Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells

Dicer is a key enzyme involved in the maturation of microRNAS (miRNAs). miRNAs have been shown to be regulators of gene expression participating in the control of a wide range of physiological pathways. To assess the role of Dicer and consequently the importance of miRNAs in the biology and functions of human endothelial cells (EC) during angiogenesis, we globally reduced miRNAs in ECs by specific silencing Dicer using siRNA and examined the effects on EC phenotypes in vitro. The knockdown of Dicer in ECs altered the expression (mRNA and/or protein) of several key regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, Tie-1, endothelial nitric oxide synthase and IL-8. Although, Dicer knockdown increased activation of the endothelial nitric oxide synthase pathway it reduced proliferation and cord formation of EC in vitro. The miRNA expression profile of EC revealed 25 highly expressed miRNAs in human EC and using miRNA mimicry, miR-222/221 regulates endothelial nitric oxide synthase protein levels after Dicer silencing. Collectively, these results indicate that maintenance and regulation of endogenous miRNA levels via Dicer mediated processing is critical for EC gene expression and functions in vitro.     

Activation of the mitogen-activated protein kinase cascade is necessary but not sufficient for basic fibroblast growth factor- and epidermal growth factor-stimulated expression of endothelial nitric oxide synthase in ovine fetoplacental artery endothelial cells

Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.     

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.     

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.     

Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis

AIMS: Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-related disorders; these states are associated with an increased incidence of cardiovascular disease. We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-dependently up-regulated gene expression and protein production of VEGF and MMPs and down-regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose- and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt) and ERK(1/2) (extracellular signal-regulated kinase) pathways. The functional effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling pathways. Inhibition of PI3K/Akt and ERK(1/2) pathways led to significant decrease of visfatin-induced MMP and VEGF production and activation, along with significant reduction in endothelial proliferation and capillary tube formation. CONCLUSION: Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP production and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt signalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our findings provide novel insights into visfatin-induced endothelial angiogenesis.     

VEGF-A stimulates ADAM17-dependent shedding of VEGFR2 and crosstalk between VEGFR2 and ERK signaling

Vascular endothelial growth factor (VEGF)-A and the VEGF receptors are critical for regulating angiogenesis during development and homeostasis and in pathological conditions, such as cancer and proliferative retinopathies. Most effects of VEGF-A are mediated by the VEGFR2 and its coreceptor, neuropilin (NRP)-1. Here, we show that VEGFR2 is shed from cells by the metalloprotease disintegrin ADAM17, whereas NRP-1 is released by ADAM10. VEGF-A enhances VEGFR2 shedding by ADAM17 but not shedding of NRP-1 by ADAM10. VEGF-A activates ADAM17 via the extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase pathways, thereby also triggering shedding of other ADAM17 substrates, including tumor necrosis factor alpha, transforming growth factor alpha, heparin-binding epidermal growth factor-like growth factor, and Tie-2. Interestingly, an ADAM17-selective inhibitor shortens the duration of VEGF-A-stimulated ERK phosphorylation in human umbilical vein endothelial cells, providing evidence for an ADAM17-dependent crosstalk between the VEGFR2 and ERK signaling. Targeting the sheddases of VEGFR2 or NRP-1 might offer new opportunities to modulate VEGF-A signaling, an already-established target for treatment of pathological neovascularization.     

Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis

The functions of caveolae and/or caveolins in intact animals are beginning to be explored. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in several postnatal vascular paradigms. First, increasing levels of Cav-1 do not increase caveolae number in the endothelium in vivo. Second, despite a lack of quantitative changes in organelle number, endothelial-specific expression of Cav-1 impairs endothelial nitric oxide synthase activation, endothelial barrier function, and angiogenic responses to exogenous VEGF and tissue ischemia. In addition, VEGF-mediated phosphorylation of Akt and its substrate, endothelial nitric oxide synthase, were significantly reduced in VEGF-treated Cav-1 transgenic mice, compared with WT littermates. The inhibitory effect of Cav-1 expression on the Akt-endothelial nitric oxide synthase pathway was specific because VEGF-stimulated phosphorylation of mitogen-activated protein kinase (ERK1/2) was elevated in the Cav-1 transgenics, compared with littermates. These data strongly support the idea that, in vivo, Cav-1 may modulate signaling pathways independent of its essential role in caveolae biogenesis.     

The BTB-Kelch protein KLEIP controls endothelial migration and sprouting angiogenesis

Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1alpha. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF- and basic fibroblast growth factor-induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF- or basic fibroblast growth factor-mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases.     

Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo

Activated protein C (APC), a natural anticoagulant, has recently been demonstrated to activate the mitogen-activated protein kinase (MAPK) pathway in endothelial cells in vitro. Because the MAPK pathway is implicated in endothelial cell proliferation, it is possible that APC induces endothelial cell proliferation, thereby causing angiogenesis. We examined this possibility in the present study. APC activated the MAPK pathway, increased DNA synthesis, and induced proliferation in cultured human umbilical vein endothelial cells dependent on its serine protease activity. Antibody against the endothelial protein C receptor (EPCR) inhibited these events. Early activation of the MAPK pathway was inhibited by an antibody against protease-activated receptor-1, whereas neither late and complete activation of the MAPK pathway nor endothelial cell proliferation were inhibited by this antibody. APC activated endothelial nitric oxide synthase (eNOS) via phosphatidylinositol 3-kinase-dependent phosphorylation, followed by activation of protein kinase G, suggesting that APC bound to EPCR might activate the endothelial MAPK pathway by a mechanism similar to that of VEGF. APC induced morphogenetic changes resembling tube-like structures of endothelial cells, whereas DIP-APC did not. When applied topically to the mouse cornea, APC clearly induced angiogenesis in wild-type mice, but not in eNOS knockout mice. These in vitro events induced by APC might at least partly explain the angiogenic activity in vivo. This angiogenic activity of APC might contribute to maintain proper microcirculation in addition to its antithrombotic activity.     

Soluble receptor-mediated selective inhibition of VEGFR and PDGFRbeta signaling during physiologic and tumor angiogenesis

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.     

Vascular endothelial growth factor-induced elimination of the type 1 interferon receptor is required for efficient angiogenesis

Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) and antagonized by type 1 interferons, including IFN-α/β. On engaging their respective receptors (VEGFR2 and IFNAR), both stimuli activate protein kinase D2 (PKD2) and type 1 IFNs require PKD2 activation and recruitment to IFNAR1 to promote the phosphorylation-dependent ubiquitination, down-regulation, and degradation of the cognate receptor chain, IFNAR1. Data reveal that PKD2 activity is dispensable for VEGF-stimulated down-regulation of VEGFR2. Remarkably, VEGF treatment promotes the recruitment of PKD2 to IFNAR1 as well as ensuing phosphorylation, ubiquitination, and degradation of IFNAR1. In cells exposed to VEGF, phosphorylation-dependent degradation of IFNAR1 leads to an inhibition of type 1 IFN signaling and is required for efficient VEGF-stimulated angiogenesis. Importance of this mechanism for proangiogenic or antiangiogenic responses in cells exposed to counteracting stimuli and the potential medical significance of this regulation are discussed.     

Nifedipine-induced vascular endothelial growth factor secretion from coronary smooth muscle cells promotes endothelial tube formation via the kinase insert domain-containing receptor/fetal liver kinase-1/NO pathway.

Endothelial cells (ECs) are the critical cellular element responsible for postnatal angiogenesis. Since the calcium channel blocker (CCB) nifedipine indirectly upregulates endothelial superoxide dismutase expression by stimulating the production of vascular endothelial growth factor (VEGF) from smooth muscle cells (SMCs), we examined whether nifedipine would induce human coronary artery endothelial cell (HCEC) tube formation via an increase in VEGF production from human coronary artery SMCs (HCSMCs) in an in vitro model. Nifedipine stimulated VEGF production from HCSMCs, and this stimulation was abolished by protein kinase C (PKC) inhibitors and a bradykinin B2 receptor antagonist. In addition, supernatant derived from nifedipine-treated HCSMCs induced HCEC tube formation. This tube formation was inhibited by pretreatment with a specific inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) tyrosine kinase and an inhibitor of nitric oxide (NO) synthase. In conclusion, nifedipine increases VEGF secretion through PKC activation via the B2 receptor. The VEGF secretion directly induces HCEC tube formation via the KDR/Flk-1/NO pathway. CCBs may thus have novel beneficial effects in improving coronary microvascular blood flow in addition to their main effect of reducing blood pressure.     

Induction of cyclooxygenase-1 and -2 modulates angiogenic responses to engagement of alphavbeta3

Prostaglandins and cyclooxygenase (COX) have been implicated in the angiogenesis that occurs around tumours, but how they are induced is unclear. Prostaglandin formation is regulated by the availability of arachidonic acid and/or COX activity that in turn are controlled by activation of G-protein-coupled receptors or kinase receptors. Adhesion receptors provide another potential level of control as they transduce a variety of "outside-in" signals implicated in inflammation. We examined whether engagement of the vitronectin receptor (alphavbeta3) modulated prostacyclin (PGI2) formation in human umbilical vein endothelial cells (EC). Engagement of EC alphavbeta3 by vitronectin (versus fibronectin or gelatin) or by monoclonal antibodies (mAbs) LM609 and LIBS6, enhanced PGI2 generation and also induced expression of both COX-1 and -2 isoforms. Alphavbeta3 engagement also led to vascular endothelial cell growth factor (VEGF) generation and EC proliferation that was attenuated by inhibition of both COX-1 and COX-2. COX-1 inhibition also prevented new vessel formation in an in vitro model of angiogenesis that is alphavbeta3 dependent. Inhibition of angiogenesis by the COX-1 inhibitor was partially reversed by removal of the inhibitor or by addition of the stable analogue of PGI2, iloprost. These findings strongly indicate that alphavbeta3-mediated angiogenesis is partly due to induction of both isoforms of COX.     

Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells

The Notch ligand, Dll4, is essential for angiogenesis during embryonic vascular development and is involved in tumour angiogenesis. Several recent publications demonstrated that blockade of Dll4 signalling inhibits tumour growth, suggesting that it may constitute a good candidate for anti-cancer therapy. In order to understand the role of Dll4 at the cellular level, we performed an analysis of Dll4-regulated genes in HUVECs. The genes identified included several angiogenic signalling pathways, such as VEGF, FGF and HGF. In particular we identified downregulation (VEGFR2, placenta growth factor PlGF) of VEGF pathway components resulting in the overall effect of limiting the response of HUVEC to VEGF. However extensive upregulation of VEGFR1 was observed allowing continued response to its ligand PlGF but the soluble form of the VEGFR1, sVEGFR1 was also upregulated. PlGF enhanced tubulogenesis of HUVEC suggesting that downregulation of PlGF and upregulation of VEGFR1 including sVEGFR1 are important mechanisms by which Dll4 attenuates PlGF and VEGF signalling. Dll4-stimulated HUVECs had impaired ERK activation in response to VEGF and HGF indicating that Dll4 signalling negatively regulates these pathways. Dll4 expression reduced vessel sprout length in a 3D tubulogenesis assay confirming that Dll4 signalling inhibits angiogenesis. Altogether, our data suggest that Dll4 expression acts as a switch from the proliferative phase of angiogenesis to the maturation and stabilisation phase by blocking endothelial cell proliferation and allowing induction of a more mature, differentiated phenotype. The regulation of sVEGFR1 provides a novel mechanism for Dll4 signalling to regulate cells at distance, not just in adjacent cells.     

Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS

VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.     

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.     

Vasoactive and permeability effects of vascular endothelial growth factor-165 in the term in vitro dually perfused human placental lobule

Vascular endothelial growth factor (VEGF) is an important vasodilator and effector of permeability in systemic blood vessels. Molecular and tissue culture techniques have provided evidence for its placental synthesis and release. Using an in vitro dual-perfusion model of the term placental lobule from normal pregnancy, we report here the relative secretion of total VEGF, soluble VEGF receptor (VEGFR)-1, and free VEGF into the maternal and fetoplacental circulations of the placenta. We tested the hypothesis that VEGF has vasomotor and permeability effects in the fetoplacental circulation of the human placenta, and we examined the broad intracellular pathways involved in the vasodilatory effect that we found. We show that total VEGF is released into the fetal and maternal circulations in a bipolar fashion, with a bias toward maternal side output. Soluble VEGFR-1 was also secreted into both circulations with bias toward the maternal side. Consequently, free VEGF (12.8 +/- 2.4 pg/ml, mean +/- se) was found only in the fetoplacental circulation. VEGF-165 was found to be a potent vasodilator of the fetoplacental circulation (maximum response: 77% of previous steady-state fetal-side inflow hydrostatic pressure after preconstriction with U46619; EC(50) = 71 pm). This vasodilatory effect was mediated by the VEGFR-2 receptor and nitric oxide in a manner-independent of the involvement of prostacyclin and the src-family tyrosine kinases. However, nitric oxide could explain only 50% of the vasodilatory effect. Finally, we measured the permeability of the perfused placenta to inert hydrophilic tracers and found no difference in the presence and absence of VEGF.     

Oxidative stress and diabetic complications

Oxidative stress plays a pivotal role in the development of diabetes complications, both microvascular and cardiovascular. The metabolic abnormalities of diabetes cause mitochondrial superoxide overproduction in endothelial cells of both large and small vessels, as well as in the myocardium. This increased superoxide production causes the activation of 5 major pathways involved in the pathogenesis of complications: polyol pathway flux, increased formation of AGEs (advanced glycation end products), increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C isoforms, and overactivity of the hexosamine pathway. It also directly inactivates 2 critical antiatherosclerotic enzymes, endothelial nitric oxide synthase and prostacyclin synthase. Through these pathways, increased intracellular reactive oxygen species (ROS) cause defective angiogenesis in response to ischemia, activate a number of proinflammatory pathways, and cause long-lasting epigenetic changes that drive persistent expression of proinflammatory genes after glycemia is normalized ("hyperglycemic memory"). Atherosclerosis and cardiomyopathy in type 2 diabetes are caused in part by pathway-selective insulin resistance, which increases mitochondrial ROS production from free fatty acids and by inactivation of antiatherosclerosis enzymes by ROS. Overexpression of superoxide dismutase in transgenic diabetic mice prevents diabetic retinopathy, nephropathy, and cardiomyopathy. The aim of this review is to highlight advances in understanding the role of metabolite-generated ROS in the development of diabetic complications.     

Comparative analysis of the in vivo angiogenic properties of stable prostacyclin analogs: a possible role for peroxisome proliferator-activated receptors

OBJECTIVE: Until recently, prostacyclin (PGI2) biological activities were thought to be exclusively mediated by cell surface receptors named IP. Recent studies have instead identified a novel pathway of PGI2 signaling, occurring through activation of peroxisome proliferator-activated receptors (PPARs) located in the nucleus. The availability of stable PGI2 analogs with different affinity for IP receptors and PPARs provides the possibility to test the importance and function of this dual pathway in vitro and in vivo. In this study, the in vivo angiogenic properties of different PGI2 analogs and the potential relationship between PPAR-mediated pathways, vascular endothelial growth factor (VEGF), and angiogenesis were investigated. METHODS AND RESULTS: By using the murine corneal model of angiogenesis, we found that PGI2 analogs able to act on nuclear PPARs, such as iloprost and carbaprostacyclin (cPGI), induce angiogenesis in vivo. In contrast, cicaprost, a PGI2 analog that only acts on IP receptors, has no in vivo angiogenic activity. Interestingly, angiogenesis induced by iloprost and cPGI does not differ in extent and morphology from that induced by VEGF and is associated with local increment of VEGF mRNA expression and protein levels. Finally, iloprost-induced angiogenesis is significantly decreased by systemic inhibition of VEGF activity, obtained by gene transfer of a soluble form of the VEGF receptor Flt-1. CONCLUSIONS: These data demonstrate that stable PGI2 analogs may have angiogenic properties in vivo, depending on their ability to act on PPARs. The resulting angiogenic process appears to be mediated by VEGF. These findings indicate that important physiological activities in the cardiovascular system, such as angiogenesis and VEGF induction, may be modulated by PGI2 through specific activation of the PPAR signaling pathway in vivo, with potentially important fundamental and clinical implications.