Emiliano Duranti

Vascular Generation of Tumor Necrosis Factor-α Reduces Nitric Oxide Availability in Small Arteries From Visceral Fat of Obese Patients

OBJECTIVES The aim of this study was to assess whether small arteries from visceral fat of obese patients show a reduced nitric oxide (NO)-dependent relaxation, as compared with lean control subjects, focusing on the role of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. BACKGROUND Visceral obesity is characterized by endothelial dysfunction. METHODS Small arteries from 14 obese (body mass index 48.4 ± 11 kg/m(2)) and 14 control subjects (body mass index 24.9 ± 2 kg/m(2)), dissected after a visceral fat biopsy (laparoscopy), were evaluated on a pressurized micromyograph. Endothelium-dependent relaxation was assessed by acetylcholine. The NO availability, superoxide production, and inflammation were assessed by testing acetylcholine under the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methylester, tempol (superoxide scavenger), and infliximab (monoclonal anti-TNF-α antibody), respectively. The roles of nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase (iNOS) were assessed by their selective inhibitors apocynin and S-methylisothiourea (SMT), respectively. Vascular superoxide generation (dihydroethidium staining) protein expression of TNF-α and NOS isoforms (Western Blot) and TNF-α localization (immunohistochemistry) were assessed. RESULTS Vessels from obese patients displayed a blunted relaxation to acetylcholine and a reduced inhibitory effect of N(ω)-nitro-L-arginine methylester. These alterations were normalized by tempol or infliximab while being partly ameliorated by apocynin and SMT. Vascular superoxide generation was increased (p < 0.01) in obese patients. This condition was abrogated by both tempol and infliximab and partly (p < 0.05 vs. control subjects) reduced by apocynin or SMT. Enhanced TNF-α and iNOS expression together with increased TNF-α localization in the vascular media were detected. CONCLUSIONS Small arteries from visceral fat of obese patients are characterized by an increased TNF-α production, which reduces NO availability by promoting superoxide generation via nicotinamide adenine dinucleotide phosphate oxidase and iNOS activation.

Atorvastatin Prevents Endothelial Dysfunction in Mesenteric Arteries From Spontaneously Hypertensive Rats: Role of Cyclooxygenase 2-Derived Contracting Prostanoids

We investigated the effect of atorvastatin on cyclooxygenase (COX) contribution to endothelial dysfunction in spontaneously hypertensive rat (SHR) mesenteric resistance arteries. Atorvastatin (10 mg/kg per day, oral gavage) or its vehicle was administered for 2 weeks to male SHR or Wistar-Kyoto rats. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In Wistar-Kyoto rats, relaxation to acetylcholine was inhibited by NG-nitro-l-arginine methyl ester and unaffected by SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), or ascorbic acid. In SHRs, the response to acetylcholine was attenuated, less sensitive to NG-nitro-l-arginine methyl ester, unaffected by SC-560, and enhanced by DuP-697 or SQ-29548 (thromboxane-prostanoid receptor antagonist) to a similar extent. Endothelium-dependent relaxation was normalized by ascorbic acid or apocynin (NADPH oxidase inhibitor), which also restored the inhibition by NG-nitro-l-arginine methyl ester. In atorvastatin-treated SHRs, relaxation to acetylcholine was normalized, fully sensitive to NG-nitro-l-arginine methyl ester, and not affected by SC-560, DuP-697, SQ 29548, or antioxidants. Dihydroethidium assay showed an increased intravascular superoxide generation in SHRs, which was abrogated by atorvastatin. RT-PCR revealed a COX-2 induction in SHR arteries, which was downregulated by atorvastatin. The release of prostacyclin and 8-isoprostane was higher from SHR than Wistar-Kyoto mesenteric vessels. COX-2 inhibition and apocynin decreased 8-isoprostane without affecting prostacyclin levels. Atorvastatin increased phosphorylated extracellular signal–regulated kinase 1/2, pAkt, peNOS1177, and inducible NO synthase levels in SHR mesenteric vessels and decreased 8-isoprostane release. In conclusion, COX-2–derived 8-isoprostane contributes to endothelial dysfunction in SHR mesenteric arteries. Atorvastatin restores NO availability by increasing phosphorylated extracellular signal–regulated kinase 1/2, pAkt, peNOS1177, and inducible NO synthase levels and by abrogating vascular NADPH oxidase–driven superoxide production, which also results in a downregulation of COX-2–dependent 8-isoprostane generation.

Oxidative Stress and Vascular Damage in Hypertension: Role of Angiotensin II.

Reactive oxygen species are oxygen derivates and play an active role in vascular biology. These compounds are generated within the vascular wall, at the level of endothelial and vascular smooth muscle cells, as well as by adventitial fibroblasts. In healthy conditions, ROS are produced in a controlled manner at low concentrations and function as signaling molecules regulating vascular contraction-relaxation and cell growth. Physiologically, the rate of ROS generation is counterbalanced by the rate of elimination. In hypertension, an enhanced ROS generation occurs, which is not counterbalanced by the endogenous antioxidant mechanisms, leading to a state of oxidative stress. In the present paper, major angiotensin II-induced vascular ROS generation within the vasculature, and relative sources, will be discussed. Recent development of signalling pathways whereby angiotensin II-driven vascular ROS induce and accelerate functional and structural vascular injury will be also considered.

Cyclooxygenase-1 Is Involved in Endothelial Dysfunction of Mesenteric Small Arteries From Angiotensin II–Infused Mice

Angiotensin II induces endothelial dysfunction by reducing NO availability and increasing reactive oxygen species. We assessed whether cyclooxygenase (COX)-1 or COX-2 participate in the angiotensin II-induced endothelial dysfunction in murine mesenteric small arteries and examined the role of reduced nicotinamide-adenine dinucleotide phosphate-dependent reactive oxygen species production. Mice received angiotensin II (600 ng/kg per minute, SC), saline (controls), angiotensin II + apocynin (reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, 2.5 mg/day), or apocynin alone for 2 weeks. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In controls, acetylcholine-induced relaxation was inhibited by NG-monomethyl-l-arginine and unaffected by DFU (COX-2 inhibitor), SC-560 (COX-1 inhibitor), or ascorbic acid. In angiotensin II-infused animals, the attenuated response to acetylcholine was less sensitive to NG-monomethyl-l-arginine, unaffected by DFU, and enhanced by SC-560 and, similarly, by SQ-29548, a thromboxane-prostanoid receptor antagonist. Moreover, response to acetylcholine was unchanged by ozagrel, a thromboxane synthase inhibitor, and normalized by ascorbic acid. Apocynin prevented the angiotensin II-induced vascular dysfunctions. In angiotensin II-infused mice, RT-PCR analysis showed a significant COX-2 downregulation, whereas COX-1 expression was upregulated. These changes were unaffected by apocynin. Modulation of COX isoform by angiotensin II was also documented by immunohistochemistry. In small mesenteric vessels, the reduced NO availability and oxidant excess, which characterize endothelial dysfunction secondary to angiotensin II, are associated with a reduced COX-2 and an increased COX-1 function and expression. Angiotensin II causes an oxidative stress-independent COX-1 overexpression, whereas angiotensin II-mediated oxidant excess production stimulates COX-1 activity to produce a contracting prostanoid endowed with agonist activity on thromboxane-prostanoid receptors.

Endothelial dysfunction in small arteries of essential hypertensive patients: role of cyclooxygenase-2 in oxidative stress generation.

Essential hypertensive patients show a reduced nitric oxide availability secondary to oxidative stress generation in peripheral microcirculation. Cyclooxygenase (COX) contributes to reduce nitric oxide availability. We assessed the possible vascular sources of oxidative stress, including COX-1, COX-2, and nicotinamide adenine dinucleotide phosphate oxidase, as determinants of endothelial dysfunction in small arteries isolated from essential hypertensive patients or normotensive controls. Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under NG-nitro-l-arginine methyl ester, SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), ascorbic acid, or the nicotinamide adenine dinucleotide phosphate oxidase inhibitors apocynin or diphenylene iodonium. Vascular oxidative stress generation (fluorescent dihydroethidium), COX-1 and COX-2 expression (Western blot), and localization (immunohistochemistry) were also assessed. In controls, response to acetylcholine was blunted by NG-nitro-l-arginine methyl ester ( P <0.001) and unmodified by SC-560, DuP-697, or ascorbic acid. In hypertensive patients, relaxation to acetylcholine was blunted, resistant to NG-nitro-l-arginine methyl ester or SC-560, and enhanced ( P <0.01) by DuP-697, apocynin, or diphenylene iodonium ( P <0.05). Furthermore, in hypertensive patients, response to acetylcholine was normalized by ascorbic acid or apocynin+DuP-697. Intravascular oxidative stress generation was enhanced in hypertensive patients, decreased ( P <0.01) by DuP-697, partly attenuated by apocynin or diphenylene iodonium, and prevented by ascorbic acid. Enhanced COX-2 expression and localization in the vascular media of hypertensive patients were also detected. In small resistance arteries of essential hypertensive patients, COX-2 is overexpressed and reduces nitric oxide availability. COX-2 represents a major source of oxidative stress generation, whereas nicotinamide adenine dinucleotide phosphate oxidase plays a minor, but significant, role in promoting superoxide generation. # Novelty and Significance {#article-title-32}Essential hypertensive patients show a reduced nitric oxide availability secondary to oxidative stress generation in peripheral microcirculation. Cyclooxygenase (COX) contributes to reduce nitric oxide availability. We assessed the possible vascular sources of oxidative stress, including COX-1, COX-2, and nicotinamide adenine dinucleotide phosphate oxidase, as determinants of endothelial dysfunction in small arteries isolated from essential hypertensive patients or normotensive controls. Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under NG-nitro-L-arginine methyl ester, SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), ascorbic acid, or the nicotinamide adenine dinucleotide phosphate oxidase inhibitors apocynin or diphenylene iodonium. Vascular oxidative stress generation (fluorescent dihydroethidium), COX-1 and COX-2 expression (Western blot), and localization (immunohistochemistry) were also assessed. In controls, response to acetylcholine was blunted by NG-nitro-L-arginine methyl ester (P<0.001) and unmodified by SC-560, DuP-697, or ascorbic acid. In hypertensive patients, relaxation to acetylcholine was blunted, resistant to NG-nitro-L-arginine methyl ester or SC-560, and enhanced (P<0.01) by DuP-697, apocynin, or diphenylene iodonium (P<0.05). Furthermore, in hypertensive patients, response to acetylcholine was normalized by ascorbic acid or apocynin+DuP-697. Intravascular oxidative stress generation was enhanced in hypertensive patients, decreased (P<0.01) by DuP-697, partly attenuated by apocynin or diphenylene iodonium, and prevented by ascorbic acid. Enhanced COX-2 expression and localization in the vascular media of hypertensive patients were also detected. In small resistance arteries of essential hypertensive patients, COX-2 is overexpressed and reduces nitric oxide availability. COX-2 represents a major source of oxidative stress generation, whereas nicotinamide adenine dinucleotide phosphate oxidase plays a minor, but significant, role in promoting superoxide generation.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue

AIMS We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. METHODS AND RESULTS Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. CONCLUSIONS In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

Effect of aliskiren treatment on endothelium-dependent vasodilation and aortic stiffness in essential hypertensive patients

AIMS Aliskiren is a new oral non-peptide renin inhibitor. Its effects on vascular function in human hypertension are unknown. We assessed whether aliskiren may improve peripheral endothelial function and arterial stiffness in essential hypertensive patients (EH), when compared with the angiotensin-converting enzyme-inhibitor ramipril. METHODS AND RESULTS Fifty EH received treatment with aliskiren (150-300 mg/daily) or ramipril (5-10 mg/daily) for 12 weeks, according to a randomized, open with blind endpoints, parallel group design. We studied the forearm blood flow (straingauge plethysmography) response to intrabrachial acetylcholine, repeated under the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) (4 μmol/min), or the antioxidant ascorbic acid (8 mg/100 mL/min). Carotid-to-femoral pulse wave velocity (PWV), central blood pressure and augmentation index (AIx) were obtained by applanation tonometry. Brachial blood pressure was similarly normalized by aliskiren (from 149/94 to 136/86 mmHg) and ramipril (from 148/92 to 135/85 mmHg), as well as central blood pressure. Aliskiren increased (P < 0.001) the vasodilation to acetylcholine and restored the inhibitory effect of l-NMMA on acetylcholine. Ascorbic acid, which at baseline potentiated the response to acetylcholine, no longer improved endothelium-dependent relaxation after aliskiren treatment. In contrast, ramipril failed to affect the response to acetylcholine, the lacking inhibitory effect of l-NMMA, or the potentiating effect of ascorbic acid. Pulse wave velocity was significantly (P < 0.05) and similarly reduced by both drugs. Aliskiren induced a significantly (P < 0.05) greater AIx reduction than ramipril. CONCLUSION Aliskiren increased nitric oxide availability in the forearm resistance arterioles of EH, an effect probably determined by an antioxidant activity, which can also contribute to improved peripheral wave reflection.

Microvascular Endothelial Dysfunction in Obesity and Hypertension

Endothelium plays a crucial role in modulating vascular function and structure, mainly by production of nitric oxide which protects the vasculature against the development of atherosclerosis and thrombosis. Traditional cardiovascular risk factors are characterized by endothelial dysfunction caused by an enhanced production of oxidative stress leading to destroy NO thus reducing its availability. A reduced endothelium-dependent relaxation is a predictor of cardiovascular events in high risk patients. Abdominal obesity is associated with microvascular endothelial dysfunction, through indirect mechanisms, such as insulin-resistance and the association with risk factors (including diabetes mellitus, hypertension and dyslipidemia), and directly, among others, by the production of adipokines and pro-inflammatory cytokines which in turn induce oxidative stress leading to a reduced NO availability. Several systems are amplified by the concomitant obesity and hypertension, thus generating a perpetual vicious circle which further contribute to the pathogenesis/progression of microvascular disease. Weight loss and modification of life-style ameliorate endothelial function in obese patients. It is conceivable that endothelial dysfunction might represent a complementary but crucial objective of a modern therapeutical approach leading to improve the prognosis in many patients, including obese patients, exposed to a high cardiovascular risk.

Cholecalciferol administration blunts the systemic renin–angiotensin system in essential hypertensives with hypovitaminosis D

Introduction: Vitamin D plasma levels are negatively associated with blood pressure and cardiovascular mortality, and vitamin D supplementation reduces cardiovascular events. Renin–angiotensin system (RAS) suppression may be one of the mechanisms involved. However, there are no interventional prospective studies demonstrating a reduction in circulating RAS components after vitamin D treatment. Methods: Fifteen consecutive drug-free patients with essential hypertension and hypovitaminosis D underwent therapy with an oral dose of 25000 I.U. of cholecalciferol once a week for two months, while maintaining a constant-salt diet. In basal conditions and at the end of the study, RAS activity (plasma angiotensinogen, renin, PRA, angiotensin II, aldosterone and urinary angiotensinogen) was investigated, in addition to blood pressure and plasma vitamin D levels (25(OH)D). Results: After cholecalciferol administration, all patients exhibited normalized plasma 25(OH)D values. At the end of the study, a reduction (p < 0.05) in plasma renin and aldosterone, and a decrement, although not significant, of PRA and angiotensin II, was observed. No difference was found in plasma and urinary angiotensinogen or blood pressure values. Conclusions: Our data indicate that in essential hypertensives with hypovitaminosis D, pharmacological correction of vitamin D levels can blunt systemic RAS activity.

Inducible Nitric Oxide Synthase Is Involved in Endothelial Dysfunction of Mesenteric Small Arteries from Hypothyroid Rats

The time-dependent effects of mild hypothyroidism on endothelial function were assessed in rat mesenteric arteries. Male Wistar rats were treated with methimazole (MMI; 0.003%) or placebo up to 16 wk. Endothelial function of mesenteric small arteries was assessed by pressurized myograph. MMI-treated animals displayed a decrease in serum thyroid hormones, an increment of plasma TSH and inflammatory cytokines, and a blunted vascular relaxation to acetylcholine, as compared with controls. Endothelial dysfunction resulted from a reduced nitric oxide (NO) availability caused by oxidative excess. Vascular-inducible NO synthase (iNOS) expression was up-regulated. S-methylisothiourea (an iNOS inhibitor) normalized endothelium-dependent relaxations and restored NO availability in arteries from 8-wk MMI-animals and partly ameliorated these alterations in 16-wk MMI rats. Similar results were obtained when MMI-induced hypothyroidism was prevented by T(4) replacement. Among controls, an impaired NO availability, secondary to oxidative excess, occurred at 16 wk, and it was less pronounced than in age-matched MMI animals. Both endothelial dysfunction and oxidant excess secondary to aging were prevented by apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor). Mesenteric superoxide production was reduced by S-methylisothiourea and T(4) replacement in MMI animals and abolished by apocynin in controls (dihydroethidium staining). MMI-induced mild hypothyroidism is associated with endothelial dysfunction caused by a reduced NO availability, secondary to oxidative excess. It is suggested that in this animal model, characterized by TSH elevation and low-grade inflammation, an increased expression and function of iNOS, resulting in superoxide generation, accounts for an impaired NO availability.