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(Circulation Research. 2000;87:999.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Vasodilator-Stimulated Phosphoprotein Serine 239 Phosphorylation as a Sensitive Monitor of Defective Nitric Oxide/cGMP Signaling and Endothelial Dysfunction

Matthias Oelze, Hanke Mollnau, Nina Hoffmann, Ascan Warnholtz, Martin Bodenschatz, Albert Smolenski, Ulrich Walter, Mikhail Skatchkov, Thomas Meinertz, Thomas Münzel

From the Abteilung für Kardiologie (M.O., H.M., N.H., A.W., M.B., M.S., T.M., T.M.), Universitäts-Krankenhaus Eppendorf, University of Hamburg, Hamburg; and Department of Clinical Biochemistry and Pathobiochemistry (A.S., U.W.), University of Würzburg, Germany

Correspondence to Thomas Münzel, MD, Abteilung für Kardiologie, Universitäts-Krankenhaus Eppendorf, Martinistr. 52, D-20246 Hamburg. E-mail muenzel{at}uke.uni-hamburg.de

	Abstract

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Abstract—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 AT₁ 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.

Key Words: cGMP-dependent kinase • VASP • nitric oxide • hyperlipidemia • AT₁ receptor blockade

	Introduction

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The endothelium-derived relaxing factor, identified as NO or a closely related compound, induces vasorelaxation by activating the target enzyme soluble guanylyl cyclase (sGC) and by increasing tissue levels of the second messenger cGMP.¹ Cyclic GMP in turn activates cGMP-dependent protein kinase types I and II (cGK-I and -II) of which cGK-I is highly expressed in vascular smooth muscle cell levels.^{2 3 4} cGK-I has mediated NO/cGMP-caused vasorelaxation, which involves phosphorylation of proteins that affect myosin light chains and intracellular Ca²⁺ levels.^{2 3 4 5} Studies with cGK-I-deficient mice demonstrated a complete disruption of the NO/cGMP signaling pathway in the vascular smooth muscle.⁶ Therefore, the activity and/or expression of cGK-I critically modulate NO-induced vasorelaxation. Recently, Smolenski and colleagues^{7 8} provided evidence that analysis of the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239 (P-VASP) is a useful biomonitor of cGK activity and therefore NO effects in intact cells such as platelets and cultured endothelial and smooth muscle cells. Whether this is also applicable to intact vascular tissue must still be established.

Depressed vasodilation to endothelium-dependent vasodilators such as acetylcholine and to authentic NO is a hallmark of early stages of atherosclerosis.⁹ The mechanisms underlying endothelial dysfunction are likely to be multifactorial but seem to be, at least in part, secondary to increased NO degradation caused by activation of superoxide-producing enzymes,¹⁰ such as the xanthine oxidase,¹¹ and a NADH-driven oxidase.¹² Pritchard et al¹³ demonstrated that incubation of cultured endothelial cells with native LDL increased vascular superoxide production, a phenomenon that was blocked by administration of the NOS inhibitor N^G-nitro-Ld-arginine methyl ester. These findings indicate that a NOS in an uncoupled state may also contribute to vascular superoxide production in the setting of hyperlipidemia. In vivo studies with animals and patients have shown that chronic AT₁ receptor blockade improves endothelial function,^{12 14} reduces plaque formation,^{12 15 16} and vascular superoxide production.¹² However, it remains to be established whether prevention of NOS uncoupling by AT₁ receptor blocker treatment contributes to these beneficial effects.

On the basis of these considerations, the present study was designed to (1) characterize the influence of endothelium, endothelium-derived NO and superoxide on cGK-I, and P-VASP in the vasculature of rabbits; (2) test whether NOS uncoupling and impairment occurs in the NO/cGMP/cGK-I/P-VASP pathway in the setting of hypercholesterolemia; (3) test whether treatment of hypercholesterolemic animals with the AT-1 receptor blocker irbesartan improves vascular NO bioavailability and simultaneously affects P-VASP.

	Materials and Methods

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Animals and Protocol
Twenty-five New Zealand White rabbits (NZWR) and 25 hyperlipidemic Watanabe rabbits (WHHL) were studied. Ten of each group received concomitant treatment with the AT₁ receptor antagonist irbesartan, which was mixed in the diet to achieve a daily dose of 10 mg · kg^–1 · d^–1.

Vessel Preparation and Organ Chamber Studies
Aortic rings were suspended in organ chambers as described previously.¹⁷ Vasodilator responses were determined after preconstriction with phenylephrine to 50% to 70% of maximal (KCl-induced) tone. Vasoconstrictor responses to angiotensin II were expressed as percent of a maximal KCl response (80 mmol/L).

Estimation of Vascular Superoxide Production
Vascular O₂^·- was estimated using lucigenin-derived chemiluminescence (LDCL; lucigenin concentration, 5 µmol/L) as described previously.¹⁸ To address the influence of endothelial (NOS III-derived) NO and NOS-mediated superoxide production on vascular LDCL, vessels were incubated with N^G-nitro-Ld-arginine (L-NNA, 1 mmol/L)¹⁸ for 30 minutes as described.¹⁹ The specificity for low-dose lucigenin for detecting superoxide has been validated recently by the demonstration of a good correlation between lucigenin chemiluminescence and SOD-inhibitable ferricytochrome C reduction.²⁰

Detection of cGK-I and VASP Expression and P-VASP
Aortic tissue from control and hyperlipidemic WHHL with and without irbesartan treatment was frozen and homogenized in liquid nitrogen. The pulverized tissue was vortexed with ice-cold homogenization buffer and centrifuged at 3000g for 5 minutes to remove insoluble material. SDS-PAGE electrophoresis and electroblotting were performed. The membrane was then divided horizontally at 65 kDa. For the upper part, immunoblotting was performed with a polyclonal antibody against cGK-I,²¹ and for the lower part, mouse monoclonal P-VASP phosphoserine 239 antibodies (16C2)⁸ were used. A second blot was used to detect VASP expression by using a monoclonal antibody against VASP (IE273).²² Immunodetections were accomplished with anti–rabbit/mouse secondary antibodies. All bands (VASP, P-VASP, and cGK-I) were standardized against ß-tubulin. The intensity of the P-VASP/VASP/cGK-I bands in the treated samples was expressed as percent of the P-VASP/VASP/cGK-I bands in the control samples. Then, all VASP serine 239 phosphorylation data are expressed as P-VASP/VASP ratio. This ratio indicates the extent of VASP serine 239 phosphorylation in the tissue extract examined and corrects for variable VASP expression levels and recoveries caused by the experimental procedures. As positive controls for cGK-I, we used 10 ng recombinant cGK-I. As a positive control for P-VASP, we used 1 µg protein of SNP-stimulated human platelets.

In separate experiments, the effects of endothelial removal, inhibition of NOS, and the effects of oxidative stress on P-VASP were studied. The endothelium was removed by exposing the vessel lumen to collagenase (0.1% for 10 minutes at 37°C). To inhibit NOS, aortic rings of NZWR were incubated for 30 minutes with the NOS inhibitor N^G-nitro-Ld-arginine (L-NNA, 3 mmol/L). To induce oxidative stress we inhibited CuZn SOD by incubating vessels with diethyldithiocarbamate (DETC) as described recently.²³

Electron Spin Resonance Studies: Detection of Vascular Nitric Oxide
Concentrations of NO in rabbit aorta were assayed using ESR spectroscopy and the spin-trap iron (II)-proline-dithiocarbamate [Fe(PrTC)₂] which has been shown to trap NO with high efficacy by forming an ESR-detectable paramagnetic complex Fe(NO)(PrTC)_2.¹⁹

Materials
Proline dithiocarbamate was obtained from Alexis (Grünberg, Germany). All chemicals were purchased from Sigma Chemical (Deisenhofen, Germany).

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Effects of Endothelial Removal, L-NNA, DETC, and Hyperlipidemia on P-VASP, Total VASP and cGK-I, and Vascular Superoxide Production
In control vessels, endothelial removal markedly reduced P-VASP and to a lesser extent total VASP without altering cGK-I (Figure 1). All phosphorylation data are subsequently reported as P-VASP/VASP ratios, which corrects for variable VASP recoveries and expression levels. Inhibition of NOS by L-NNA reduced P-VASP (Figure 1) without altering total VASP and cGK-I, which results in an L-NNA-caused reduction of the P-VASP/VASP ratio. Inhibition of CuZn SOD with DETC increased vascular superoxide and simultaneously reduced P-VASP without altering total VASP and cGK-I (Figure 2). In hyperlipidemic WHHL, increased vascular superoxide levels were associated with reduced P-VASP, whereas total VASP (as detected with the IE273 antibody) was reduced only moderately (Figure 2). Incubation of vessels from NZWR with sodium nitroprusside (SNP, 10^–5 M) for 30 minutes increased P-VASP. Likewise, incubation of vessels from WHHL with SNP increased P-VASP to a similar degree (Figure 3).

View larger version (37K): [in this window] [in a new window]   Figure 1. Figure 1. Effects of endothelial removal and incubation with the NOS inhibitor NG-nitro-Ld-arginine (L-NNA) on cGK-I, P-VASP (detected with the antibody 16C2), and total VASP (detected with the antibody IE273). Top, Original blots; bottom, densitometric quantification. Endothelial removal (left) markedly decreased P-VASP (5±1% of control), significantly decreased total VASP (61±6% of control), and did not affect the level of cGK-I (100±2 vs 112±3%). NOS inhibition caused by incubation with L-NNA (right) significantly reduced P-VASP (35±4% of control) without affecting total VASP and cGK-I levels. Densitometric phosphorylation data are mean±SEM of 5 to 7 experiments and are expressed as P-VASP/total VASP ratios. *P<0.05 vs vessels with endothelium.

View larger version (38K): [in this window] [in a new window]   Figure 2. Figure 2. Effects of oxidative stress in vitro (induced by DETC; left) and in vivo (induced by hyperlipidemia; right) on P-VASP and superoxide production. Top, Original blots; bottom, densitometric quantification. Inhibition of CuZn SOD with DETC significantly increased vascular steady-state superoxide levels and simultaneously decreased P-VASP (47±5% of control) without altering cGK-I (91±6% of control) and total VASP (98±4% of control). Likewise, in vessels from hyperlipidemic WHHL we found increased superoxide levels, decreased P-VASP (to 24±6% of control), slightly reduced total VASP (82±8% of control), and no change in cGK-I (99±8% of control). Data are mean±SEM from 6 to 8 experiments and are expressed as P-VASP/total VASP ratios. *P<0.05 vs control.

View larger version (26K): [in this window] [in a new window]   Figure 3. Figure 3. Effects of in vitro incubations with the NO donor SNP (10 µmol/L for 30 minutes) on the phosphorylation of the VASP serine 239 (P-VASP) in aortas from control and hyperlipidemic WHHL. Incubation of aortas from control and hyperlipidemic animals with SNP resulted in a similar degree of P-VASP. Densitometric phosphorylation data are presented as mean±SEM of 5 experiments and are expressed as P-VASP/total VASP ratios. +P<0.05 vs control.

Effects of AT₁ Receptor Blockade on Vasodilator Responses to Acetylcholine and Nitroglycerin
In hyperlipidemic Watanabe rabbits, the sensitivity to endothelium-dependent (ACh) and endothelium-independent (NTG) relaxation was reduced significantly (Table 1). AT₁ receptor blockade for 8 weeks had no effect on the ACh and NTG dose-response relationship of control animals but significantly improved it in WHHL (Table 1), compatible with an improved sensitivity to NO-mediated vasodilation. AT₁ receptor blockade for 8 weeks inhibited sensitivity and potency of angiotensin II in vessels from control and from hypercholesterolemic animals, compatible with a sufficient blockade of the AT₁ receptor (Table 2).

View this table: [in this window] [in a new window]   Table 1. Effects of AT1 Receptor Blockade on ED50 and Maximal Relaxation Values to Endogenous and Exogenous Nitrovasodilators in Aortas from NZWR and Hyperlipidemic WHHL

View this table: [in this window] [in a new window]   Table 2. Effects of AT1 Receptor Blockade on EC50 and Maximal Constriction Values to Angiotensin II in Aortas from NZWR and Hyperlipidemic WHHL

Effects of AT₁ Receptor Blockade on Vascular Superoxide and NOS III Uncoupling in Hyperlipidemic Watanabe Rabbits
Hyperlipidemia led to a significant increase in LDCL compared with controls (Figure 4). Treatment of control animals with irbesartan had a slight but significant effect on LDCL. AT₁ receptor blockade markedly reduced vascular superoxide levels in hyperlipidemic WHHL. Incubation of control vessels with the NOS inhibitor L-NNA increased the LDCL signals but paradoxically decreased it in vessels from hyperlipidemic animals. Incubation of vessels from hyperlipidemic animals treated with both irbesartan and L-NNA increased LDCL comparable with control vessels.

View larger version (14K): [in this window] [in a new window]   Figure 4. Figure 4. Effects of AT1 receptor blockade with irbesartan (10 mg · kg–1 · d–1, PO for 8 weeks) on vascular superoxide production as measured by lucigenin-derived chemiluminescence in control and hypercholesterolemic Watanabe rabbits. Hypercholesterolemia caused a marked increase in vascular superoxide levels compared with controls, which was normalized by AT1 receptor blockade. Incubation of control vessels with L-NNA increased vascular LDCL. In contrast, incubation of vessels from hyperlipidemic animals with L-NNA caused a marked decrease in LDCL identifying the NOS III as a significant superoxide source. Incubation of vessels from WHHL treated with irbesartan responded to L-NNA-like control vessels indicating that AT1 receptor blockade is able to prevent NOS III uncoupling. Data are presented as mean±SEM of 6 to 8 experiments. *P<0.05 vs control; +P<0.05 vs without L-NNA; and P<0.05 vs WHHL without irbesartan treatment.

Effects of AT₁ Receptor Blockade on P-VASP in Control and Hyperlipidemic WHHL
The effects of AT₁ receptor blockade on cGK-I and VASP expression as well as on P-VASP are depicted in Figure 5 (original blots and quantitative densitometry). Treatment of control animals with irbesartan had no effect on cGK-I, VASP expression, and P-VASP. In hyperlipidemic animals we found no significant decrease in cGK-I and a minor decrease of total VASP, but P-VASP was strikingly reduced. Treatment of hyperlipidemic animals with irbesartan significantly increased P-VASP.

View larger version (28K): [in this window] [in a new window]   Figure 5. Figure 5. Effects of hypercholesterolemia and AT1 receptor blockade with irbesartan (irb, 10 mg · kg–1 · d–1, PO for 8 weeks) on the expression of cGK-I and VASP and on the level P-VASP in rabbit aortas. As a positive control for P-VASP we used 10 µg protein of SNP-stimulated human platelets. Treatment of hyperlipidemic animals with irbesartan significantly increased P-VASP. Top, original blots; bottom, densitometric evaluation. All bands (VASP, P-VASP, and cGK-I) were standardized against ß-tubulin. The intensities of the P-VASP and VASP bands were expressed as a P-VASP/VASP ratio. C indicates control group; C+I, control group+irbesartan; WHHL, hyperlipidemic animals; WHHL+I, hyperlipidemic animals+irbesartan. Data obtained from quantitative densitometry are presented as mean±SEM of 6 independent experiments, and in the phosphorylation, data are expressed as P-VASP/VASP ratios. *P<0.05 vs control; P<0.05 vs without irbesartan.

Effects of AT₁ Receptor Blockade on Vascular NO Bioavailability of Hyperlipidemic WHHL
The effects of irbesartan treatment on vascular NO bioavailability in vascular tissue as assessed with ESR spectroscopy are depicted in Figure 6. The representative ESR recording of the signal of the iron-nitrosyl dithiocarbamate complex Fe(NO)(PrTC)₂ was strikingly reduced in vessels from hyperlipidemic WHHL animals as compared with spectra obtained from vessels from WHHL treated with irbesartan.

View larger version (26K): [in this window] [in a new window]   Figure 6. Figure 6. Effects of AT1 receptor blockade with irbesartan (irb, 10 mg · kg–1 · d–1, PO for 8 weeks) on the bioavailability of vascular NO as assessed with ESR spectroscopy. Left, original spectra obtained with a vessel from WHHL rabbit (top) and a vessel from a WHHL rabbit treated with irbesartan (bottom). Right, average amount of NO trapped by the spin-trap iron (II)-proline-dithiocarbamate [Fe(PrTC)2] in control and irbesartan-treated animals (n=5 for each group). Irbesartan treatment markedly increased NO bioavailability in vessels from hyperlipidemic WHHL.

	Discussion

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P-VASP as Indicator of Vessel Wall Integrity and cGK-I Activity
The present study demonstrates that the level of vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP) in the rabbit aortic vessel wall is an indicator of both cGK-I activity and endothelium integrity under physiological and pathophysiological conditions. It is now well established that the NO-cGMP pathway is a key regulator of vascular tone and that cGK-I mediates many of these NO/cGMP effects. Studies with cGK-I-deficient human cells and mice demonstrated that cGK-I ablation disrupts the NO/cGMP pathway in vascular cells and tissues.^{3 6} Gene-targeted loss of murine cGK-I abolished NO/cGMP-dependent relaxation of smooth muscle resulting in severe vascular and intestinal dysfunctions, whereas cAMP-mediated smooth muscle relaxation was not impaired.^{5 6} These recent developments highlight the importance of assessing cGK expression and/or cGK activity in the presence of endothelial dysfunction. However, cGMP-independent NO effects in vascular tissues exist which are not addressed by our present study.

This study shows that vascular wall cGK-I activity can be estimated by the analysis of VASP phosphorylation. VASP is a well-characterized substrate for both cGK and cAMP-dependent protein kinase (cAK) in platelets, vascular endothelial and smooth muscle cells, and many other cell types and tissues.^{24 25 26 27} Functionally, VASP regulates microfilaments and enhances spatially confined actin polymerization.^{22 28} In platelets, VASP and VASP phosphorylation have recently been demonstrated to be involved in the inhibition of agonist-induced platelet aggregation and, in particular, integrin _Iibß₃ activation.^{29 30} In smooth muscle function and relaxation, however, the precise role of VASP and VASP phosphorylation remains to be established.^{29 30} In platelets and many primary and cultured cell types, activation of cAK and cGK can be monitored by antibodies, which are specific for the distinct VASP phosphorylation sites (serine 157, serine 239, and threonine 278,^{8 22} other data not shown). Although all 3 sites can be phosphorylated by both protein kinases, serine 157 and serine 239 are the preferred phosphorylation sites for cAK or cGK, respectively.^{7 8 22 31} Studies with cGK-I-deficient cells established that the NO/cGMP-mediated VASP phosphorylation is mediated by cGK-I.^{3 26} It remains to be established, however, whether a similar approach can be used with intact vascular tissue.

Here, we found that removal of the rabbit aorta endothelium strikingly reduced P-VASP (detected by the monoclonal antibody 16C2) to 5±1% and moderately reduced (down to 61±5%) total vessel VASP (detected by the monoclonal antibody IE273), which collectively also resulted in a substantially reduced P-VASP/VASP-ratio (down to 15±7%). The cGK-I content did not change significantly. Although cGK-I is clearly expressed in endothelial cells,^{3 26} our present data indicate that the endothelium contributes only a minor amount to the overall content of the rabbit aortic vessel cGK-I. In contrast, the endothelial layer and medial layer of the rabbit aorta appear to contribute about one third and two thirds, respectively, of the total content of vessel wall VASP. These data agree with previous immunohistochemical studies, which demonstrated high levels of VASP in medial, intimal, and neointimal layers of the rat carotid artery.²⁷ The data also show that the presence of P-VASP in this vessel almost exclusively depends on the presence of the endothelium. Incubation of aortic pieces with the NOS inhibitor L-NNA decreased P-VASP to 35±4% indicating that endothelium-derived NO is a major contributor to VASP phosphorylation in vascular tissue. The half-life of NO and therefore its biological activity is determined decisively by oxygen-derived free radicals such as superoxide.³² To analyze the effects of oxidative stress on P-VASP, we incubated vascular tissue in vitro with the inhibitor of the superoxide-scavenging enzyme CuZn-SOD DETC. Incubation of vascular tissue with DETC markedly increases steady-state superoxide levels and simultaneously causes endothelial dysfunction.²³ Increased superoxide inactivates NO derived from endothelial cells via formation of peroxynitrite, which has been much less potent in stimulating the downstream target soluble guanylyl cyclase.³³

Accordingly, we found that P-VASP in DETC incubated vessels was strikingly reduced, whereas total VASP content and cGK-I were not modified. Similar changes were observed in an in vivo model of oxidative stress such as hypercholesterolemia. In hyperlipidemic WHHL we established endothelial dysfunction, increased vascular superoxide production, and accordingly decreased P-VASP production. Total VASP (determined with the IE273 antibody) did not decrease significantly indicating that decreased NO/cGMP signaling (as measured by the level of NO/cGMP-increased P-VASP) rather than decreased expression of the cGK-I substrate accounts for this phenomenon. Incubation of control and tissue from hyperlipidemic animals with the NO donor SNP revealed similar increases in the phosphorylation of VASP (Figure 3), which demonstrates that the cGMP signaling pathway downstream of the soluble guanylyl cyclase per se is not impaired. These findings further indicate that NO derived from endothelial cells or NO left after interactions with superoxide (vascular NO bioavailability) is closely monitored by the degree of VASP phosphorylation (P-VASP). In addition, endothelial dysfunction in hyperlipidemic animals is not likely to be secondary to decreases in the expression of either cGK-I or its substrate VASP. cGK-I and VASP are also well expressed in neointimal cells of the injured rat carotid artery.²⁷

Evidence for NOS Uncoupling in Hyperlipidemia
Our current findings are supported by previous observations showing that hypercholesterolemia is associated with endothelial dysfunction and increased vascular superoxide production.¹² In hyperlipidemic Watanabe rabbits, vascular superoxide was increased about 2-fold compared with controls. Treatment of hyperlipidemic WHHL with an AT₁ receptor blocker improves endothelial dysfunction and simultaneously reduces vascular superoxide production.¹²

It has become clear from studies that both NOS I and NOS III may become "uncoupled" in the absence of Ld-arginine or tetrahydrobiopterin. In such uncoupled state, electrons flowing from the reductase domain to the oxygenase domain are diverted to molecular oxygen rather than to Ld-arginine^{34 35} resulting in production of superoxide rather than NO. Recent in vitro studies proposed that oxidized LDL in particular is able to decrease endothelial Ld-arginine uptake ultimately leading to both local depletion of Ld-arginine and NOS III uncoupling.³⁶ Hypercholesterolemia also has increased vascular formation of superoxide¹⁰ leading to increased formation of the NO/superoxide reaction product peroxynitrite.³⁷ Peroxynitrite in turn rapidly oxidizes the active NOS cofactor tetrahydrobiopterin to cofactor inactive molecules such as dihydrobiopterin,³⁸ leading to NOS III uncoupling.

With the present studies we provide evidence that an uncoupled NOS III is at least partially involved in the increased superoxide production seen in vessels from hyperlipidemic WHHL. When control aortas were exposed to the NOS inhibitor L-NNA, a significant increase in the LDCL signal was observed. This indicates that the amount of NO formed in normal vascular endothelium under basal conditions is sufficiently high to compete for the reaction of superoxide with lucigenin.¹⁸ In contrast, incubation of aortic tissue from WHHL with L-NNA decreased rather than increased steady-state vascular superoxide levels identifying NOS III as a significant superoxide source. These data obtained from in vivo cholesterol-fed hyperlipidemic animals coincide exactly with recent in vitro observations. In these experiments, Pritchard et al¹³ demonstrated that L-NAME (the methyl ester of L-NNA) increased steady-state superoxide levels in cultured endothelial cells. After incubation of the endothelial cells with native LDL, superoxide levels increased markedly, a phenomenon that was largely blocked by L-NAME identifying NOS III as an important superoxide source. We cannot entirely exclude, however, that the inhibitory effects of L-NNA on superoxide levels in hyperlipidemia are secondary to some stimulatory NO effects on vascular oxidases.

AT₁ Receptor Blockade Prevents NOS Uncoupling and Increases P-VASP
Next we studied the effects of AT₁ receptor blockade with irbesartan on endothelial function, vascular superoxide, NOS-mediated superoxide production, vascular NO/cGMP signaling as measured by the content of P-VASP. Treatment of hyperlipidemic animals with the AT₁ receptor blocker irbesartan significantly improved endothelial function without altering plasma cholesterol levels (data not shown). These data agree with our previous observations that blockade of the AT₁ receptor beneficially influences vascular function in hyperlipidemic animals¹² and patients with coronary artery disease.¹⁴ Irbesartan significantly improved endothelial dysfunction of hyperlipidemic WHHL and reduced vascular steady-state superoxide levels. Incubation of vessels from irbesartan-treated hyperlipidemic animals with the NOS inhibitor increased vascular superoxide rather than decreased it (compatible with changes observed in controls), which indicates that AT₁ receptor blockade may reduce oxidative stress at least in part by preventing NOS III uncoupling (see Figure 4). Hypercholesterolemia has been associated with increases in vascular AT₁ receptor expression^{12 39} and increased formation of angiotensin II in vascular tissue.⁴⁰ Acute incubation of vessels with angiotensin II stimulates peroxynitrite formation.⁴¹ Taking into account the above-mentioned mechanisms of NOS uncoupling, we therefore hypothesize that chronic AT₁ receptor blockade may reduce NOS III-mediated superoxide production by reducing vascular peroxynitrite formation and therefore less oxidation of the redox-sensitive NOS III cofactor tetrahydrobiopterin. The reduction of oxidative stress within vascular tissue [eg, also via inhibition of the NAD(P)H oxidase] decreases oxidation of LDL, which in turn may favorably influence intracellular Ld-arginine availability.³⁶

The inhibition of superoxide production in vessels from WHHL treated with irbesartan led to a significant increase in vascular NO bioavailability as assessed by ESR measurements. Accordingly, we found that P-VASP, which was markedly depressed in WHHL, was significantly enhanced after concomitant AT₁ receptor blocker treatment (Figure 6).

Conclusions
The present studies demonstrate that the level of P-VASP in the rabbit aorta is strikingly regulated by the vascular NO/cGMP signaling pathway. Endothelial removal and inhibition of NOS or increases in oxidative stress in vitro and in vivo markedly decreases the VASP phosphorylation at the cGK-preferred phosphorylation site serine 239. Treatment of hypercholesterolemic animals with the AT₁ receptor blocker irbesartan improved endothelial dysfunction, reduced vascular superoxide production, increased vascular NO bioavailability, and accordingly increased P-VASP. Therefore, P-VASP seems to be a new biochemical marker capable of monitoring the NO/sGC/cGK-I pathway in rabbit aorta, which presents a major aspect of NO bioavailability in vascular tissues. Considering the established inhibitory role of VASP and VASP phosphorylation on platelet activation, VASP phosphorylation may also contribute to the vasoprotective effects of the NO/cGMP pathway in the healthy and diseased vessel wall.

	Acknowledgments

 

This work was supported by the Deutsche Forschungsgemeinschaft (Mu, 1079/2-2; SFB 355) and in part by a vascular biology grant from Bristol Myers Squibb.

Received June 27, 2000; revision received September 26, 2000; accepted September 27, 2000.

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