Characterization of a Novel Caveolin Modulator That Reduces Vascular Permeability and Ocular Inflammation.

Purpose: Caveolin (Cav) regulates various aspect of endothelial cell signaling and cell-permeable peptides (CPPs) fused to domains of Cav can reduce retinal damage and inflammation in vivo. Thus, the goal of the present study was to identify a novel CPP that improves delivery of a truncated Cav modulator in vitro and in vivo. Methods: Phage display technology was used to identify a small peptide (RRPPR) that was internalized into endothelial cells. Fusions of Cav with the peptide were compared to existing molecules in three distinct assays, vascular endothelial growth factor-A (VEGF) induced nitric oxide (NO) release, VEGF induced vascular leakage, and in a model of immune mediated uveitis. Results: RRPPR was internalized efficiently and was potent in blocking NO release. Fusing RRPPR with a minimal Cav inhibitory domain (CVX51401) dose-dependently blocked NO release, VEGF induced permeability, and retinal damage in a model of uveitis. Conclusions: CVX51401 is a novel Cav modulator that reduces VEGF and immune mediated inflammation. Translational Relevance: CVX51401 is an optimized Cav modulator that reduces vascular permeability and ocular inflammation that is poised for clinical development.

Transcatheter Aortic Heart Valves: Histological Analysis Providing Insight to Leaflet Thickening and Structural Valve Degeneration.

OBJECTIVES: This study investigated processes causing leaflet thickening and structural valve degeneration (SVD). BACKGROUND: Although transcatheter aortic valve replacement (TAVR) has changed the treatment of aortic stenosis, concerns remain regarding SVD, potentially related to valve thrombosis and thickening, based on studies using computed tomography (CT). Detailed histological analyses are provided to help attain insights into these processes. METHODS: Explanted transcatheter heart valves (THVs) were evaluated for thrombosis, fibrosis, and calcification for quantification of leaflet thickness. Immunohistochemical and microscopy approaches were used to investigate SVD-associated mechanisms. RESULTS: THVs (n = 23) were obtained from 22 patients (median 81 years of age; 50% male) from 0 to 2,583 days post TAVR. Maximal leaflet thickness increased relative to implant duration (ρ = 0.427; p = 0.027). THVs explanted after >2 years were thicker than those explanted after <2 years (p = 0.007). All THVs had adherent thrombus on both aortic and ventricular sides, which beyond 60 days was seen in combination with fibrosis and beyond 4 years had calcification. Early thrombus formation (<60 days) occurred despite rapid endothelialization with an abnormal hyperplastic phenotype. Fibrosis was observed in 6 patients on both the aortic and the ventricular THV surfaces, remodeled over time, and was associated with matrix metalloproteinase-1 expression. Five THVs showed overt calcification associated with adherent thrombus and fibrosis. CONCLUSIONS: There is a time-dependent degeneration of THVs consisting of thrombus formation, endothelial hyperplasia, fibrosis, tissue remodeling, proteinase expression, and calcification. Future investigation is needed to further understand these mechanisms contributing to leaflet thickening and SVD.

Tropoelastin enhances nitric oxide production by endothelial cells.

AIMS: This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells. MATERIALS AND METHODS: Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin. RESULTS: Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W. CONCLUSION: Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.

Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance.

Caveolin-1 (Cav-1) gene inactivation interferes with caveolae formation and causes a range of cardiovascular and pulmonary complications in vivo. Recent evidence suggests that blunted Cav-1/endothelial nitric-oxide synthase (eNOS) interaction, which occurs specifically in vascular endothelial cells, is responsible for the multiple phenotypes observed in Cav-1-null animals. Under basal conditions, Cav-1 binds eNOS and inhibits nitric oxide (NO) production via the Cav-1 scaffolding domain (CAV; amino acids 82-101). Although we have recently shown that CAV residue Phe-92 is responsible for eNOS inhibition, the "inactive" F92A Cav-1 mutant unexpectedly retains its eNOS binding ability and can increase NO release, indicating the presence of a distinct eNOS binding domain within CAV. Herein, we identified and characterized a small 10-amino acid CAV subsequence (90-99) that accounted for the majority of eNOS association with Cav-1 (Kd = 49 nM), and computer modeling of CAV(90-99) docking to eNOS provides a rationale for the mechanism of eNOS inhibition by Phe-92. Finally, using gene silencing and reconstituted cell systems, we show that intracellular delivery of a F92A CAV(90-99) peptide can promote NO bioavailability in eNOS- and Cav-1-dependent fashions. To our knowledge, these data provide the first detailed analysis of Cav-1 binding to one of its most significant client proteins, eNOS.

Caveolin as a potential drug target for cardiovascular protection.

Caveolae and caveolin are key players in a number of disease processes. Current research indicates that caveolins play a significant role in cardiovascular disease and dysfunction. The far-reaching roles of caveolins in disease and dysfunction make them particularly notable therapeutic targets. In particular, caveolin-1 (Cav-1) and caveolin-3 (Cav-3) have been identified as potential regulators of vascular dysfunction and heart disease and might even confer cardiac protection in certain settings. Such a central role in vascular health therefore makes manipulation of Cav-1/3 function or expression levels clear therapeutic targets in a variety of cardiovascular related disease states. Here, we highlight the role of Cav-1 and Cav-3 in cardiovascular health and explore the potential of Cav-1 and Cav-3 derived experimental therapeutics.

Targeting endothelial dysfunction in vascular complications associated with diabetes.

Cardiovascular complications associated with diabetes remain a significant health issue in westernized societies. Overwhelming evidence from clinical and laboratory investigations have demonstrated that these cardiovascular complications are initiated by a dysfunctional vascular endothelium. Indeed, endothelial dysfunction is one of the key events that occur during diabetes, leading to the acceleration of cardiovascular mortality and morbidity. In a diabetic milieu, endothelial dysfunction occurs as a result of attenuated production of endothelial derived nitric oxide (EDNO) and augmented levels of reactive oxygen species (ROS). Thus, in this review, we discuss novel therapeutic targets that either upregulate EDNO production or increase antioxidant enzyme capacity in an effort to limit oxidative stress and restore endothelial function. In particular, endogenous signaling molecules that positively modulate EDNO synthesis and mimetics of endogenous antioxidant enzymes will be highlighted. Consequently, manipulation of these unique targets, either alone or in combination, may represent a novel strategy to confer vascular protection, with the ultimate goal of improved outcomes for diabetes-associated vascular complications.

ß-receptor antagonist treatment prevents activation of cell death signaling in the diabetic heart independent of its metabolic actions.

We have previously shown that metoprolol improves function in the diabetic heart, associated with inhibition of fatty acid oxidation and a shift towards protein kinase B signaling. The aim of this study was to determine the relative importance of these metabolic and signaling effects to the prevention of cellular damage. Diabetes was induced in male Wistar rats by a single IV injection of 60mg/kg streptozotocin, and treated groups received 15mg/kg/day metoprolol delivered subcutaneously by osmotic pumps. Echocardiography was performed 6weeks after streptozotocin injection, and the hearts immediately excised for histological and biochemical measurements of lipotoxicity, apoptosis, signaling and caveolin/caspase interactions. Metoprolol improved stroke volume and cardiac output, associated with attenuation of TUNEL staining and a more modest attenuation of caspase-3; however, the positive TUNEL staining was not associated with an increase in apoptosis or cell regeneration markers. Metoprolol inhibited CPT-1 without affecting CD36 translocation, associated with increased accumulation of triglycerides and long chain acyl CoA in the cytoplasm, and no effect on oxidative stress. Metoprolol induced a shift from protein kinase A to protein kinase B-mediated signaling, associated with a shift in the phosphorylation patterns of BCl-2 and Bad which favored BCl-2 action. Metoprolol also increased the interaction of activated caspase-3 with caveolins 1 and 3 outside caveolae. The actions of metoprolol on fatty acid oxidation do not prevent lipotoxicity; its beneficial effect is more likely to be due to pro-survival signaling and sequestration of activated caspase-3 by caveolins.

New insights into caveolae, caveolins and endothelial function.

A growing body of evidence suggests that the cholesterol-rich invaginations of the plasma membrane, known as caveolae, are essential to cardiovascular homeostasis. The considerable attention devoted to these major plasmalemma macrostructures is attributable to their involvement in compartmentalization and clustering of signalling molecules that contribute to their function in the pathophysiology of the cardiovascular system. In light of recent developments in caveolae research, a better comprehension of the role of caveolae in the vasculature and how they mediate their activity is needed.

Myoferlin is critical for endocytosis in endothelial cells.

Myoferlin is a member of the ferlin family of proteins that promotes endomembrane fusion with the plasma membrane in muscle cells and endothelial cells. In addition, myoferlin is necessary for the surface expression of vascular endothelial growth factor receptor 2 through the formation of a protein complex with dynamin-2 (Dyn-2). Since Dyn-2 is necessary for the fission of endocytic vesicles from the plasma membrane, we tested the hypothesis that myoferlin may regulates aspects of receptor-dependent endocytosis. Here we show that myoferlin gene silencing decreases both clathrin and caveolae/raft-dependent endocytosis, whereas ectopic myoferlin expression in COS-7 cells increases endocytosis by up to 125%. Interestingly, we have observed that inhibition of Dyn-2 activity or caveolin-1 (Cav-1) expression impairs endocytosis as well as membrane resealing after injury, indicating that Dyn-2 and Cav-1 also participate in both membrane fission and fusion processes. Mechanistically, myoferlin partially colocalizes with Dyn-2 and Cav-1 and forms a protein complex with Cav-1 solubilized from tissue extracts. Together, these data describe a new role for myoferlin in receptor-dependent endocytosis and an overlapping role for myoferlin-Dyn-2-Cav-1 protein complexes in membrane fusion and fission events.

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo.

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.

Myoferlin regulates vascular endothelial growth factor receptor-2 stability and function.

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase.

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides.

In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS-dependent activities. Peptides containing the Cav-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO-dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B- and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cav-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cav-1. These data highlight the importance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cav-1.

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.

Sphingosine 1-phosphate effect on endothelial cell PAF synthesis: role in cellular migration.

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) are two inflammatory mediators capable of promoting endothelial cell (EC) migration and angiogenesis. As VEGF inflammatory effect is mediated by the synthesis of endothelial platelet-activating factor (PAF) which is also contributing to VEGF chemotactic activity, we wanted to assess if S1P can trigger PAF synthesis in EC and if S1P-induced migration is PAF-dependent. Treatment of bovine aortic EC (BAEC) with S1P (10(-10)-10(-6) M) increased dose- and time-dependently the synthesis of PAF by up to 3.3-fold above the basal level, with a maximal amount of PAF detected at 20 min post-stimulation. This biological response was attenuated by inhibiting p38 mitogen-activated protein kinase (MAPK), cytosolic or secreted phospholipase A(2) (cPLA(2), sPLA(2)) activity, suggesting that p38 MAPK activation by S1P promotes the conversion of membrane phospholipids into PAF through the combined activation of cPLA(2) and sPLA(2). Interestingly, pretreatment of BAEC with extracellular PAF receptor antagonists (BN52021, 10(-5) M and CV3988, 10(-6) M) reduced by up to 42% the cellular migration induced by S1P (10(-6) M). These data demonstrate the capacity of S1P to induce PAF synthesis, which contributes in part to S1P chemotactic activity.

Antisense inhibition of Flk-1 by oligonucleotides composed of 2'-deoxy-2'-fluoro-beta-D-arabino- and 2'-deoxy-nucleosides.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA-DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2'F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-beta-D-arabinose and 2'-deoxyribose sugars (S-2'F-ANA-DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.

Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC and PLC pathways.

1. The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2. Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-phosphorylation mediated by VEGF, but did not alter immediate (0-10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4. Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5. In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.

Estrogen regulation of endothelial and smooth muscle cell migration and proliferation: role of p38 and p42/44 mitogen-activated protein kinase.

OBJECTIVE: Restenosis is a major limitation of percutaneous coronary intervention. Migration and proliferation of vascular cells remain a cornerstone in neointimal formation. The cardioprotection of estrogen is well recognized, but the intracellular mechanisms related to these beneficial effects are not completely understood. METHODS AND RESULTS: We investigated the effects of 17beta-estradiol (17betaE) on mitogen-activated protein kinase (MAPK) activity and the migration and proliferation of porcine aortic endothelial cells (PAECs) and porcine smooth muscle cells (PSMCs). Treatment with 17betaE (10(-8) mol/L) abrogated p38 and p42/44 MAPK phosphorylation mediated by platelet-derived growth factor-BB as well as the migration and proliferation of PSMCs. In contrast, treatment with 17betaE (10(-8) mol/L) induced the phosphorylation of p38 and p42/44 MAPK and the migration and proliferation of PAECs. Interestingly, the effects of 17betaE on PSMCs and PAECs were reversed by selective estrogen receptor antagonists (tamoxifen, 4-OH-tamoxifen, and raloxifen). These results suggest that in PSMCs, 17betaE inhibits chemotactic and mitogenic effects of platelet-derived growth factor-BB as well as p38 and p42/44 MAPK phosphorylation. In contrast, 17betaE promotes in PAECs the phosphorylation of p42/44 and p38 MAPK as well as the migration and proliferation of these cells. CONCLUSIONS: Treatment with 17betaE has a dual beneficial effect: the improvement of vascular healing and the prevention of restenosis after angioplasty.

Relative effects of VEGF-A and VEGF-C on endothelial cell proliferation, migration and PAF synthesis: Role of neuropilin-1.

Vascular endothelial growth factor (VEGF-A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet-activating factor (PAF). Recently, neuropilin-1 (NRP-1) has been described as a coreceptor of KDR which potentiates VEGF-A activity. However, the role of NRP-1 in numerous VEGF-A activities remains unclear. To assess the contribution of NRP-1 to VEGF-A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt-1, NRP-1, KDR or KDR and NRP-1. Cells were stimulated with VEGF-A, which binds to Flt-1, KDR and NRP-1, and VEGF-C, which binds to KDR only. VEGF-A was 12.4-fold more potent than VEGF-C in inducing KDR phosphorylation in PAEC-KDR. VEGF-A and VEGF-C showed similar potency to mediate PAEC-KDR proliferation, migration, and PAF synthesis. On PAEC-KDR/NRP-1, VEGF-A was 28.6-fold more potent than VEGF-C in inducing KDR phosphorylation and PAEC-KDR/NRP-1 proliferation (1.3-fold), migration (1.7-fold), and PAF synthesis (4.6-fold). These results suggest that cooperative binding of VEGF-A to KDR and NRP-1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP-1 receptors. In contrast, stimulation of PAEC-Flt-1 and PAEC-NRP-1 with VEGF-A or VEGF-C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP-1 on EC preferentially increases KDR activation by VEGF-A as well as KDR-mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF-A and VEGF-C have equipotent biological activities on EC in absence of NRP-1.