Introduction

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The vascular endothelium plays a key role in cardiovascular homeostasis through its diverse influences on blood vessel structure and function. In addition to its importance as a physical permeability barrier and as a site for metabolism of certain vasoactive substances, the endothelium elaborates proteins and other factors that confer antithrombogenic properties to the vessel wall (Mombouli and Vanhoutte, 1999). The endothelium also releases a host of paracrine and autocrine factors that not only influence vascular tone and permeability, but also play an important role in long-term vascular growth and remodeling. Among the various endothelium-derived molecules, nitric oxide (NO), a molecule produced in the endothelium by NO synthase (NOS) from L-arginine, has been demonstrated to play a pivotal role in the maintenance of normal cardiovascular function under physiologic conditions and the adaptation of the cardiovascular system under pathologic conditions (Arnal et al., 1999).

Numerous studies have demonstrated that endothelium-dependent vasorelaxation is markedly decreased in hypertensive patients and in experimental hypertensive animal models (Dominiczak and Bohr, 1995) (Grunfeld et al., 1995). However, the underlying mechanisms of this endothelial dysfunction are unknown, and the existing explanations are often controversial and seem to vary depending on the model studied. In Dahl salt-sensitive rats, the decrease in endothelium-dependent relaxation is associated with impaired endothelial NOS (eNOS) activity and decreased NO production (Boulanger, 1999). However, in other animal models of hypertension [such as the spontaneously hypertensive rat of the stroke-prone strain (SHRSP)] and in patients subjected to essential hypertension, endothelium-dependent vasorelaxation is markedly decreased even though eNOS expression and NO production have been found to be significantly increased (McIntyre et al., 1999). These results suggest that endothelial-generated NO may have been inactivated before it could reach its desired target (e.g., guanylate cyclase in vascular smooth muscle cells), thus resulting in a decreased bioactive NO concentration. In this regard, several studies have recently demonstrated that production of superoxide anion (O₂), a free radical that reacts with NO in a diffusion-limited rate, is markedly increased in vascular endothelial cells from SHRSP and essential hypertensive patients (Tschudi et al., 1996; Swei et al., 1997; Kerr et al., 1999). However, direct evidence demonstrating the inactivation of NO by superoxide anion and its concurrent production of peroxynitrite (ONOO), an extremely cytotoxic radical, in vascular tissue subjected to hypertension is not yet available.

Carvedilol, 1-[carbazoyl-(4)-oxy]-3-[(2-methoxy-phenoxyethyl) amino]-propanol-(2), was originally introduced as a vasodilating -adrenoreceptor antagonist and has been used for the treatment of mild to moderate hypertension. Several recent clinical trails have consistently demonstrated that carvedilol exerts a superior cardiovascular protection over other -adrenoceptor antagonist, such as bisoprolol and metoprolol (Packer et al., 1996; Metra et al., 2000). However, the mechanisms underlying carvedilol's superior protective effects remain undefined. Moreover, we have recently demonstrated that in SHRSP with high-salt, high-fat diet (SFD), treatment with carvedilol markedly decreased cardiomyopathy (Barone et al., 1998), renal damage, and mortality (Barone et al., 1996). Once again, the mechanism underlying its protection remains unclear.

Therefore, the aims of the present study were 1) to determine the mechanism of endothelial dysfunction (i.e., decreased NO production or increased NO destruction) in SHRSP; 2) to provide direct evidence of NO inactivation by O₂ and its concurrent formation of ONOO; and 3) to determine the effects of treatment with carvedilol on the severity of endothelial dysfunction and ONOO formation in SHRSP, thus providing further insight into the mechanisms of cardiovascular injury and the end-organ protection exerted by carvedilol. Our general hypothesis was that oxidative inactivation, but not reduced production of NO, is responsible for endothelial dysfunction in SHRSP, and carvedilol, an antioxidant -adrenoreceptor antagonist, may reduce NO inactivation and improve endothelial function in these animals.

	Materials and Methods

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SHRSP. SHRSP progeny from the strain developed by Okamoto et al.(1974) were obtained from the National Institutes of Health and were bred in the Department of Laboratory Animal Science at SmithKline Beecham Pharmaceuticals. Animals were housed and cared for in accordance with the Guide for Care and Use of Laboratory Animals (Department of Health, Education, and Welfare, 1985). Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals and Thomas Jefferson University. Male SHRSP at 10 weeks of age were adapted to individual cages and fed powdered National Institutes of Health-07 diet for 2 weeks.

Plasma NOx Assay. Nitric oxide has a very short half-life (<10 s) and is oxidized to form NO₂ and NO₃ in vivo. Measurement of NOx (NO + NO₂ +NO₃) concentration in plasma has been demonstrated to reflect NO formation in vivo. When rats were prepared for sacrifice, they were anesthetized with pentobarbital sodium, 0.5 ml of blood was withdrawn from the left carotid artery, and plasma was obtained after centrifugation. To each 0.2 ml of plasma, 0.4 ml of ice-cold 100% alcohol was added and placed in ice for 30 min. The plasma-alcohol mixture was recentrifuged, and the supernatant was used to measure the concentration of NOx by using the vanadium reduction method (Ma et al., 1997). Briefly, 50 µl of sample was injected into a water-jacketed, oxygen-free purge vessel containing 5 ml of 0.1 M vanadium (III) chloride (Aldrich, Milwaukee, WI) in 2 N HCl (Sigma, St. Louis, MO). Acidic vanadium (III) at >80°C quantitatively reduces both nitrite and nitrate to NO, which is quantified by a chemiluminescence detector (SIEVERS 270B nitric oxide analyzer, SIEVERS, Boulder, CO) after reaction with ozone. Signals from the detector were collected and analyzed using a computer-based data acquisition and analyzing system (MacLab, ADInstruments, Inc., Milford, MA). Standard curves were obtained using the area under the curve after each injection of 50 µl of 0, 12.5, 25, 50, 75, and 100 µM sodium nitrate. The calculations to determine the NOx content of the plasma were done by the slope of the regression analysis using the linear formula y = a + bx.

Determination of Endothelium-Dependent, Nitric Oxide-Mediated Vasorelaxation. After blood was withdrawn, rats were then overdosed with pentobarbital. The chest was then opened and the thoracic aortas were carefully removed and placed into ice-cold Krebs-Henseleit (K-H) buffer consisting of (mmol/l) NaCl 118, KCl 4.75, CaCl₂·2H₂O 2.54, KH₂PO₄ 1.19, MgSO₄·7H₂O 1.19, NaHCO₃ 25, and glucose 10.0. Isolated vessels were cleaned of adhering fat and connective tissue and cut into rings 3 to 4 mm in length. The rings were then mounted onto stainless steel hooks, suspended in 7.5-ml tissue baths, and connected to FORT-10 force transducers (WPI, Sarasota, FL) to record changes via a MacLab data acquisition system. The baths were filled with 7.5 ml of K-H buffer and aerated at 37°C with a gas mixture of 95% O₂ and 5% CO₂. Aorta rings were initially stretched to give an optimal preload of 1 g of force and equilibrated for 60 min. During this period, the K-H buffer in the tissue bath was replaced every 20 min.

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Determination of Total NO Release in Response to ACh Stimulation in Aortic Segments. In a separate study, rat aorta were isolated as described above and cut into segments 5 to 6 mm in length. Aortic segments were weighed and then placed individually in 24-well plates containing serum-free modified Eagle's medium (1 ml/100 mg of tissue) supplemented with 2 mM L-arginine (Ing et al., 1999). To each well, ACh (final concentration: 10⁵ M) or its vehicle (PBS) was added. The plate was covered and incubated at 37°C for 24 h in a humidified cell culture incubator. At the end of incubation, NOx concentration in culture buffer was determined in a similar manner as described above, except that a larger volume of sample solution was injected into the reaction vessel (i.e., 200 µl instead of 50 µl when plasma NOx was determined), and a lower concentration of sodium nitrate (0, 0.3, 1, 3, and 10 µM) was used for constructing the standard curves. NOx concentration in vehicle-treated segments was defined as basal NO production. Differences in NOx concentration between vehicle-treated and ACh-treated segments were calculated and defined as ACh-stimulated total NO production.

Immunohistological Detection of iNOS Expression and Nitrotyrosine Formation in Aortic Vascular Tissue. In a separate study, four pentobarbital-anesthetized rats from each group were whole-body perfused with 200 ml of PBS followed by 300 ml of 10% formalin in PBS. Thoracic aorta was removed and stored in 10% formalin for less than 48 h. Fixed aortic segments were dehydrated and embedded in paraffin, and sections were cut at 6 mm and mounted onto glass slides. Immunohistochemical procedures for detecting iNOS and nitrotyrosine formation were performed according to the procedure published by Liu et al. (1997) and Beckman et al. (1994), respectively. Rabbit polyclonal antibody against nitrotyrosine was provided by Dr. Joseph Beckman as a gift (University of Alabama at Birmingham) and rabbit polyclonal antibody against iNOS was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The DAKO avidin-peroxidase kit (DAKO Corporation, Carpinteria, CA) was used for both iNOS and nitrotyrosine immunostaining.

Detection of Vascular Cell Apoptosis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin in Situ Nick-End Labeling (TUNEL). The TUNEL assay was performed in formalin-fixed and paraffin-embedded thoracic aorta tissue slides by using an apoptosis detection kit (Roche Molecular Biochemicals, Ridgefield, CT) according to the manufacturer's instructions. The digoxigenin-conjugated dUTP was incorporated to the ends of DNA fragments by terminal deoxynucleotidyl transferase. The signal of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was then detected by an anti-fluorescein antibody conjugated with alkaline phosphatase, a reporter enzyme that catalytically generates a red-colored product from Vector red substrate.

Quantification of Nitrotyrosine in Aortic Tissue. Quantification of aortic tissue nitrotyrosine levels was performed by using a modified ELISA procedure (Tanaka et al., 1998; Ronson et al., 1999). In brief, aortic tissue was homogenized in ice-cold PBS (1:10 w/v) using a PRO 200 homogenizer first (PRO Scientific Inc., Monroe, CT, 60 s at 7000 rpm) followed by sonication with a dismembrator (Fisher Scientific, Pittsburgh, PA; medium intensity, 30 s). The homogenates were centrifuged for 10 min at 12,500g at 4°C. The supernatants were collected and protein concentrations were determined by the bicinchoninic acid method. A nitrated protein solution was prepared for use as a standard by adding 8 µl (2 µl × 4) of chemically synthesized ONOO (concentration: 100-120 mM) to 3 ml of 0.04% (0.4 mg/ml) BSA in PBS. The amount of nitrotyrosine present in the peroxynitrite-treated BSA solution was measured at 430 nm (_m= 4400 M¹ cm¹) using a spectrophotometer (Beckman DU 640, Fullerton, CA) and expressed as nanograms per milliliter. The stock solution of the peroxynitrite-treated BSA was diluted with PBS (final nitrotyrosine concentration, 0.75-75 ng/ml). These standard samples, along with tissue samples from hearts (protein concentration, 4 mg/ml) were applied to disposable sterile ELISA plates (Corning Glassworks, Corning, NY) and allowed to bind for 1 h at 37°C in a microincubator shaker (Teitec Co., San Jose, CA). After blocking nonspecific binding sites with 1% BSA in PBS, the wells were incubated for 60 min at 37°C with a rabbit polyclonal anti-nitrotyrosine primary antibody (from Dr. Joseph Beckman, 1:500 in 10% goat serum PBS) and subsequently for 60 min at 37°C with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Amersham Pharmacia Biotech, Inc. Piscataway, NJ). After washing the plates, the peroxidase reaction product was generated using O-phenylenediamine dihydrochloride (2.2 mM) (Abbott Diagnostics, Abbott Park, IL). The plate was incubated for 20 min in the dark at room temperature, and the reaction was stopped by addition of 20 ml of 2 M H₂SO₄. The optical density was measured at 460 nm with a microplate reader (Bio Tek Instruments, Inc., Winooski, VT). The amount of nitrotyrosine content in tissue samples was calculated using standard curves generated from nitrated BSA containing known amounts of nitrotyrosine.

Statistical Analysis. All values in the text, table, and figures are presented as means ± S.E. of n independent experiments. All data were subjected to analysis of variance followed by the Scheffe's correction for post hoc t test comparison. For nonparametric (%) data, the x² test for independent samples was utilized. Probabilities of 0.05 or less were considered to be statistically significant.

	Results

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Systolic Blood Pressure and Mortality. Table 1 summarizes systolic blood pressure, heart rate, and morbidity/mortality for the four groups of rats at week 11. The SFD significantly increased blood pressure further in the already significantly hypertensive SHRSP. Carvedilol did not reduce the extremely elevated blood pressure induced by SFD. Carvedilol did decrease heart rate in SHRSP, probably due to its -adrenoreceptor antagonist activity. It also significantly eliminated the morbidity/mortality produced in SHRSP by SFD. These results were similar to those reported previously (Barone et al., 1996). During the next 2 weeks (weeks 12 and 13), rats were sacrificed in groups of four (i.e., one rat from each group), and in vitro tissue studies were conducted. In this manner, tissue was removed from healthy rats after group differences in morbidity were determined and tissues between groups were compared appropriately under the different experimental conditions.

Endothelium-Dependent Vasorelaxation in Aortic Rings. As summarized in Fig. 1, aortic rings isolated from WKY exhibited full relaxation to the endothelium-dependent vasodilator, ACh, as well as the endothelium-independent vasodilator, NaNO₂. ACh-induced vasorelaxation was moderately decreased in aortic rings from SHRSP when compared with rings from WKY. Vasorelaxation to an endothelium-independent vasodilator, acidified NaNO₂, was unchanged. When SHRSP were exposed to SFD, the vasorelaxation response to ACh was severely impaired. Moreover, vasoconstrictor response to U-46619 was also decreased in the rings from SHRSP on SFD (0.41 ± 0.022 versus 0.62 ± 0.018 g in WKY, p < 0.01), although percentage of vasorelaxation to an endothelium-independent vasodilator, NaNO₂, was normal (Fig. 1). Treatment with carvedilol completely reversed SFD-induced additional endothelial dysfunction in SHRSP and brought ACh-induced vasorelaxation to a level that was comparable with that of SHRSP on normal diet. These results demonstrate that aortic endothelial function was impaired in SHRSP and further exaggerated by SFD exposure. Treatment with carvedilol, a vasodilator, antioxidant, and -blocker, markedly preserved endothelial function in hypertensive rats exposed to SFD.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Concentration-relaxation response of rat aortic rings to the endothelium-dependent vasodilator, ACh (A), and to the endothelium-independent vasodilator, acidified NaNO2 (B). The rings were constricted by U-46619 (50 nM). Once a stable constriction was obtained, cumulative concentrations of ACh (10 nM to 10 µM) or acidified NaNO2 (100 nM to 100 µM) were added, and the vasorelaxation to each concentration of vasodilator was calculated as a percentage of the maximal vasoconstriction induced by U-46619. Each point represents the mean value of five to seven rats in each experimental group. CV, carvedilol. *p < 0.05, **p < 0.01 versus WKY group; ##p < 0.01 versus SHRSP group; p < 0.01 versus SHRSP on SFD group.

Nitric Oxide Production. To determine whether decreased NO production was responsible for the observed endothelial dysfunction in the aortic rings from SHRSP and SHRSP on SFD, we measured NOx concentration in plasma, a reliable index for in vivo NO production. Plasma NO concentration was not significantly changed in SHRSP on normal diet (36.6 ± 2.2 versus 37.6 ± 3.1 µM in WKY, p > 0.5), and was markedly increased in SHRSP on SFD (59.2 ± 3.7 µM, p < 0.01 versus WKY and SHRSP). In SHRSP on SFD treated with carvedilol, plasma NO concentration was significantly decreased (44 ± 2.1 µM, p < 0.01 versus SHRSP on SFD without carvedilol treatment). These results demonstrated that whole-body total NO production in the hypertensive animals was not decreased, and even significantly increased in hypertensive rats with SFD exposure.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   NOx concentrations in culture medium containing aortic segments treated with either vehicle (basal) or ACh (10 µM) for 24 h. Heights of columns are means; brackets represent ± S.E.M. n = 10 to 11 wells/group. CV, carvedilol. *p < 0.05, **p < 0.01 versus WKY group; #p < 0.05, ##p < 0.01 versus SHRSP group; p < 0.05, p < 0.01 versus SHRSP on SFD group.

Inducible NOS Expression and Nitrotyrosine Formation in Aortic Vessels. To provide further insight into the mechanisms of endothelial dysfunction in hypertensive rats, we studied the expression of iNOS (Fig. 3, top) and formation of nitrotyrosine (Fig. 3, middle), a footprint of NO oxidation by reactive oxygen species in vivo, in the aortic vessels. Immunohistological staining showed that iNOS and nitrotyrosine residues were undetectable in aortic samples from WKY, suggesting that NO generated in this tissue by eNOS was not significantly oxidized by O₂ to produce a detectable nitrotyrosine formation. However, in the aortic samples isolated from SHRSP, clear immunostaining was observed. Both iNOS and nitrotyrosine staining were primarily located on the luminal surface of the aortic segments, as well as in the adventitial tissues. Most notably, when SHRSP were exposed to SFD, there were significantly more vessels staining with greater intensity for iNOS and nitrotyrosine in aortic vascular samples. Moreover, iNOS and nitrotyrosine staining were not only observed on the endothelial side of the vessel, but also observed in the vascular wall, suggesting that ONOO was present in the vascular smooth muscle cells. Treatment with carvedilol, an antioxidant -blocker, in SHRSP on SFD markedly decreased iNOS expression and nitrotyrosine staining.

View larger version (85K): [in this window] [in a new window]   Fig. 3.   Photomicrographs of iNOS (top), nitrotyrosine immunoperoxidase staining (middle), and in situ detection of DNA fragments (bottom) in thoracic aorta from WKY (A), SHRSP (B), SHRSP with SFD receiving vehicle (C), and SHRSP with SFD treated with carvedilol (D). Arrowheads signify positive staining. L, lumen.

Vascular Cell Apoptosis. Data from the NOx production study in isolated aortic segments showed that although basal NOx production was markedly increased in aortic segments from SHRSP on SFD, ACh-stimulated NOx production in these vessel segments was significantly decreased. This result suggested that endothelial cells might have been injured by the high concentration of ONOO. As illustrated in Fig. 3 (bottom), no significant TUNEL positive cells were observed in the aortic section from either control WKY or SHRSP. However, a large number of cells were TUNEL positive in the aortic segments from SHRSP on SFD, which was significantly inhibited by carvedilol treatment.

	Discussion

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The major novel findings of the current study are that 1) in SHRSP on SFD, ACh-stimulated vasorelaxation and vascular NO production were markedly decreased, but whole-body NO production and vascular basal NO production were significantly enhanced; 2) high salt and high fat intake in SHRSP resulted in a marked induction of iNOS expression, massive formation of the highly toxic peroxynitrite, and significant endothelial as well as smooth muscle cell death, likely via apoptosis; and 3) carvedilol treatment in SHRSP on SFD significantly suppressed iNOS expression, decreased nitrotyrosine formation, improved endothelial function, and reduced vascular cell death.

Growing evidence suggests that an increased breakdown of NO by free radicals is a major mechanism for hypertension-induced impairment of NO-mediated vasorelaxation (Harrison, 1997). Our present study confirms previously published results that in SHRSP on normal diet, endothelium-dependent vasorelaxation is significantly decreased but total plasma NOx concentration is unchanged (McIntyre et al., 1999). Because plasma NOx reflects whole-body total NO production and is not vascular specific, we further directly measured the basal and ACh-stimulated NO production in aortic segments. Our results showed, for the first time, that ACh-stimulated NO production was normal in vascular segments from SHRSP, although ACh-induced vasorelaxation was significantly decreased in the same preparation. In addition, our immunohistochemical results demonstrate that clear nitrotyrosine staining can be observed in vessels from SHRSP, but not from WKY. Quantitative assays indicate that nitrotyrosine content in vessels from SHRSP was 3 times higher than that in vessels from WKY. These results provide additional evidence that oxidative inactivation, not decreased production, of NO is responsible for endothelial dysfunction in SHRSP on normal diet.

Elevating dietary sodium intake for SHRSP over a prolonged period (>6 weeks) has been consistently shown to accelerate the onset of stroke and increase mortality (Camargo et al., 1993). In our previous study, we provided a high-fat and high-salt diet to SHRSP of a specific age range and established optimal conditions for accelerated morbidity and mortality (Barone et al., 1996, 1998). The mechanisms underlying these profound deleterious effects of adverse diet are not completely understood. Although SFD further increased blood pressure, it seems unlikely that the lethal effects of SFD could be completely attributed to the systolic blood pressure change since carvedilol does not affect this severe hypertension. It has been recently reported that in hypertensive rats, high-salt diet (4 weeks) further increased O₂ generation in the microvessels of the mesentery (Swei et al., 1997), suggesting that overproduction of reactive oxygen species may play an important role. However, effect of high-salt loading on NO production is not clear, and previous results obtained from short-term high-salt loading (<2 weeks) are controversial (Shultz and Tolins, 1993; Campese et al., 1996; Higashi et al., 1996; Fujiwara et al., 2000). More importantly, whether highly toxic ONOO was formed in a large quantity under this condition, thus contributing to the end-organ damage and high mortality, has not been previously studied.

In the present study, we have demonstrated that sustained exposure of SHRSP to SFD markedly increased iNOS expression and total NOx concentrations in the plasma. Moreover, although basal NOx concentration was markedly increased, ACh-stimulated increase in NOx production was significantly decreased in the incubation buffer containing aortic segments isolated from these animals. ACh-induced vasorelaxation, a measure of the level of NO bioactivity, decreased to a level that was only about 50% of WKY and 70% of SHRSP without SFD exposure. These results suggest that NO production from the normal constitutive eNOS was significantly reduced, whereas NO production from inducible calcium/calmodulin-independent nitric-oxide synthase (i.e., iNOS) that was highly expressed in the vascular wall in these animals was dramatically increased. Therefore, the severe endothelial dysfunction observed in hypertensive animals exposed to SFD probably results from a combination of increased inactivation of NO by reactive oxygen species and decreased NO production from eNOS. Our present experiment has provided clear evidence supporting this conclusion. In vascular segments from SHRSP on SFD, nitrotyrosine content was dramatically increased (21-fold versus WKY control animals and 7.5-fold versus SHRSP on normal diet), indicating that increased oxidative NO inactivation occurred in these vessels. On the other hand, positive TUNEL staining, an index of DNA fragmentation and cell death, was significantly increased in vascular endothelial cells in the vascular segments isolated from SHRSP on SFD, indicating that not only functional, but also structural, injury has occurred in the vascular endothelial cells.

Superoxide and NO react at a diffusion-limited rate (k = 6.7 × 10⁹ M¹ s¹) (Beckman and Koppenol, 1996). This bi-radical reaction can be harmful in at least two ways, first by removing the beneficial effects of NO, and second by producing a highly toxic product, ONOO. In in vitro and cell culture studies, ONOO has been shown to be highly reactive with a wide variety of molecules, including deoxyribose, cellular lipids, and protein sulfhydryl moieties, and it causes direct oxidative tissue damage apparently similar to that caused by ·OH in vitro (Beckman and Koppenol, 1996). In a recent study, Arstall et al. (1999) demonstrated that iNOS expression and subsequent ONOO formation plays a pivotal role in myocyte apoptosis induced by cytokine stimulation. In the present study, we have provided evidence that iNOS expression and ONOO formation is probably a major cause of vascular endothelial as well as smooth muscle cell death occurring in SHRSP on SFD and may thus play a significant role in the end-organ injury and morbidity/mortality demonstrated in this animal model.

In our previous studies using the same animal model, we demonstrated that administration of carvedilol in SHRSP on SFD prevented hypertensive cardiomyopathy, attenuated renal damage, and reduced mortality (Barone et al., 1996, 1998). The present study provides direct evidence that the organ-protective effects of carvedilol observed in our previous study may be related to its vascular protective effects. We have demonstrated that carvedilol treatment markedly reduced nitrotyrosine formation, decreased vascular cell death, and improved endothelial function. In addition, we have provided the first evidence that in addition to its well defined superoxide anion scavenging property, carvedilol markedly inhibits iNOS expression in vascular segments. Therefore, carvedilol can inhibit ONOO formation by reducing not only O₂, but also NO production from iNOS. However, treatment with carvedilol failed to reduce the systolic blood pressure in these animals. This was likely due to the extreme hypertension in SHRSP on SFD. Therefore, -adrenoreceptor blockade and endothelial functional improvement afforded by carvedilol treatment was not sufficient to effectively reduce the systolic blood pressure in these animals. Moreover, we recently demonstrated that endothelin plays a significant role in the premature morbidity/mortality exhibited in SHRSP on SFD; mixed inhibition of endothelin A and endothelin B receptors reduced end-organ damage (Barone et al., 2000). It is thus possible that the combined treatment of carvedilol with endothelin receptor inhibitor may exert the best protection in SHRSP on SFD.

In summary, our present experimental results suggest that oxidative inactivation, but not reduced production of NO, is responsible for endothelial dysfunction in SHRSP on normal diet. However, when SHRSP animals were exposed to SFD, a severe endothelial dysfunction occurred, probably because of a combination of increased NO inactivation and a decreased NO production from eNOS. A massive production of ONOO in the vascular wall under this pathologic condition is a major cause of vascular endothelial and smooth muscle cell injury. Pharmacological intervention to inhibit ONOO formation is important and may be useful in the prevention and treatment of a host of cardiovascular diseases common to the Western world.