Phenotypic switching prevention and proliferation/migration inhibition of vascular smooth muscle cells by the ruthenium nitrosyl complex trans-[Ru(NO)Cl(cyclam](PF6 )2.

OBJECTIVES: Vascular smooth muscle cell (VSMC) migration and proliferation at sites of vascular injury are both critical steps in the development of intimal hyperplasia (IH). Local delivery of nitric oxide (NO) largely prevents these events. Among the NO donors, tetraazamacrocyclic nitrosyl complexes, such as trans-[Ru(NO)Cl(cyclam)](PF6 )2 (cyclamNO), gained attention for their features, which include the possibility of being embedded in solid matrices, and ability to participate in a nitrite/NO catalytic conversion cycle. METHODS: Methods used to evaluate cyclamNO activity: safety margin by NR and MTT; cell proliferation by 3H-thymidine incorporation and proliferating cell nuclear antigen (PCNA) expression; antimigratory properties by transwell and wound healing; prevention of cell phenotypic switching under platelet-derived growth factor type BB (PDGF-BB) stimuli by analysis of alpha smooth muscle actin (α-SMA) expression. KEY FINDINGS: Cell proliferation and migration induced by PDGF-BB were significantly inhibited by cyclamNO. The ~60% reduction on expression of contractile protein α-SMA induced by PDGF-BB revealed VSMC phenotypic switching which is significantly prevented by cyclamNO. Compared to the NO donor sodium nitroprusside, cyclamNO showed to be significantly less cytotoxic. CONCLUSIONS: With great potential to maintain VSMC functionality and prevent IH-associated events, cyclamNO might be a promissory drug for several applications in cardiovascular medicine, as in stents.

Aerobic exercise training protects against endothelial dysfunction by increasing nitric oxide and hydrogen peroxide production in LDL receptor-deficient mice.

BACKGROUND: Endothelial dysfunction associated with hypercholesterolemia is an early event in atherosclerosis characterized by redox imbalance associated with high superoxide production and reduced nitric oxide (NO) and hydrogen peroxide (H2O2) production. Aerobic exercise training (AET) has been demonstrated to ameliorate atherosclerotic lesions and oxidative stress in advanced atherosclerosis. However, whether AET protects against the early mechanisms of endothelial dysfunction in familial hypercholesterolemia remains unclear. This study investigated the effects of AET on endothelial dysfunction and vascular redox status in the aortas of LDL receptor knockout mice (LDLr(-/-)), a genetic model of familial hypercholesterolemia. METHODS: Twelve-week-old C57BL/6J (WT) and LDLr(-/-) mice were divided into sedentary and exercised (AET on a treadmill 1 h/5 × per week) groups for 4 weeks. Changes in lipid profiles, endothelial function, and aortic NO, H2O2 and superoxide production were examined. RESULTS: Total cholesterol and triglycerides were increased in sedentary and exercised LDLr(-/-) mice. Endothelium-dependent relaxation induced by acetylcholine was impaired in aortas of sedentary LDLr(-/-) mice but not in the exercised group. Inhibition of NO synthase (NOS) activity or H2O2 decomposition by catalase abolished the differences in the acetylcholine response between the animals. No changes were noted in the relaxation response induced by NO donor sodium nitroprusside or H2O2. Neuronal NOS expression and endothelial NOS phosphorylation (Ser1177), as well as NO and H2O2 production, were reduced in aortas of sedentary LDLr(-/-) mice and restored by AET. Incubation with apocynin increased acetylcholine-induced relaxation in sedentary, but not exercised LDLr(-/-) mice, suggesting a minor participation of NADPH oxidase in the endothelium-dependent relaxation after AET. Consistent with these findings, Nox2 expression and superoxide production were reduced in the aortas of exercised compared to sedentary LDLr(-/-) mice. Furthermore, the aortas of sedentary LDLr(-/-) mice showed reduced expression of superoxide dismutase (SOD) isoforms and minor participation of Cu/Zn-dependent SODs in acetylcholine-induced, endothelium-dependent relaxation, abnormalities that were partially attenuated in exercised LDLr(-/-) mice. CONCLUSION: The data gathered by this study suggest AET as a potential non-pharmacological therapy in the prevention of very early endothelial dysfunction and redox imbalance in familial hypercholesterolemia via increases in NO bioavailability and H2O2 production.

Preventive and therapeutic moderate aerobic exercise programs convert atherosclerotic plaques into a more stable phenotype.

UNLABELLED: The mechanisms by which exercise affects atherosclerotic plaque stability remain incompletely understood. We evaluated the effects of two training protocols on both atherosclerotic plaque structure and the signaling pathways involved in plaque rupture. METHODS: Male low-density lipoprotein (LDL) receptor knockout mice were fed a high-fat, high-cholesterol diet (HFD). One group was subjected to moderate exercise using a treadmill for 14weeks (preventive protocol). The other group started an exercise regimen after 16weeks of the HFD (therapeutic group). Atherosclerotic plaques within the aorta were evaluated for lipid and collagen contents, as well as for inflammatory markers. Plasma cholesterol and cytokine levels were also determined. RESULTS: The mice receiving a HFD developed hypercholesterolemia and atherosclerotic plaques within the aorta. The aortas from the animals in the preventive protocol exhibited smaller lipid cores and higher collagen content. These animals also exhibited lower CD40 expression within the plaques. The aortas of the mice in the therapeutic group exhibited higher collagen content, but no differences in either lipid core size or plaque size were noted. No differences in blood pressure, plasma cholesterol, cytokine levels, plaque size or metalloproteinase 9 expression were observed in the trained animals compared with the sedentary animals. CONCLUSION: Moderate aerobic exercise modified atherosclerotic plaque characteristics and converted the plaques into a more stable phenotype, increasing the collagen content in response to both exercise programs. Furthermore, moderate aerobic exercise reduced the animals' fat content and decreased the activity of the CD40-CD40L signaling pathway in the preventive group.

In vitro evaluation of the safe margin, antithrombotic and antiproliferative actions for the treatment of restenosis: Nitric oxide donor and polymers.

Drug-eluting stents (DES) were developed to combat the problem of in-stent restenosis, and evaluating the biological activity from DES systems is critical for its safety and efficacy. To test the cytotoxicity of nitric oxide (NO) donor-containing polymers for their potential use in DES applications, S-nitrosoglutathione (GSNO) or in combination with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) in an aqueous polymeric solution (PVA/PVP/GSNO) was investigated using Balb/c 3T3 and Rabbit arterial smooth muscle (RASM) cells. The sensitivity of 3T3 cells to the cytotoxicity effects induced by GSNO was higher than that of RASM cells, while RASM cells were more susceptible to alterations in membrane permeability. Cell growth assays showed that GSNO and PVA/PVP/GSNO induced antiproliferative effects in RASM cells. Moreover, the presence of polymers can reduce the cytotoxicity and enhance the antiproliferative effects of GSNO. Dose-dependent inhibition of platelet aggregation was similar for both PVA/PVP/GSNO (EC50 of 3.4 ± 2.3 µM) and GSNO (EC50 of 2.8 ± 1.1 µM) solutions. Platelet adhesion assays showed that the inhibition caused by GSNO (EC50 of 5.0 mM) was dependent on the presence of plasma. These results demonstrate that the methodology adopted here is suitable to establish safety margins and evaluate the antithrombotic potential and antiproliferative effects of NO-eluting biomaterials and polymeric solutions for the new cardiovascular devices, and also to emphasize the importance of using more specific cell lines in these evaluations.

Telmisartan regresses left ventricular hypertrophy in caveolin-1-deficient mice.

The role of angiotensin II (Ang II) in promoting cardiac hypertrophy is well known; however, its role in a spontaneous model of hypertrophy in mice lacking the protein caveolin-1 (Cav-1 KO) has not been explored. In this study, WT and Cav-1 KO mice were treated with angiotensin receptor blocker (ARB), telmisartan (Telm), and cardiac function was assessed by echocardiography. Treatment of Cav-1 KO mice with Telm significantly improved cardiac function compared with age-matched vehicle-treated Cav-1 KO mice, whereas Telm did not affect cardiac function in WT mice. Both left ventricular (LV) weight to body weight ratios and LV to tibial length ratios were also reverted by Telm in Cav-1 KO but not in WT mice. LV hypertrophy was associated with increased expression of natriuretic peptides A and B, ß-myosin heavy chain and TGF-ß, and Telm treatment normalized the expression of these genes. Telm reduced the expression of collagen genes (Col1A and Col3A) and associated perivascular fibrosis in intramyocardial vessels in Cav-1 KO mice. In conclusion, Telm treatment reduces indexes of cardiac hypertrophy in this unique genetic model of spontaneous LV hypertrophy.

Glu298Asp eNOS gene polymorphism causes attenuation in nonexercising muscle vasodilatation.

The influence of Glu298Asp endothelial nitric oxide synthase (eNOS) polymorphism in exercise-induced reflex muscle vasodilatation is unknown. We hypothesized that nonexercising forearm blood flow (FBF) responses during handgrip isometric exercise would be attenuated in individuals carrying the Asp298 allele. In addition, these responses would be mediated by reduced eNOS function and NO-mediated vasodilatation or sympathetic vasoconstriction. From 287 volunteers previously genotyped, we selected 33 healthy individuals to represent three genotypes: Glu/Glu [n = 15, age 43 +/- 3 yr, body mass index (BMI) 22.9 +/- 0.3 kg/m(2)], Glu/Asp (n = 9, age 41 +/- 3 yr, BMI 23.7 +/- 1.0 kg/m(2)), and Asp/Asp (n = 9, age 40 +/- 4 yr, BMI 23.5 +/- 0.9 kg/m(2)). Heart rate (HR), mean blood pressure (MBP), and FBF (plethysmography) were recorded for 3 min at baseline and 3 min during isometric handgrip exercise. Baseline HR, MBP, FBF, and forearm vascular conductance (FVC) were similar among genotypes. FVC responses to exercise were significantly lower in Asp/Asp when compared with Glu/Asp and Glu/Glu (Delta = 0.07 +/- 0.14 vs. 0.64 +/- 0.20 and 0.57 +/- 0.09 units, respectively; P = 0.002). Further studies showed that intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA) did not change FVC responses to exercise in Asp/Asp, but significantly reduced FVC in Glu/Glu (Delta = 0.79 +/- 0.14 vs. 0.14 +/- 0.09 units). Thus the differences between Glu/Glu and Asp/Asp were no longer observed (P = 0.62). l-NMMA + phentolamine increased similarly FVC responses to exercise in Glu/Glu and Asp/Asp (P = 0.43). MBP and muscle sympathetic nerve activity increased significant and similarly throughout experimental protocols in Glu/Glu and Asp/Asp. Individuals who are homozygous for the Asp298 allele of the eNOS enzyme have attenuated nonexercising muscle vasodilatation in response to exercise. This genotype difference is due to reduced eNOS function and NO-mediated vasodilatation, but not sympathetic vasoconstriction.

S-nitroso-N-acetylcysteine (SNAC) prevents myocardial alterations in hypercholesterolemic LDL receptor knockout mice by antiinflammatory action.

We investigated the ability of S-nitroso-N-acetylcyseine (SNAC) to prevent structural and functional myocardial alterations in hypercholesterolemic mice. C57BL6 wild-type (WT) and LDL-R-/- male mice (S) were fed a standard diet for 15 days. LDL-R-/- mice (S) showed an 11% increase in blood pressure, 62% decrease in left atrial contractility, and lower CD40L and eNOS expression relative to WT. LDL-R-/- mice fed an atherogenic diet for 15 days (Chol) showed significant increased left ventricular mass compared to S, which was characterized by: (1) 1.25-fold increase in the LV weight/body weight ratio and cardiomyocyte diameter; (2) enhanced expression of the NOS isoforms, CD40L, and collagen amount; and (3) no alteration in the atrial contractile performance. Administration of SNAC to Chol mice (Chol + SNAC) (0.51 micromol/kg/day for 15 day, IP) prevented increased left ventricular mass, collagen deposit, NOS isoforms, and CD40L overexpression, but it had no effect on the increased blood pressure or atrial basal hypocontractility. Deletion of the LDL receptor gene in mice resulted in hypertension and a marked left atrial contractile deficit, which may be related to eNOS underexpression. Our data show that SNAC treatment has an antiinflammatory action that might contribute to prevention of structural and functional myocardial alterations in atherosclerotic mice independently of changes in blood pressure.

Dissociation of blood pressure and sympathetic activation of renin release in sinoaortic-denervated rats.

1. Blood pressure (BP) and heart rate (HR) increase 6 and 24 h after sinoaortic baroreceptor denervation (SAD), whereas plasma renin activity (PRA) and renal renin mRNA levels remain unchanged. We postulated that a simultaneous rise in BP could offset the expected activation of renin associated with an increased renal sympathetic discharge secondary to SAD. 2. To test this hypothesis, the increase in BP associated with the onset of SAD was prevented by a continuous infusion of sodium nitroprusside (SNP; 30 microg/kg per h). Changes were measured in five groups of conscious adult male Wistar rats: (i) sham; (ii) SAD; (iii) SAD rats in which the BP was prevented from increasing by infusion of SNP; (iv) sham rats in which the BP was increased by 30% by infusion of phenylephrine (PE; 1.5-2.0 mL/h); and (v) SNP + PE for 3 h by infusion as above. 3. As expected, BP and heart rate (HR) increased significantly following SAD compared with sham rats (152 +/- 4 vs 116 +/- 3 mmHg, respectively, for BP and 503 +/- 6 vs 345 +/- 13 b.p.m., respectively for HR; n = 5; P < 0.05) but remained unchanged when SNP was infused for 3 h (106 +/- 1 mmHg and 455 +/- 9 b.p.m., respectively; n = 5; P < 0.05). 4. Similarly, BP and HR increased with PE infusion compared with PE + SNP (138 +/- 9.9 vs 113 +/- 2.3 mmHg for BP, respectively, and 325 +/- 9 vs 423 +/- 18 b.p.m. for HR, respectively; n = 5; P < 0.05). 5. Plasma renin activity remained unchanged in SAD compared with sham rats (1.67 +/- 0.35 vs 1.05 +/- 0.17 ng angiotensin (Ang) I/mL per h), but increased significantly when hypertension was prevented (5.86 +/- 0.77 ng AngI/mL per h; n = 5; P < 0.05). Renin mRNA levels in the kidneys were unchanged in all groups. 6. These results show that an elevation in BP appears to offset increased renal sympathetic discharge with no change in PRA.

Evidence for two atypical conformations of beta-adrenoceptors and their interaction with Gi proteins.

In this study, we investigated whether the responses of right atria from sinoaortic denervated rats to CGP12177 (4(3-t-butylamino-2-hydroxypropoxy benzidimidazole-2 one, hydrochloride)), isoprenaline and norepinephrine desensitized in parallel and whether CGP12177 interacted with distinct conformations of beta-adrenoceptors. Right atria from rats 48 h after sinoaortic denervation were subsensitive to isoprenaline, norepinephrine and CGP12177. One week after sinoaortic denervation, the sensitivity to CGP12177 had recovered whereas the responses to isoprenaline and norepinephrine were still subsensitive, suggesting that the binding sites for these molecules showed independent behavior. In atria from 48 h sinoaortic-denervated rats, propranolol or 3 microM CGP20712A (2-hydroxy-5(2-((2-hydroxy-3-(4-((methyl-4-trifluormethyl)1H imidazole-2-yl)-phenoxypropyl) amino) ethoxy)-benzamide monomethane sulphonate)) blocked the responses to 10 nM-1 microM CGP12177 and steepened the curves. The concentration-response curves to CGP12177 in the presence of ICI118,551 (erythro-DL-1(-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol) were biphasic, suggesting that CGP12177 interacted with at least two conformations of beta-adrenoceptors that showed negative cooperativism, one acting through beta(2)-adrenoceptor-Gi and the other via beta(1)-adrenoceptor-Gs. This hypothesis was confirmed in right atria from sinoaortic-denervated rats treated with pertussis toxin.

In vivo effects of the controlled NO donor/scavenger ruthenium cyclam complexes on blood pressure.

Ruthenium(II/III) complexes able to bind and release NO* were tested in vivo, in conscious Wistar rats instrumented for continuous blood pressure (BP) measurement and administration of in bolus injections (5 to 100 nmol/Kg i.v.) of trans-[Ru(II)Cl(NO+)(cyclam)](PF6)2 (cyclam-NO) or sodium nitroprusside (SNP). For normotensive rats, cyclam-NO produced a sustained 10% BP reduction of basal MAP during 7 +/- 0.4 to 11 +/- 0.3 min. In acute hypertensive rats, cyclam-NO produced BP reduction 3-fold larger than in normotensive rats and similar to that of SNP (maximal effect: 41 +/- 1.3 vs. 45 +/- 2.2 mmHg, respectively). However, the duration of the effect of cyclam-NO was 13 to 21-fold longer than that of SNP. The hypotensive effect of cyclam-NO was fully blocked in presence of continuous infusion of a NO* scavenger, carboxy-PTIO (6 mmol/Kg/min), or of the inhibitor of cGMP activation, methylene blue (83 nmol/Kg/min), or of the cyclam-NO precursor, trans-[RuCl(tfins)(cyclam)](tfms) (cyclam-tfms) (500 mmol/Kg/min). The long lasting BP reduction of cyclam-NO can be interpreted in terms of a slower rate of NO* release (k-NO = 2.2 x 10(-3) S(-1) at 35 degrees C) following chemical reduction (E(0') = 0.10 V vs NHE). In summary, cyclam-NO showed an hypotensive effect around 20 times longer than SNP in either normotensive or hypertensive rats, which was completely inhibited by methylene blue or carboxy-PTIO. Continuous infusion of cyclam-tfms completely blocked the hypotensive effect of cyclam-NO by scavenging the NO* released by the reduced cyclam-NO.