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Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(l,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses. cGMP; vessels: sprouting; leakage; p38 doi:10.1152/ajpregu.00222.2009

Human bladder contraction mainly depends on [Ca.sup.2+] influx via L-type voltage-gated [Ca.sup.2+] channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type [Ca.sup.2+] channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type [Ca.sup.2+] channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [[Ca.sup.2+]], which was abrogated by the L-type [Ca.sup.2+] channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated [Ca.sup.2+] currents ([I.sub.Ca]), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol ([10.sup.-7] M) increased L-type [I.sub.Ca] size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol ([10.sup.-8] M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type [[alpha]sub.IC]-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30[micro]g*[kg.sup.-1]*[day.sup.-1], 2 wk). In conclusion, elocalcitol upregulated [Ca.sup.2+] entry through L-type [Ca.sup.2+] channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms. vitamin D analogue; human bladder smooth muscle cells; voltage-gated calcium channel; overactive bladder

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of [[alpha].sub.1]-adrenoceptors ([[alpha].sub.1]-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% [O.sub.2]) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce atrophic effect in human endothelial cells through stimulation of the [[alpha].sub.1D]-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective [[alpha].sub.1D]-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that [[alpha].sub.1D]-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by [N.sup.G]-nitro-L-arginine methyl ester. Conversely, the [[alpha].sub.1A]-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the [[alpha].sub.1B]-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of [[alpha].sub.1D]-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of [[alpha].sub.1D]-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury. vascular remodeling; low oxygen tension; endothelial cell growth and migration; catecholamines; nitric oxide

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fins-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% [O.sub.2]) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% [O.sub.2]) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A. monocyte chemotactic protein-1; endogenous vascular endothelial growth factor-A; remodeling; autocrine proliferation; chemokine