BM-573 inhibits the development of early atherosclerotic lesions in Apo E deficient mice by blocking TP receptors and thromboxane synthase

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2α) have recently received a lot of attention. This study aimed to investigate the effect of a dual thromboxane synthase inhibitor and thromboxane receptor antagonist (BM-573) and ASA on lesion formation in apolipoprotein E-deficient mice. The combination of ASA and BM-573 was also studied. Plasma measurements demonstrated that the treatments did not affect body weight or plasma cholesterol levels. BM-573, but not ASA, significantly decreased atherogenic lesions as demonstrated by macroscopic analysis. Both treatments alone inhibited TXB(2) synthesis but only BM-573 and the combination therapy were able to decrease firstly, plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) and secondly, the expression of these proteins in the aortic root of Apo E. These results were confirmed in endothelial cell cultures derived from human saphenous vein endothelial cells (HSVECs). In these cells, BM-573 also prevented the increased mRNA expression of ICAM-1 and VCAM-1 induced by U-46619 and 8-iso-PGF(2α). Our results show that a molecule combining receptor antagonism and thromboxane synthase inhibition is more efficient in delaying atherosclerosis in Apo E(-/-) mice than sole inhibition of TXA(2) formation.     

Effects of BM-573, a thromboxane A2 modulator on systemic hemodynamics perturbations induced by U-46619 in the pig

The aim of our study was to evaluate the effects of thromboxane A2 (TXA2) agonist, U-46619, on systemic circulatory parameters in the pigs before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Twelve anesthetized pigs were randomly assigned in two groups: in Ago group (n=6), the animals received six consecutive injections of U-46619 at 30 min interval, while in Anta group (n=6) they received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. The effects of each dose of BM-573 on ex vivo platelet aggregation induced by arachidonic acid, collagen or ADP were also evaluated. Vascular properties such as characteristic impedance, peripheral resistance, compliance, arterial elastance were estimated using a windkessel model. Intravenous injections of 0.500 mg/ml of BM-573 and higher doses resulted in a complete inhibition of platelet aggregation induced by arachidonic acid. In the same conditions, BM-573 completely blocked the increase of arterial elastance, and stabilized both mean aortic blood pressure and mean systemic blood flow. In conclusion, BM-573 could therefore be a promising therapeutic approach in pathophysiological states where TXA2 plays a main role in the increase of vascular resistance like in pathologies such as systemic hypertension.     

Sodium nitrite downregulates vascular NADPH oxidase and exerts antihypertensive effects in hypertension

Dietary nitrite and nitrate are important sources of nitric oxide (NO). However, the use of nitrite as an antihypertensive drug may be limited by increased oxidative stress associated with hypertension. We evaluated the antihypertensive effects of sodium nitrite given in drinking water for 4 weeks in two-kidney one-clip (2K1C) hypertensive rats and the effects induced by nitrite on NO bioavailability and oxidative stress. We found that, even under the increased oxidative stress conditions present in 2K1C hypertension, nitrite reduced systolic blood pressure in a dose-dependent manner. Whereas treatment with nitrite did not significantly change plasma nitrite concentrations in 2K1C rats, it increased plasma nitrate levels significantly. Surprisingly, nitrite treatment exerted antioxidant effects in both hypertensive and sham-normotensive control rats. A series of in vitro experiments was carried out to show that the antioxidant effects induced by nitrite do not involve direct antioxidant effects or xanthine oxidase activity inhibition. Conversely, nitrite decreased vascular NADPH oxidase activity. Taken together, our results show for the first time that nitrite has antihypertensive effects in 2K1C hypertensive rats, which may be due to its antioxidant properties resulting from vascular NADPH oxidase activity inhibition.     

Hemodynamic changes in apolipoprotein E-knockout mice

Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P < 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P < 0.001), and pulse-wave velocity (+13%, P < 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios.     

Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human alpha- and beta(S)-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E(2) (PGE(2)) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.     

Tempol attenuates atherosclerosis associated with metabolic syndrome via decreased vascular inflammation and NADPH-2 oxidase expression

Abstract

Oxidative stress is an important factor in the generation of vascular injury in atherosclerosis. Chronic administration of fructose in rodents is able to facilitate oxidative damage. In the present study we evaluated the role of Tempol, a superoxide dismutase mimetic, on the effect of high fructose intake in apolipoprotein E-deficient (ApoE-KO) mice. Rodents were fed with fructose overload (FF, 10% w/v) for 8 weeks and treated with Tempol 1 mg/kg/day the latest 4 weeks. Tempol revert the pro-oxidant effects caused by FF, diminished lipid peroxidation and impaired vascular NADPH oxidase system through the downregulation of p47phox expression in the vascular wall. Tempol inhibited the expression of vascular adhesion molecule 1 (VCAM-1) in aorta and reduced the development of atheroma plaques. Our results indicate that tempol attenuates oxidative stress by interfering with the correct assembly of Nox2 oxidase complex in the vascular wall and is able to reduce atherosclerosis. Thus tempol represents a potential therapeutic target for preventing risk factors associated with metabolic syndrome.     

Decreased levels of lipid peroxidation-induced DNA damage in the onset of atherogenesis in apolipoprotein E deficient mice

Increased oxidative stress and subsequent lipid peroxidation (LPO) are thought to be critical events in the formation of atherosclerotic lesions in apolipoprotein E deficient mice (ApoE-KO). LPO derived reactive aldehydes react with DNA to form exocyclic etheno-DNA adducts. These pro-mutagenic DNA lesions are known to be involved in the initiation of carcinogenesis, but their role in the development of atherosclerosis is unknown. In the present study we show that levels of the LPO derived 1,N(6)-ethenodeoxyadenosine (varepsilondA) and 3,N(4)-ethenodeoxycytidine (varepsilondC) were both significantly lower in aorta of 12 weeks old ApoE-KO mice as compared to their wild type controls (1.6+/-0.3 versus 3.2+/-0.8 varepsilondA per 10(8) parent nucleotides, P=0.04 and 4.8+/-0.8 versus 9.2+/-2.1 for varepsilondC, P=0.02). Moreover, levels of both DNA adduct types were inversely related with total plasma cholesterol levels. Consequently, lowest etheno-DNA adduct levels were observed in ApoE-KO mice on a high fat diet. Hypercholesterolemia has previously been associated with increased expression of base excision repair (BER) enzymes, which could explain the lower levels of etheno-DNA adducts in ApoE-KO mice as compared to wild type controls. Indeed, increased staining for the BER-specific DNA repair enzyme apurinic/apyrimidinic endonuclease (Ape1/Ref1) was observed by immunohistochemistry in the endothelium and the first layers of arterial smooth muscle cells of ApoE-KO mice as compared to their wild type counterparts. A high fat diet further increased overall Ape1/Ref1 protein expression in ApoE-KO mice. Although these data suggest no role for increased LPO derived DNA damage in the onset of atherogenesis in ApoE-KO mice, the potentially modulating role of Ape1/Ref1 in the arterial wall deserves further attention.     

Reduction of blood pressure,plasma cholesterol,and atherosclerosis by elevated endothelial nitric oxide

In the vascular system, nitric oxide is generated by endothelial NO synthase (eNOS). NO has pleiotropic effects, most of which are believed to be atheroprotective. Therefore, it has been argued that patients suffering from cardiovascular disease could benefit from an increase in eNOS activity. However, increased NO production can cause oxidative damage, cell toxicity, and apoptosis and hence could be atherogenic rather than beneficial. To study the in vivo effects of increased eNOS activity, we created transgenic mice overexpressing human eNOS. Aortic blood pressure was approximately 20 mm Hg lower in the transgenic mice compared with control mice because of lower systemic vascular resistance. The effects of eNOS overexpression on diet-induced atherosclerosis were studied in apolipoprotein E-deficient mice. Elevation of eNOS activity decreased blood pressure ( approximately 20 mm Hg) and plasma levels of cholesterol ( approximately 17%), resulting in a reduction in atherosclerotic lesions by 40%. We conclude that an increase in eNOS activity is beneficial and provides protection against atherosclerosis.     

Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles.

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.     

Chronic Cadmium Treatment Promotes Oxidative Stress and Endothelial Damage in Isolated Rat Aorta

Cadmium is a highly toxic metal that is present in phosphate fertilizers, and the incidence of cadmium poisoning in the general population has increased, mainly due to cigarette smoking. Once absorbed, cadmium accumulates in the tissues, causing harmful effects including high blood pressure, endothelial damage and oxidative stress. Oxidative stress is known to efficiently produce oxidized low-density lipoprotein and consequently atherosclerosis, mainly in the aorta. However, the mechanisms through which endothelial damage is induced by cadmium have not been elucidated. Thus, the aim of this study was to investigate the effects of this metal in the isolated aorta and the possible role of oxidative stress. Rats received 100 mg.L⁻¹ cadmium chloride (CdCl₂) in the drinking water or distilled water alone for four weeks. The pressor effect of cadmium was followed throughout the exposure period by tail plethysmography. At the end of the fourth week, the blood cadmium content was established, and the vascular reactivity of the isolated aorta to phenylephrine, acetylcholine and sodium nitroprusside was analyzed in the context of endothelium denudation and incubation with L-NAME, apocynin, losartan, enalapril, superoxide dismutase (SOD) or catalase. We observed an increased response to phenylephrine in cadmium-treated rats. This increase was abolished by catalase and SOD incubation. Apocynin treatment reduced the phenylephrine response in both treatment groups, but its effect was greater in cadmium-treated rats, and NOX2 expression was greater in the cadmium group. These results suggested that cadmium in blood concentrations similar to those found in occupationally exposed populations is able to stimulate NOX2 expression, contributing to oxidative stress and reducing NO bioavailability, despite enhanced eNOS expression. These findings suggest that cadmium exposure promotes endothelial damage that might contribute to inflammation, vascular injury and the development of atherosclerosis.     

Antioxidant N-acetylcysteine restores systemic nitric oxide availability and corrects depressions in arterial blood pressure and heart rate in diabetic rats

Increased oxidative stress and reduced nitric oxide (NO) bioactivity are key features of diabetes mellitus that eventually result in cardiovascular abnormalities. We assessed whether N-acetylcysteine (NAC), an antioxidant and glutathione precursor, could prevent the hyperglycaemia induced increase in oxidative stress, restore NO availability and prevent depression of arterial blood pressure and heart rate in vivo in experimental diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were treated or not treated with NAC in drinking water for 8 weeks, initiated 1 week after induction of diabetes. At termination, plasma levels of free 15-F2t-isoprostane, a specific marker of oxygen free radical induced lipid peroxidation, was increased while the plasma total antioxidant concentration was decreased in untreated diabetic rats as compared to control rats (P<0.05). This was accompanied by a significant reduction of plasma levels of nitrate and nitrite, stable metabolites of NO, (P<0.05, D vs. C) and a reduced endothelial NO synthase protein expression in the heart and in aortic and mesenteric artery tissues. Systolic, diastolic and mean arterial blood pressures (SBP, DBP and MAP) and heart rate (HR) were reduced in diabetic rats (P<0.05 vs. C) and NAC normalised the changes that occurred in the diabetic rats. The protective effects may be attributable to restoration of NO bioavailability in the circulation.     

Mineralocorticoid receptor blockade improves diastolic function independent of blood pressure reduction in a transgenic model of RAAS overexpression

There is emerging evidence that aldosterone can promote diastolic dysfunction and cardiac fibrosis independent of blood pressure effects, perhaps through increased oxidative stress and inflammation. Accordingly, this investigation was designed to ascertain if mineralocorticoid receptor blockade improves diastolic dysfunction independently of changes in blood pressure through actions on myocardial oxidative stress and fibrosis. We used young transgenic (mRen2)27 [TG(mRen2)27] rats with increases in both tissue ANG II and circulating aldosterone, which manifests age-related increases in hypertension and cardiac dysfunction. Male TG(mRen2)27 and age-matched Sprague-Dawley rats were treated with either a low dose (～1 mg·kg(-1)·day(-1)) or a vasodilatory, conventional dose (～30 mg·kg(-1)·day(-1)) of spironolactone or placebo for 3 wk. TG(mRen2)27 rats displayed increases in systolic blood pressure and plasma aldosterone levels as well as impairments in left ventricular diastolic relaxation without changes in systolic function on cine MRI. TG(mRen2)27 hearts also displayed hypertrophy (left ventricular weight, cardiomyoctye hypertrophy, and septal wall thickness) as well as fibrosis (interstitial and perivascular). There were increases in oxidative stress in TG(mRen2)27 hearts, as evidenced by increases in NADPH oxidase activity and subunits as well as ROS formation. Low-dose spironolactone had no effect on systolic blood pressure but improved diastolic dysfunction comparable to a conventional dose. Both doses of spironolactone caused comparable reductions in ROS/3-nitrotryosine immunostaining and perivascular and interstitial fibrosis. These data support the notion mineralocorticoid receptor blockade improves diastolic dysfunction through improvements in oxidative stress and fibrosis independent of changes in systolic blood pressure.     

Chronic tempol treatment attenuates the renal hemodynamic effects induced by a heme oxygenase inhibitor in streptozotocin diabetic rats

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in protecting the kidney from oxidant-mediated damage in the streptozotocin (STZ) rat model of type-1 diabetes mellitus (DM-1). HO-derived metabolites, presumably carbon monoxide (CO), mediate vasodilatory influences in the renal circulation, particularly in conditions linked to elevated HO-1 protein expression or diminished nitric oxide (NO) levels. We tested the hypothesis that diabetes increases oxidative stress and induces HO-1 protein expression, which contributes to regulate renal hemodynamics in conditions of low NO bioavailability. Two weeks after the induction of diabetes with STZ (65 mg/kg iv), Sprague-Dawley rats exhibited higher renal HO-1 protein expression, hyperglycemia, and elevated renal nitrotyrosine levels than control normoglycemic animals. In anesthetized diabetic rats, renal vascular resistance (RVR) was increased, and in vivo cortical NO levels were reduced (P < 0.05) compared with control animals. Acute administration of the HO inhibitor Stannous mesoporphyrin (SnMP; 40 μmol/kg iv) did not alter renal hemodynamics in control rats, but greatly decreased glomerular filtration rate and renal blood flow, markedly increasing RVR in hyperglycemic diabetic rats. Chronic oral treatment with the SOD mimetic tempol prevented the elevation of nitrotyrosine, the HO-1 protein induction, and the increases in RVR induced by SnMP in the diabetic group, without altering basal NO concentrations or RVR. Increasing concentrations of a CO donor (CO-releasing molecule-A1) on pressurized renal interlobar arteries elicited a comparable relaxation in vessels taken from control or diabetic animals. These results suggest that oxidative stress-induced HO-1 exerts vasodilatory actions that partially maintain renal hemodynamics in uncontrolled DM-1.     

Effects of U-46619 on pulmonary hemodynamics before and after administration of BM-573, a novel thromboxane A2 inhibitor

We studied the effects on pulmonary hemodynamics of U-46619, a thromboxane A2 (TXA2) agonist, before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Six anesthetized pigs (Ago group) received 6 consecutive injections of U-46619 at 30-min interval and were compared with six anesthetized pigs (Anta group) which received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. Consecutive changes in pulmonary hemodynamics, including characteristic resistance, vascular compliance, and peripheral vascular resistance, were continuously assessed during the experimental protocol using a four-element Windkessel model. At 2 mg/kg, BM-573 completely blocked pulmonary hypertensive effects of U-46619 but pulmonary vascular compliance still decreased. This residual effect can probably be explained by a persistent increase in the tonus of the pulmonary vascular wall smooth muscles sufficient to decrease vascular compliance but not vessel lumen diameter. Such molecule could be a promising therapeutic approach in TXA2 mediated pulmonary hypertension as it is the case in pulmonary embolism, hyperacute lung rejection and endotoxinic shock.     

Evaluation of BM-573, a novel TXA2 synthase inhibitor and receptor antagonist, in a porcine model of myocardial ischemia-reperfusion

AIMS: To investigate whether BM-573 (N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), an original combined thromboxane A2 synthase inhibitor and receptor antagonist, prevents reperfusion injury in acutely ischemic pigs. METHODS: Twelve animals were randomly divided in two groups: a control group (n = 6) intravenously infused with vehicle, and a BM-573-treated group (n = 6) infused with BM-573 (10 mg kg(-1) h(-1)). In both groups, the left anterior descending (LAD) coronary artery was occluded for 60 min and reperfused for 240 min. Either vehicle or BM-573 was infused 30 min before LAD occlusion and throughout the experiment. Platelet aggregation induced by arachidonic acid ex vivo measured was prevented by BM-573. RESULTS: In both groups, LAD occlusion decreased cardiac output, ejection fraction, slope of stroke work--end-diastolic volume relationship, and induced end-systolic pressure-volume relationship (ESPVR) rightward shift, while left ventricular afterload increased. Ventriculo-arterial coupling and mechanical efficiency decreased. In both groups, reperfusion further decreased cardiac output and ejection fraction, while ESPVR displayed a further rightward shift. Ventriculo-arterial coupling and mechanical efficiency remained impaired. Area at risk, evidenced with Evans blue, was 33.2+/-3.4% of the LV mass (LVM) in both groups, and mean infarct size, revealed by triphenyltetrazolium chloride (TTC), was 27.3+/-2.6% of the LVM in the BM-573-treated group (NS). Histological examination and immunohistochemical identification of desmin revealed necrosis in the anteroseptal region similar in both groups, while myocardial ATP dosages and electron microscopy also showed that BM-573 had no cardioprotective effect. CONCLUSIONS: These data suggest that BM-573 failed to prevent reperfusion injury in acutely ischemic pigs.     

Effects of rosuvastatin on cardiovascular morphology and function in an ApoE-knockout mouse model of atherosclerosis

This study investigated the effects of rosuvastatin on plaque progression and in vivo coronary artery function in apolipoprotein E-knockout (ApoE-KO) mice, using noninvasive high-resolution ultrasound techniques. Eight-week-old male ApoE-KO mice (n = 20) were fed a high-fat diet with or without rosuvastatin (10 micromol.kg(-1).day(-1)) for 16 wk. When compared with control, rosuvastatin reduced total cholesterol levels (P < 0.05) and caused significant retardation of lesion progression in the brachiocephalic artery, as visualized in vivo using an ultrasound biomicroscope (P < 0.05). Histological analysis confirmed the reduction of brachiocephalic atherosclerosis and also revealed an increase in collagen content in the statin-treated group (P < 0.05). Coronary volumetric flow was measured by simultaneous recording of Doppler velocity signals and left coronary artery morphology before and during adenosine infusion. The hyperemic flow in response to adenosine was significantly greater in left coronary artery following 16 wk of rosuvastatin treatment (P < 0.001), whereas the baseline flow was similar in both groups. In conclusion, rosuvastatin reduced brachiocephalic artery atherosclerotic plaques in ApoE-KO mice. Coronary artery function assessed using recently developed in vivo ultrasound-based protocols, also improved.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

Low-dose chronic lead exposure increases systolic arterial pressure and vascular reactivity of rat aortas

Chronic lead exposure induces hypertension affecting endothelial function. We investigated whether low-concentration lead exposure alters blood pressure and vascular reactivity, focusing on the roles of NO, oxidative stress, cyclooxygenase-derived vasoconstrictor prostanoids, and the local angiotensin–renin system. Aortic rings from 3-month-old Wistar rats were treated daily with lead acetate (first dose 4 mg/100 g, subsequent doses 0.05 mg/100 g, im) or vehicle for 30 days. Treatment increased lead blood levels (12 μg/dl), blood pressure, and aortic ring contractile response to phenylephrine (1 nM–100 mM). Contractile response after L-NAME administration increased in both groups but was higher after lead treatment. Lead effects on R_max decreased more after apocynin and superoxide dismutase administration compared to control. Indomethacin reduced phenylephrine response more after lead treatment than in controls. The selective COX-2 inhibitor NS398, thromboxane A₂/prostaglandin H2 receptor antagonist SQ 29,548, TXA₂ synthase inhibitor furegrelate, EP₁ receptor antagonist SC 19220, and ACE inhibitor and AT₁ receptor antagonist losartan reduced phenylephrine responses only in vessels from lead-treated rats. Basal and stimulated NO release was reduced and local O₂⁻ liberation increased in the lead-treated group compared to controls. eNOS, iNOS, and AT₁ receptor protein expression increased with lead exposure, but COX-2 protein expression decreased. This is the first demonstration that blood Pb²⁺ (12 µg/dl) concentrations below the WHO-established values increased systolic blood pressure and vascular phenylephrine reactivity. This effect was associated with reduced NO bioavailability, increased reactive oxygen species production, increased participation of COX-derived contractile prostanoids, and increased renin–angiotensin system activity.     

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O₂ ^{. -} production in aorta, but significantly increased NO and reduced O₂ ^{. -} productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production.