Signaling pathways mediating VEGF165-induced calcium transients and membrane depolarization in human endothelial cells.

Cytosolic calcium and membrane potential were monitored simultaneously in quiescent human umbilical vein endothelial cells (HUVEC) exposed to vascular endothelial growth factor (VEGF)165 using the fluorescent indicators indo-1 AM and DiSBAC2(3), respectively. Application of VEGF165 to cells elicits a rapid rise in cytosolic calcium followed by a slower decline toward control values. Peak calcium is associated with a slight membrane hyperpolarization; however, as calcium falls toward control, a strong depolarization develops and is sustained throughout a 10-min period of VEGF165 stimulation. Both the VEGF165-mediated rise in cytosolic calcium and membrane depolarization are eliminated by inhibitors of VEGFR-2, tyrosine kinase, src kinase and inositol-1,4,5 triphosphate-operated calcium channels. Calcium entry, which is initially facilitated by transient hyperpolarization, is restricted by a substantial, sustained depolarization that developed during the downstroke of the calcium spike. Inhibition of plasmalemmal calcium channels diminished the magnitude and duration of the calcium spike, suggesting that extracellular calcium influx, secondary to stores release, is a significant component of the calcium transient. Inhibition of chloride channels substantially reduced membrane depolarization. In addition, the depolarization is modulated by PI3 kinase in a ras-independent manner. In summary, intracellular calcium and membrane potential are influenced by several key signaling cascades of VEGFR-2 activation in HUVEC.

Role of ephrin B2 in human retinal endothelial cell proliferation and migration.

This study was designed to determine the presence of Eph B4 or ephrin B2 in human retinal endothelial cells (REC) and their signal transduction. Human retinal endothelial cells were stimulated with an Eph B4/Fc chimera and probed for phosphorylation of phosphatidylinositol-3-kinase (PI3K), Src, and mitogen-activated protein kinase (MAPK) pathways. Proliferation and migration were investigated after Eph B4/Fc stimulation in the presence of various pathway inhibitors. Human retinal endothelial cells express ephrin B2, with little expression of Eph B4. Treatment with EphB4/Fc chimera resulted in activation of PI3K, Src, and MAPK pathways. Eph B4-stimulated endothelial cell proliferation was mediated via PI3K, nitric oxide synthase, and extracellular signal-regulated kinase 1/2 (ERK1/2). Blockade of Src-PI3K pathways produced significant attenuation of Eph B4/Fc-stimulated migration. These results demonstrate for the first time that ephrin B2 is present in human retinal endothelial cells. Additionally, it appears that vascular growth may be modulated in the retina through activation of the PI3K pathway and its downstream components.

Nerve growth factor regulates human choroidal, but not retinal, endothelial cell migration and proliferation.

We have previously shown that sympathetic denervation results in significant blood vessel growth of the choroid and retina. The mechanism of this growth remains unclear. Since sympathetic denervation can result in increased nerve growth factor (NGF) levels, it was the goal of this study to determine if choroidal and retinal endothelial cells in culture would respond to nerve growth factor and if nerve growth factor promote endothelial cell migration and proliferation, two components of angiogenesis. Western blotting with phospho-specific antibodies, cell migration, and cell proliferation assays were employed to determine NGF effects on both choroidal and retinal cell growth. NGF treatment produced phosphorylation of TrkA in choroidal and retinal endothelial cells. NGF stimulation resulted in activation of ERK1/2, Akt, and Src in choroidal endothelial cells, while little phosphorylation was noted following NGF treatment in retinal endothelial cells. NGF increased choroidal endothelial cell migration by 50% over control and this was inhibited by pretreatment with LY294002 (PI3K inhibitor), Akt inhibitor, and MMP2/9 inhibitor. KT5823, PD98059, and PP2 did not affect choroidal cell migration. NGF also produced a 47% increase in choroidal endothelial cell proliferation, which was blocked by PP2, LY294002, Akt inhibitor, KT5823, and PD98059. NGF stimulation did not alter retinal endothelial cell migration or proliferation. Thus, it appears that increased NGF levels that may be noted after sympathectomy are capable of producing some aspects of vascular remodeling via different signaling cascades in choroidal endothelial cells in culture.

Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function.

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.

Fibroblast growth factor-2 mediates Angiotensin-converting enzyme inhibitor-induced angiogenesis in coronary endothelium.

The beneficial effect exerted by angiotensin-converting enzyme inhibitors (ACEI) on vascular endothelium has been attributed to restoration of endothelial cell survival properties and improvement of angiogenesis. Fibroblast growth factor (FGF)-2 is an angiogenic factor for the microvascular endothelium, which tonically promotes endothelial cell growth and survival through an autocrine/paracrine mechanism. Here, we formulate the hypothesis that FGF-2 might contribute to the prosurvival/proangiogenic effect of ACEI. We investigated zofenoprilat and, in selected experiments, lisinopril, as representatives of ACEI. These compounds induced formation of pseudocapillaries in vessel fragments isolated from porcine coronary and human umbilical arteries by increasing endothelial cell growth up to 5-fold. Angiogenesis was abolished by inhibitors of nitric-oxide synthase (NOS) pathway and by anti-FGF-2 antibodies. Consistently, in cultured coronary endothelial cells (CVECs), ACEI up-regulated endothelial NOS (eNOS) and FGF-2 and induced mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 activation. The overexpression of eNOS/FGF-2 produced, at the functional level, enhanced cell proliferation and migration, the latter effect being dose-dependent and maximal at 0.1 microM zofenoprilat. The importance of FGF-2 for the acquisition of the angiogenic phenotype elicited by ACEI was clearly demonstrated by the impairment of endothelial functions following transfection of CVECs with small interference RNA for FGF-2. Moreover, FGF-2 silencing greatly affected the nuclear translocation of the FGF receptor (FGFR)-1, highlighting the autocrine mode of action of FGF-2. At the endothelial membrane level, zofenoprilat appeared to activate the bradykinin B1 receptor, a known stimulant of FGF-2 expression. In conclusion, we show that ACEI exert protective/proangiogenic effects in microvascular coronary endothelial cells by activating the endogenous FGF-2/FGFR-1 system.

The protein kinase MEK1/2 mediate vascular endothelial growth factor- and histamine-induced hyperpermeability in porcine coronary venules.

Mitogen-activated protein kinases (MAPKs) have been implicated in the signal transduction of the endothelial response to growth factors and inflammatory stimuli. The objective of this study was to test the hypothesis that the p42/44 MAPK pathway plays a common role in mediating the microvascular hyperpermeability response to vascular endothelial growth factor (VEGF) and histamine. The apparent permeability coefficient of albumin was measured in isolated and perfused coronary venules. Application of VEGF induced a rapid increase in venular permeability, and the effect was blocked by PD98059 and UO126, selective inhibitors of the mitogen-activated protein kinase kinase MEK1/2, in a dose-dependent pattern. The same MEK1/2 inhibitors dose-dependently attenuated the increase in venular permeability caused by histamine. In addition, the increases in venular permeability caused by agents that are known to activate the nitric oxide pathway, including the calcium ionophore ionomycin, the nitric oxide donor S-nitroso-N-acetylpenicillamine, and the protein kinase G activator 8-bromo-cGMP, were significantly attenuated in venules pretreated with the MEK1/2 inhibitors. Furthermore, transfection of venules with active MEK1 increased baseline permeability. In contrast, transfection of active ERK1, a downstream target of MEK1/2, did not significantly alter the basal permeability of venules. Moreover, inhibition of ERK1/2 with a specific inhibiting peptide did not prevent the hyperpermeability response to VEGF or histamine. The results suggest that activation of MEK1/2 may play a central role in the signal transduction of microvascular hyperpermeability in response to growth factors and inflammatory mediators.

Beta 3-adrenergic receptors mediate choroidal endothelial cell invasion, proliferation, and cell elongation.

Beta(3)-adrenergic receptors have been reported to function primarily in adipose tissues to regulate thermogenesis. In this study, we determined if beta-adrenergic receptors are present on human choroidal endothelial cells and examined their ability to promote invasion, proliferation, and/or cell elongation. Using western blotting techniques and assays of cell invasion, cell proliferation, and endothelial cell elongation, we were able to determine that human choroidal endothelial cells do possess all three subtypes of beta-adrenergic receptors. Stimulation of the beta(3)-adrenergic receptor with BRL37344, a specific beta(3)-adrenergic receptor agonist, resulted in phosphorylation of Src, Akt, and ERK1/2. BRL37344 treatment also increased choroidal endothelial cell invasion by 103% above control values; the invasion response was inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), Akt inhibitor (Akt-I), and matrix metalloproteinase 2/9 inhibitor (MMP-I). Invasion was not affected by PD98059 (mek inhibitor) or KT5823 (protein kinase G inhibitor). BRL37344 produced a significant increase in the total elongation of choroidal endothelial cells formed on Matrigel over a 24hr period. BRL37344 did significantly increase proliferation, although not to the same level as invasion. Stimulation of choroidal endothelial cells with dobutamine to activate beta(1)/beta(2)-adrenergic receptors did not affect invasion, proliferation, or endothelial cell elongation. In conclusion, beta(3)-adrenergic receptors may play a role in choroidal endothelial cell invasion and elongation, while playing a more limited function in regulation of cell proliferation.

Focal adhesion kinase in neutrophil-induced microvascular hyperpermeability.

OBJECTIVE: Recent experimental evidence indicates an essential role of focal adhesion kinase (FAK) in mediating endothelial adhesion, contraction, and migration under physical stress and chemical stimulation. However, the functional impact of FAK on microvascular barrier property during inflammation has not been revealed. The aim of this study was to explore the potential contribution of FAK to neutrophil-dependent microvascular hyperpermeability. METHODS: The apparent permeability coefficient of albumin was measured in intact, isolated porcine coronary venules during stimulation by C5a-activated neutrophils. In parallel, the transendothelial flux of albumin was quantified in cultured venular endothelial cell monolayers exposed to C5a-activated neutrophils. Western blotting and immunocytochemistry were performed to assess FAK tyrosine phosphorylation and distribution in endothelial cells, respectively. To specify the signaling effect of FAK on neutrophil-elicited endothelial hyperpermeability, FAK-related nonkinase (FRNK) was expressed, purified, and directly transfected into the endothelium of venules, and the permeability response to neutrophils was measured during inhibition of FAK. RESULTS: C5a-activated neutrophils induced a time- and concentration-dependent increase in venular permeability. Transfection of venules with FRNK did not alter the basal barrier function but greatly attenuated neutrophil-induced hyperpermeability in a dose-related manner. A similar permeability response to neutrophils was observed in venular endothelial cell monolayers, which was diminished after FRNK transfection. In addition, Western blot analysis showed that activated neutrophils caused a concentration-dependent increase in FAK tyrosine phosphorylation with a time course correlating with that of venular hyperpermeability. Transfection of FRNK blocked neutrophil-evoked FAK tyrosine phosphorylation. Furthermore, immunofluorescence microscopy revealed a significant morphological change of FAK from a punctuated, dot-like pattern under normal conditions to an elongated, dash-like staining that aligned with the longitudinal axis of cells upon neutrophil stimulation. CONCLUSION: The results suggest that focal adhesion kinase significantly contributes to the endothelial hyperpermeability response to neutrophil activation. Phosphorylation of FAK may play an important signaling role in the regulation of microvascular barrier function during inflammation.

Transference of recombinant VE-cadherin cytoplasmic domain alters endothelial junctional integrity and porcine microvascular permeability.

VE-cadherin constitutes endothelial adherens junctions through a homophilic binding of its extracellular domain and by the anchoring of its intracellular domain to actin cytoskeleton via catenins. The aim of this study was to determine the functional importance of VE-cadherin-cytoskeleton association in the maintenance of endothelial junctional integrity. A recombinant VE-cadherin cytoplasmic domain (rVE-cad CPD) was expressed in E. coli and purified through Ni-NTA spin columns. Immunoprecipitation assays showed that rVE-cad CPD was able to bind beta-catenin in vitro and to compete with endogenous VE-cadherin for binding of beta-catenin in human umbilical vein endothelial cells. A significant increase in the transendothelial flux of albumin was observed in the endothelial cell monolayers transfected with rVE-cad CPD. Importantly, transfection of rVE-cad CPD into intact isolated coronary venules markedly elevated the albumin permeability of the venular endothelium. In addition, immunofluorescence microscopic analysis revealed a conformational change of VE-cadherin from a uniform, continuous distribution along the cell membrane under control conditions to a diffuse, stitch-like pattern after rVE-cad CPD transfection. The effects were likely due to an attenuated anchorage of endogenous VE-cadherin to the cytoskeleton, as evidenced by a decreased partitioning of VE-cadherin in the detergent-insoluble cytoskeletal pool. The results suggest that the intracellular association of VE-cadherin with beta-catenin-linked cytoskeleton is essential to the maintenance of endothelial junctional integrity and microvascular permeability.

Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor.

Focal adhesion kinase (FAK) is known to mediate endothelial cell adhesion and migration in response to vascular endothelial growth factor (VEGF). The aim of this study was to explore a potential role for FAK in VEGF regulation of microvascular endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Treating the vessels with VEGF induced a time- and concentration-dependent increase in Pa. Inhibition of FAK through direct delivery of FAK-related non-kinase (FRNK) into venular endothelium did not alter basal barrier function but significantly attenuated VEGF-elicited hyperpermeability. Furthermore, cultured human umbilical vein endothelial monolayers displayed a similar hyperpermeability response to VEGF which was greatly attenuated by FRNK. Western blot analysis showed that VEGF promoted FAK phosphorylation in a time course correlating with that of venular hyperpermeability. The phosphorylation response was blocked by FRNK treatment. In addition, VEGF stimulation caused a significant morphological change of FAK from a punctate pattern to an elongated, dash-like staining that aligned with the longitudinal axis of the cells. Taken together, the results suggest that FAK contributes to VEGF-elicited vascular hyperpermeability. Phosphorylation of FAK may play an important role in the signal transduction of vascular barrier response to VEGF.

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway.

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.

Beta 3-adrenergic receptors regulate retinal endothelial cell migration and proliferation.

Sympathetic nerves may play a role in vascular disorders of the eye. In the present study, we hypothesized that activation of beta3-adrenergic receptors on retinal endothelial cells would promote migration and proliferation of these cells, two markers of an angiogenic phenotype. We show, for the first time, expression of beta3-adrenergic receptors on cultured retinal endothelial cells. Activation of these receptors with BRL37344, a specific beta3-adrenergic receptor agonist, promoted migration that was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K), the mitogen activated protein kinase component MEK, and matrix metalloproteinases (MMPs) 2 and 9. BRL37344 stimulated proliferation, which could be blocked by inhibitors of Src, PI3K, and MEK. These cells also express the beta1-adrenergic receptor with no beta2-adrenergic receptor expression observed. Stimulation of the beta1-adrenergic receptor with xamoterol, a specific partial agonist, did not promote proliferation or migration. These results support the hypothesis that beta3-adrenergic receptors play a role in proliferation and migration of cultured human retinal endothelial cells.