances, it may be pro-angiogenic. We have examined this issue using the transient ocular microvessel network called the pupillary membrane as a unique in vivo model for studying the effects of vascular regulators. We show that in vivo, in the presence of endogenous vascular endothelial growth factor (VEGF)-A, Ang2 promotes a rapid increase in capillary diameter, remodeling of the basal lamina, proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from anti- to pro-angiogenic.     

ADAMTS13 promotes angiogenesis and modulates VEGF-induced angiogenesis

Plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 and its interactions with VEGF-mediated angiogenesis, the effects of ADAMTS13 on human umbilical vein endothelial cell (HUVEC) were studied in Matrigel tube formation, proliferation, cell migration, and scratch wound assays. Treatment of endothelial cells with exogenous recombinant full-length ADAMTS13 alone promoted angiogenesis in a dose-dependent manner. HUVEC incubated with 200 ng/mL ADAMTS13 (1.4 nM) resulted in a 65% increase in cell tube formation when compared to the EBM-2 control. HUVEC treated with 30 ng/mL ADAMTS13 (204.1 pM) resulted in an 83% increase in proliferation in a visual counting assay, whereas HUVEC treated with 10 ng/mL ADAMTS13 (68.0 pM) yielded a 295% increase in EC migration in a Boyden chamber assay. In contrast, ADAMTS13 inhibited VEGF-induced angiogenesis in a dose-dependent manner, with 200ng/mL inhibiting tube formation by 35%. HUVEC co-incubated with ADAMTS13 and an antibody to the ADAMTS13 thrombospondin domains 5-7 reversed the inhibition of tube formation. HUVEC treated with 30 ng/mL ADAMTS13 and 6.2 ng/mL (323.0 pM) VEGF proliferated 40% slower than the VEGF control after 24 h of incubation as measured by visual counting assay. Treatment of HUVEC with 6.2 ng/mL VEGF and 10 ng/mL ADAMTS13 inhibited cell migration by 48%, compared to the VEGF control. Substitution of ADAMTS13 with truncated ADAMTS13 (deletion of C-terminal TSP1 domain) did not significantly increase angiogenesis or suppress VEGF-induced angiogenesis, suggesting that the TSP1 domain is involved in ADAMTS13 angiogenic activities. Co-immunoprecipitation experiments provided further evidence that ADAMTS13 binds to VEGF via its TSP1 domain.     

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

BACKGROUND/AIMS: We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo. METHODS: The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining. RESULTS: VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors. CONCLUSIONS: The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.     

Anti-angiogenesis effects of aldosterone antagonist diuretics

Angiogenesis requires endothelial cell proliferation and their vascular rearrangement. A report of inhibiting effect of spironolactone on smooth muscle cell proliferation led us to study in vitro the effects of this drug on the endothelial cell proliferation and migration. Spironolactone (10 to 100 microM) and one of its active metabolite, canrenone (10 to 100 microM), are added to human umbilical vein endothelial cells (HUVEC). Their effect on cellular proliferation is evaluated by measuring the amount of the cellular nucleic acids using a fluorometric assay (CyQuant). Cell migration is measured using a multiwell chamber assay (Transwell). In further experiments, we investigated their effect on the capillary-like tube formation in vitro generated by HUVEC seeded in a three-dimensional biological gel (Matrigel). The VEGF (10 ng/mL) and the bFGF (10 ng/mL) were used as mitotic and cell differentiation factors. Effect on cell cycle distribution is investigated by flow cytometry analysis. Spironolactone inhibits HUVEC proliferation but canrenone does not have any significant effect. The growth promoters VEGF or bFGF do not modify inhibiting effect of spironolactone. Spironolactone (50 microM) and canrenone (50 microM) are without effect on cell migration. Capillary-like networks on Matrigel is not modified by spironolactone or canrenone. Spironolactone inhibits progression through S phase of the cell cycle. Spironolactone inhibits the proliferation of the endothelial cells in vitro but shows no effect on their migration and their rearrangement in capillary-like structures. These data should be confirmed in models of angiogenesis in vivo.     

Aggretin, a snake venom-derived endothelial integrin alpha 2 beta 1 agonist, induces angiogenesis via expression of vascular endothelial growth factor

Aggretin, a collagen-like alpha 2 beta 1 agonist purified from Calloselasma rhodostoma venom, was shown to increase human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC migration toward immobilized aggretin was also increased. These effects were blocked by A2-IIE10, an antibody raised against integrin alpha 2. Aggretin bound to HUVECs in a dose-dependent and saturable manner, which was specifically inhibited by A2-IIE10, as examined by flow cytometry. Aggretin elicited significant angiogenic effects in both in vivo and in vitro angiogenesis assays, and incubation of HUVECs with aggretin activated phosphatidylinositol 3-kinase (PI3K), Akt, and extracellular-regulated kinase 1/2 (ERK1/2); these effects were blocked by A2-IIE10 or vascular endothelial growth factor (VEGF) monoclonal antibody (mAb). The angiogenic effect induced by aggretin may be via the production of VEGF because the VEGF level was elevated and VEGF mAb pretreatment inhibited Akt/ERK1/2 activation as well as the in vivo angiogenesis induced by aggretin. The VEGF production induced by aggretin can be blocked by A2-IIE10 mAb pretreatment. In conclusion, aggretin induces endothelial cell proliferation, migration, and angiogenesis by interacting with integrin alpha 2 beta 1 leading to activation of PI3K, Akt, and ERK1/2 pathways, and the increased expression of VEGF may be responsible for its angiogenic activity.     

Angiostatin diminishes activation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular endothelial cells

Angiostatin is an endogenous inhibitor of angiogenesis that was isolated from tumor-bearing mice. It has been established that angiostatin inhibits endothelial cell proliferation; however, the underlying mechanisms remain to be elucidated. Here we report that angiostatin reduces transiently the phosphorylation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular cells, but not in human vascular smooth muscle cells or human dermal fibroblasts. We demonstrate that angiostatin diminishes ERK activation by basic fibroblast growth factor and vascular endothelial growth factor. Dephosphorylation of ERK and other tyrosine-phosphorylated proteins was blocked by pretreatment of the cells with sodium meta-vanadate, an inhibitor of protein tyrosine phosphatases, indicating that angiostatin signaling may require the activity of a tyrosine phosphatase. Concentrations of angiostatin that inhibited ERK activation also inhibited basic fibroblast growth factor-stimulated collagen gel invasion by endothelial cells, but did not affect endothelial cell proliferation. We thus show that angiostatin inhibits primarily the invasion of endothelial cells and exerts minimal (if any) effects on their proliferation. Invasion is a process that involves proteolysis, adhesion and migration, all of which have been linked to ERK signaling.     

Natriuretic peptides suppress vascular endothelial cell growth factor signaling to angiogenesis

Vascular endothelial cell growth factor (VEGF) is essential for angiogenesis. Atrial natriuretic peptide (ANP) inhibits the production of VEGF, but whether this important vascular peptide also inter- rupts VEGF signaling to angiogenesis is unknown. In cultured bovine aortic endothelial cells, VEGF significantly stimulated extracellular signal-regulated protein kinase activity and phosphorylation, which was inhibited 60% by coincubation with ANP or a natriuretic peptide clearance receptor specific ligand (NPRC), C-type NAP-(4-23) [C-ANP-(4-23)]. VEGF also stimulated c-Jun N-terminal kinase (JNK) and p38 activities/phosphorylation that were prevented by the two natriuretic peptides (NP). A specific NP guanylate cyclase (GC) receptor antagonist, HS-142-1, blocked the actions of ANP [but not those of C-ANP-(4-23)], supporting the involvement of both GC and NPRC receptors. VEGF and expression of constituitively active JNK each stimulated the synthesis of cyclin D1 and increased the activity of the cyclin-dependent kinase-4, which was inhibited 55% by ANP. VEGF induced endothelial cell proliferation and migration, which was significantly blocked by NP or by expressing a dominant negative JNK-1. VEGF stimulated human microvascular endothelial cells to form capillary tubes, which was significantly inhibited by expressing dominant negative JNK-1 and by NP. Therefore, VEGF induction of critical steps in angiogenesis is enhanced through JNK activation. The actions are significantly prevented by NP, which act through both the NPRC and GC receptors to block growth factor signaling. Thus, NP are candidate antiangiogenesis factors that inhibit both the synthesis and function of VEGF.     

Targeting the vascular endothelial growth factor in hematologic malignancies

There exists increasing evidence that apart from solid tumors, angiogenic growth factors also play important roles in the development and/or maintenance of hematolymphoid malignancies. Thus, in these cancers, angiogenesis and bone marrow microvessel density often correlate with prognosis and disease burden. Several reports speculated on the role of angiogenesis and the resulting possible therapeutic options in hematologic malignancies. The most prominent angiogenic factor, vascular endothelial growth factor (VEGF), is expressed in a number of established leukemic cell lines as well as in freshly isolated human leukemias and lymphomas, and several human leukemias express VEGF receptor 1 and/or VEGF receptor 2. VEGF/VEGF‐receptor interactions are also involved in proliferation, migration, and survival of leukemic cells by autocrine and paracrine mechanisms. As a consequence, a possible drugable effect by inhibiting VEGF signaling in different hematologic malignancies has been discussed. This review focuses on angiogenesis‐independent effects of VEGF on survival and proliferation of leukemic or lymphoma cells and on possible therapeutic approaches using anti‐VEGF/VEGF‐receptor therapies to inhibit proliferation or induce apoptosis of malignant cells in hematologic diseases.     

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) plays an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs). Here we characterize the mechanism by which PILSAP regulates the vascular endothelial growth factor (VEGF)-stimulated proliferation of ECs. The specific elimination of PILSAP expression or its enzymatic activity inhibited VEGF-stimulated G1/S transition in ECs. This G1 arrest correlated with reduced cyclin dependent kinase 4/6 (CDK4/6) activity and retinoblastoma (Rb) protein phosphorylation. Analyses of signaling molecules upstream of CDK4/6 revealed that S6 kinase (S6K) activation was affected by PILSAP, whereas that of phosphatidylinositol-3 kinase (PI3K), Akt, and extracellular signal-related kinase 1/2 (ERK1/2) was not. We further demonstrated that PILSAP bound phosphatidylinositol-dependent kinase 1 (PDK1) and removed 9 amino acids from its N-terminus, which allowed S6K to associate with PDK1 and PILSAP upon VEGF stimulation. We constructed mutant PILSAP, which lacked the aminopeptidase activity but bound PDK1. Mutant PILSAP abrogated S6K activation upon VEGF stimulation in a dominant-negative manner. An N-terminal truncated form of PDK1 abolished the dominant-negative effect of mutant PILSAP. Finally, the introduction of a mutated PILSAP gene in ECs inhibited angiogenesis and retarded tumor growth in vivo. These results indicate that PILSAP plays a crucial role in the cell cycle progression of ECs and angiogenesis via the binding and modification of PDK1.     

Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway

Lysophosphatidylcholine (lysoPC), a major lipid component of oxidized low density lipoprotein, inhibits endothelial cell (EC) migration and proliferation, which are critical processes during angiogenesis and the repair of injured vessels. However, the mechanism(s) of lysoPC-induced inhibition of EC migration and proliferation has not been clarified. In this report, we demonstrate the critical role of extracellular signal-regulated kinase (ERK) in growth factor-stimulated EC migration and proliferation as well as their inhibition by lysoPC. EC migration and proliferation stimulated by basic fibroblast growth factor (FGF-2) were blocked by inhibition of ERK activity by both the specific mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 and the overexpression of a dominant-negative mutant of MEK1. Conversely, overexpression of a constitutively active mutant of MEK1 increased EC migration and proliferation, which were comparable to those of ECs stimulated with FGF-2. LysoPC inhibited FGF-2-induced ERK activation via prevention of Ras activation without inhibiting tyrosine phosphorylation of phospholipase C-gamma. Taken together, our data demonstrate that ERK activity is required for FGF-2-induced EC migration and proliferation and suggest that inhibition of the Ras/ERK pathway by lysoPC contributes to the reduced EC migration and proliferation.     

Angiostatin selectively inhibits signaling by hepatocyte growth factor in endothelial and smooth muscle cells

Angiostatin, an inhibitor of angiogenesis, contains 3 to 4 kringle domains that are derived from proteolytic cleavage of plasminogen. The antiangiogenic effects of angiostatin occur, in part, from its inhibition of endothelial cell surface adenosine triphosphate synthase, integrin functions, and pericellular proteolysis. Angiostatin has structural similarities to hepatocyte growth factor (HGF; "scatter factor"), a promoter of angiogenesis, that induces proliferation and migration of both endothelial and smooth muscle cells via its cell surface receptor, c-met. We hypothesized that angiostatin might block HGF-induced signaling in endothelial and smooth muscle cells. Angiostatin inhibited HGF-induced phosphorylation of c-met, Akt, and ERK1/2. Angiostatin also significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) induced by HGF. In contrast, angiostatin did not inhibit vascular endothelial growth factor (VEGF)-or basic fibroblast growth factor (bFGF)-induced signaling events or HUVEC proliferation. Angiostatin bound to immobilized truncated c-met produced by A431 cells and could be immunoprecipitated as a complex with soluble c-met. HGF inhibited the binding of (125)I-angiostatin to HUVECs. Soluble c-met, produced by several tumor cell lines, could inhibit the antiangiogenic effect of angiostatin. The disruption of HGF/c-met signaling is a novel mechanism for the antiangiogenic effect of angiostatin.