Increasing survival of ischemic tissue by targeting CD47

Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.     

Thrombospondin-1 antagonizes nitric oxide-stimulated vascular smooth muscle cell responses

OBJECTIVE: Endothelial-derived nitric oxide (NO), by increasing cGMP, is a major physiological regulator of vascular tone and of vascular smooth muscle cell (VSMC) adhesion, chemotaxis, and proliferation. Thrombospondin-1 is a potent antagonist of NO/cGMP signaling in endothelial cells. Because endothelial and VSMC typically exhibit opposing responses to thrombospondin-1, we examined thrombospondin-1 effects on NO signaling in VSMC. METHODS: Effects of exogenous thrombospondin-1 on human VSMC adhesion, chemotaxis, proliferation, and cGMP signaling were examined. Endogenous thrombospondin-1 function was characterized by comparing NO signaling in VSMC from wild type and thrombospondin-1 null mice. RESULTS: Picomolar concentrations of exogenous thrombospondin-1 prevented adhesive, chemotactic, and proliferative responses of human aortic VSMC stimulated by low dose NO. A recombinant CD36-binding domain of thrombospondin-1 or antibody ligation of CD36 similarly inhibited NO-stimulated VSMC responses. Thrombospondin-1 and CD36 ligation inhibited NO responses in VSMC by preventing cGMP accumulation. Thrombospondin-1 null VSMC responses to NO and cGMP signaling were enhanced relative to wild type murine VSMC. CONCLUSIONS: In the presence of NO, thrombospondin-1 is converted from a weak stimulator to a potent inhibitor of VSMC responses. Both exogenous and endogenous thrombospondin-1 inhibit NO signaling in VSMC. This activity is mediated by the type 1 repeats and utilizes the same CD36-dependent cGMP signaling pathway in endothelial and VSMC.     

Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling

Platelet alpha-granules constitute the major rapidly releasable reservoir of thrombospondin-1 in higher animals. Although some fragments and peptides derived from thrombospondin-1 stimulate or inhibit platelet aggregation, its physiologic function in platelets has remained elusive. We now show that endogenous thrombospondin-1 is necessary for platelet aggregation in vitro in the presence of physiologic levels of nitric oxide (NO). Exogenous NO or elevation of cGMP delays thrombin-induced platelet aggregation under high shear and static conditions, and exogenous thrombospondin-1 reverses this delay. Thrombospondin-1-null murine platelets fail to aggregate in response to thrombin in the presence of exogenous NO or 8Br-cGMP. At physiologic concentrations of the NO synthase substrate arginine, thrombospondin-1-null platelets have elevated basal cGMP. Ligation of CD36 or CD47 is sufficient to block NO-induced cGMP accumulation and mimic the effect of thrombospondin-1 on aggregation. Exogenous thrombospondin-1 also reverses the suppression by NO of alphaIIb/beta3 integrin-mediated platelet adhesion on immobilized fibrinogen, mediated in part by increased GTP loading of Rap1. Thrombospondin-1 also inhibits cGMP-mediated activation of cGMP-dependent protein kinase and thereby prevents phosphorylation of VASP. Thus, release of thrombospondin-1 from alpha-granules during activation provides positive feedback to promote efficient platelet aggregation and adhesion by overcoming the antithrombotic activity of physiologic NO.     

Thrombospondin-1, endothelium and systemic vascular tone

Evaluation of: Bauer EM, Qin Y, Miller TW et al.: Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial dependent vasorelaxation. Cardiovasc. Res. 88, 471-481 (2010). Several lines of evidence, both in vivo and ex vivo, suggest that thrombospondin-1 (TSP-1) is important in maintaining systemic vascular tone. Recently published papers demonstrate that TSP-1 can inhibit vascular smooth muscle relaxation by interfering with the interaction between nitric oxide (NO) and soluble guanylyl cyclase, providing a possible mechanism of action to explain this observation. While these in vitro experiments in vascular smooth muscle cells were provocative, it is not clear how such a large protein circulating in the plasma could cross the intact endothelial basal membrane and regulate NO/cGMP signaling in smooth muscle in vivo. This raised the question of whether TSP-1 could modulate NO/cGMP signaling through another mechanism. Herein, we evaluate a recently published paper by Bauer and colleagues that examined whether TSP-1 could exert vasoactive effects without directly accessing the vascular smooth muscle. In their studies they found that TSP-1 could inhibit the NO/cGMP signaling pathway through an alternate mechanism: inhibiting the activation of endothelial NO synthase (eNOS), and therefore NO production in endothelial cells. These findings, combined with previous results from these investigators, suggest that TSP-1 can blunt NO/cGMP signaling through two different mechanisms: inhibiting NO production in endothelial cells by preventing the agonist-induced influx of Ca(2+) required to activate endothelial NO synthase and blunting the ability of endothelial-derived NO to activate soluble guanylyl cyclase in vascular smooth muscle cells. The importance of these two pathways in supporting systemic and pulmonary vascular tone in health and disease is unclear.     

Translational therapeutics of dipyridamole

Dipyridamole (DP) is a phosphodiesterase inhibitor that increases the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP) by preventing their conversion to AMP and GMP, respectively. By increasing cAMP and cGMP levels in platelets, DP reversibly inhibits platelet aggregation and platelet-mediated thrombotic disease. In addition, DP may potentiate some of the vascular protective effects of endothelium-derived nitric oxide (NO), which increases cGMP by stimulating soluble guanylyl cyclase. Endothelium-derived NO is an important regulator of vascular tone, blood flow, and tissue perfusion. Indeed, endothelial NO synthase-deficient (eNOS-/-) mice exhibit elevated systemic blood pressure and have larger myocardial and cerebral infarct size after ischemic injury. Other NO/cGMP-dependent effects that may be potentiated by DP include inhibition of vascular smooth muscle proliferation and prevention of endothelial-leukocyte interaction. In addition, DP increases local concentrations of adenosine and prostacyclin, which could affect vascular tone and inflammation. Finally, DP has antioxidant properties, which could stabilize platelet and vascular membranes as well as prevent the oxidation of low-density lipoprotein. These platelet and nonplatelet actions of DP may contribute to some of its therapeutic benefits in vascular disease.     

Diet-induced obesity and diabetes reduce coronary responses to nitric oxide due to reduced bioavailability in isolated mouse hearts

AIM: The aim of the present study was to examine nitric oxide (NO)-mediated coronary vascular responses in a mouse model of obesity and diabetes induced by a high-fat, high-carbohydrate diet. We hypothesized that endogenous NO bioavailability would be reduced in obese/diabetic mouse hearts due to enhanced superoxide anion production, and that coronary smooth muscle responses to exogenous NO would be reduced. METHODS: Age-matched, male C57BL/6J mice were fed either a control diet or a high-fat, high-carbohydrate diet. After 15 weeks, the mice were anesthetized and their hearts were removed and perfused by the Langendorff method under constant flow conditions with an oxygenated buffer solution, and changes in coronary vascular resistance were quantified. RESULTS: Mice fed the high-fat, high-carbohydrate diet became obese, hyperglycaemic and hyperinsulinaemic. Coronary vasoconstrictor responses to NO synthase inhibition by N(omega)-nitro-L-arginine methyl ester were reduced in obese/diabetic mice; normal responses were restored by pretreatment with the superoxide dismutase mimetic 2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol). Coronary endothelium-independent vasodilation to the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) was reduced; however, 8-bromo-cyclic guanosine monophosphate (cGMP)-induced vasodilation was unchanged in obese/diabetic hearts. CONCLUSIONS: These findings suggest that in a diet-induced mouse model of obesity and diabetes, NO bioavailability is reduced by increased superoxide NO scavenging leading to impaired NO-mediated vasodilation. Furthermore, the attenuation of SNAP-induced vasodilation may be due to increased reactive oxygen species scavenging of exogenous NO because normal vascular smooth muscle NO signalling is maintained as indicated by similar 8-bromo-cGMP responses in control and obese/diabetic hearts.     

Vascular system: role of nitric oxide in cardiovascular diseases

In contrast with the short research history of the enzymatic synthesis of nitric oxide (NO), the introduction of nitrate-containing compounds for medicinal purposes marked its 150th anniversary in 1997. Glyceryl trinitrate (nitroglycerin) is the first compound of this category. On October 12, 1998, the Nobel Assembly awarded the Nobel Prize in Medicine or Physiology to scientists Robert Furchgott, Louis Ignarro, and Ferid Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system. NO-mediated signaling is a recognized component in various physiologic processes (eg, smooth muscle relaxation, inhibition of platelet and leukocyte aggregation, attenuation of vascular smooth muscle cell proliferation, neurotransmission, and immune defense), to name only a few. NO has also been implicated in the pathology of many inflammatory diseases, including arthritis, myocarditis, colitis, and nephritis and a large number of pathologic conditions such as amyotrophic lateral sclerosis, cancer, diabetes, and neurodegenerative diseases. Some of these processes (eg, smooth muscle relaxation, platelet aggregation, and neurotransmission) require only a brief production of NO at low nanomolar concentrations and are dependent on the recruitment of cyclic guanosine monophosphate (cGMP)-dependent signaling. Other processes are associated with direct interaction of NO or reactive nitrogen species derived from it with target proteins and requires a more sustained production of NO at higher concentrations but do not involve the cGMP pathway.     

Myoglobin’s novel role in nitrite-induced hypoxic vasodilation

Hypoxic vasodilation represents a key physiological response of the cardiovascular system to low tissue oxygen tension, adjusting local blood flow to meet the metabolic requirements in tissue. Vasodilation occurs by nitric oxide (NO) activation of the cyclic guanosine monophosphate (cGMP) signaling pathway in vascular smooth muscle cells. Under normoxia, NO is formed by the well-known endothelial NO synthase (eNOS) system while under hypoxia NO is generated from nitrite. We have unraveled the heme-protein myoglobin in vascular smooth muscle cells as a major source of NO generation by reduction of endogenous nitrite under hypoxia. This mediates hypoxic vasodilation under physiological conditions without direct involvement of eNOS and independently of effects on cardiac function.     

Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction

Studies with cGMP-dependent protein kinase I (cGK-I)-deficient human cells and mice demonstrated that cGK-I ablation completely disrupts the NO/cGMP pathway in vascular tissue, which indicates a key role of this protein kinase as a mediator of the NO/cGMP action. Analysis of the vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP) is a useful tool to monitor cGK-I activation in platelets and cultured endothelial and smooth muscle cells. Therefore, we investigated whether endothelial dysfunction and/or vascular NO bioavailability is reflected by decreased vessel wall P-VASP and whether improvement of endothelial dysfunction restores this P-VASP. Incubation of aortic tissue from New Zealand White Rabbits with the NOS inhibitor N:(G)-nitro-Ld-arginine and endothelial removal strikingly reduced P-VASP. Oxidative stress induced by inhibition of CuZn superoxide dismutase increased superoxide and decreased P-VASP. Endothelial dysfunction in hyperlipidemic Watanabe rabbits (WHHL) was associated with increased vascular superoxide and with decreased P-VASP. Treatment of WHHL with AT(1) receptor blockade improved endothelial dysfunction, reduced vascular superoxide, increased vascular NO bioavailability, and increased P-VASP. Therefore, the level of vessel P-VASP closely follows changes in endothelial function and vascular oxidative stress. P-VASP is suggested to represent a novel biochemical marker for monitoring the NO-stimulated sGC/cGK-I pathway and endothelial integrity in vascular tissue.     

Sildenafil Citrate,a Selective Phosphodiesterase Type 5 Inhibitor:: Research and Clinical Implications in Erectile Dysfunction

Under normal physiological conditions, following sexual stimulation, release of nitric oxide (NO) from penile non-adrenergic, non-cholinergic nerves and the endothelium activates guanylyl cyclase and induces intracellular cGMP synthesis in erectile tissue trabecular smooth muscle cells. Increased cGMP levels reduce intracellular Ca²⁺ concentrations, inhibiting smooth muscle contractility and thereby initiating the erectile response. Phosphodiesterase type 5 (PDE type 5) is the predominant enzyme responsible for cGMP hydrolysis in trabecular smooth muscle. Activation of PDE type 5 terminates NO-induced, cGMP-mediated smooth muscle relaxation, resulting ultimately in restoration of basal smooth muscle contractility and penile flaccidity. Sildenafil citrate is a potent PDE type 5 reversible and selective inhibitor that blocks cGMP hydrolysis effectively (K_i ∼3 nM). Under conditions of excessive adrenergic tone or impaired neurovascular status, following sexual stimulation, sildenafil acts to enhance NO-mediated smooth muscle relaxation, resulting in improved penile erection in men with erectile dysfunction. In this review, we summarize the current state of knowledge of the physiology of penile erection and the pharmacology, metabolism and clinical experience with sildenafil citrate in the management of erectile dysfunction.     

Nitric Oxide and cGMP in Regulation of Arterial Tone

Nitric Oxide (NO) is an important factor in the control of vascular tone and peripheral resistance. Guanosine 3',5'-monophosphate (cGMP) mediates NO-induced vasorelaxation via multiple mechanisms, including decreased Ca(2+) entry and release, enhanced Ca(2+) extrusion, and inhibition of sensitization of myofilaments to Ca(2+) caused by some agonists such as norepinephrine (but not others such as ATP). This may result in differential effects of NO, depending on the agonist and the smooth muscle phenotype. In blood vessels exposed to inflammatory stimuli (for instance in endotoxemia), enhanced NO production causes loss of vascular reactivity to vasoconstrictor agents. This results from the induction of NO synthase activity in vascular cells, especially in the adventitia. The role of the adventitia may explain differences between large and small resistance arteries, in addition to the phenotype of smooth muscle cells. Protein-bound dinitrosyl non-heme iron complexes with thiols can be generated in arteries subsequent to the induction of NO synthase. Low molecular thiols can displace Fe-NO from these complexes, leading to activation of guanylyl cyclase and vasorelaxation. This may represent a novel mechanism of NO storage and release, enabling prolonged effects of NO in blood vessels and, perhaps, protection of vascular tissue against oxidative injury in sepsis and other inflammatory diseases.     

Coadministration of endothelial and smooth muscle progenitor cells enhances the efficiency of proangiogenic cell-based therapy

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.     

Nitric oxide and C-type atrial natriuretic peptide stimulate primary aortic smooth muscle cell migration via a cGMP-dependent mechanism: relationship to microfilament dissociation and altered cell morphology

Migration of aortic smooth muscle cells is thought to be of essential importance in vascular restenosis, remodeling, and angiogenesis. Recent studies have shown that NO donors inhibit the migration of subcultured aortic smooth muscle cells. However, there is evidence that NO elicits opposite effects on cell proliferation in primary versus subcultured cells, indicating fundamental differences among different models of aortic smooth muscle cell cultures. The purpose of the current study was to investigate the effect of NO donors on migration of primary cultures of rat aortic smooth muscle cells and to compare and contrast their response with those in subcultured cells. A second purpose was to investigate some of the underlying mechanisms associated with NO-induced effects on cell migration. We report that 2 NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 2, 2-(hydroxynitrosohydrazino)bis-ethanamine, stimulated the migration of primary cells in a wounded-culture model as well as in a transwell migration model. The effect of NO donors was mimicked by 2 cGMP analogues and C-type natriuretic peptide and blocked by a specific inhibitor of guanyl cyclase, 1H-(1,2,4)oxadiazolo[4,3, -a]quinoxalin-1-one, indicating the involvement of cGMP as second messenger. Moreover, neither NO donors nor cGMP analogues altered migration of primary cultures stimulated by either FBS or angiotensin II. In contrast to its effect in primary cultures, SNAP did not alter basal or stimulated migration of subcultured cells, except at a relatively high concentration of 1 mmol/L, at which migration was inhibited. The migration-stimulatory effect of NO donors and cGMP was associated with altered cell morphology and dissociation of actin filaments, consistent with recent studies indicating that cell morphology and cytoskeletal organization influence cell migration. The results suggest the possible involvement of NO-induced cell migration in vascular injury or remodeling, representing conditions in which vascular NO levels would be expected to be elevated.     

Soluble guanylate cyclase alpha(1) and beta(1) gene transfer increases NO responsiveness and reduces neointima formation after balloon injury in rats via antiproliferative and antimigratory effects

In vascular smooth muscle cells, NO stimulates the synthesis of cGMP by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1) and beta(1) subunits. NO/cGMP signal transduction affects multiple cell functions that contribute to neointima formation after vascular injury. Balloon-induced vascular injury was found to decrease sGC subunit expression and enzyme activity in rat carotid arteries. The effect of restoring sGC enzyme activity on neointima formation was investigated using recombinant adenoviruses specifying sGC alpha(1) and beta(1) subunits (Adalpha1 and Adbeta1). Coinfection of cultured rat aortic smooth muscle cells with Adalpha1 and Adbeta1 increased NO-stimulated intracellular cGMP levels 60-fold and decreased DNA synthesis and migration by 16% and 48%, respectively. Immunoreactivity for alpha(1) and beta(1) subunits colocalized in carotid arteries infected with Adalpha1 and Adbeta1. Molsidomine-stimulated carotid tissue cGMP levels were greater after coinfection with Adalpha1 and Adbeta1 than after infection with a control virus, AdRR5 (0.53+/-0.09 pmol/mg protein, mean+/-SEM, versus 0.23+/-0.09, P<0.05). Mean intima/media ratio, 2 weeks after balloon injury and twice-daily administration of 5 mg/kg molsidomine, was less in rats coinfected with Adalpha1 and Adss1 than in rats infected with AdRR5 or in uninfected rats (0.36+/-0.11 versus 0. 81+/-0.13 and 0.75+/-0.25, respectively, P<0.05). Thus, Adalpha1 and Adbeta1 gene transfer to balloon-injured rat carotid arteries increases NO responsiveness and attenuates neointima formation via a direct antiproliferative and antimigratory effect on vascular smooth muscle cells.     

Angiotensin AT2 receptors directly stimulate renal nitric oxide in bradykinin B2-receptor-null mice

Both bradykinin B2 and angiotensin II type 2 (AT2) receptors are known to stimulate renal production of nitric oxide (NO). To evaluate the individual contributions of AT2 and B2 receptors to renal NO production, we monitored renal interstitial, stable NO metabolites and cGMP by a microdialysis technique in conscious, bradykinin B2-null and wild-type mice (n=8 in each group) during low sodium intake alone or with the angiotensin AT1 or AT2 receptor blockers, valsartan (0.5 microg/min) or PD123319 (0.15 microg/min), or both. During normal salt intake, renal interstitial fluid NO and cGMP levels in B2-null mice were not different from those of wild-type mice. Low sodium intake increased NO and cGMP in wild-type mice but not in B2-null mice. Valsartan increased NO and cGMP in both wild-type and B2-null mice but to a significantly greater degree in the wild-type than in B2-null mice. PD123319 decreased NO and cGMP in both wild-type and B2-null mice. Combined valsartan and PD123319 decreased NO and cGMP in both wild-type and B2-null mice, but there was no significant difference during combined treatment from their levels after administration of PD123319 alone. Our results indicate that during ingestion of a low-salt diet, production of NO is mediated mainly via the AT2-B2 receptor cascade. Blockade of the AT1 receptor enhances the production of NO via the AT2 receptor in both wild-type and B2-null mice. We conclude that NO can be produced by 2 alternative pathways: directly through the AT2 receptor or indirectly from AT2 receptor stimulation of bradykinin via the B2 receptor.     

Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor

Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.     

Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase.

Nitric oxide (NO)-induced relaxation is associated with increased levels of cGMP in vascular smooth muscle cells. However, the mechanism by which cGMP causes relaxation is unknown. This study tested the hypothesis that activation of Ca-sensitive K (KCa) channels, mediated by a cGMP-dependent protein kinase, is responsible for the relaxation occurring in response to cGMP. In rat pulmonary artery rings, cGMP-dependent, but not cGMP-independent, relaxation was inhibited by tetraethylammonium, a classical K-channel blocker, and charybdotoxin, an inhibitor of KCa channels. Increasing extracellular K concentration also inhibited cGMP-dependent relaxation, without reducing vascular smooth muscle cGMP levels. In whole-cell patch-clamp experiments, NO and cGMP increased whole-cell K current by activating KCa channels. This effect was mimicked by intracellular administration of (Sp)-guanosine cyclic 3',5'-phosphorothioate, a preferential cGMP-dependent protein kinase activator. Okadaic acid, a phosphatase inhibitor, enhanced whole-cell K current, consistent with an important role for channel phosphorylation in the activation of NO-responsive KCa channels. Thus NO and cGMP relax vascular smooth muscle by a cGMP-dependent protein kinase-dependent activation of K channels. This suggests that the final common pathway shared by NO and the nitrovasodilators is cGMP-dependent K-channel activation.     

Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner

Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking the angiogenesis inhibitor thrombospondin-1 (TSP1) exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several proangiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated endothelial cell responses are inhibited by recombinant type 1 repeats of TSP1 and a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and proangiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis.