Single-Dose Monocrotaline Pyrrole Injection as a Model of Pulmonary Endothelial Injury In Mice

Monocrotaline (MCT) is a plant substance that induces severe pulmonary hypertension in several animals except for mice. The aim of our study was to state whether monocrotaline pyrrole (MCTp), the main monocrotaline metabolite, could induce significant injury in mouse lung when given intravenously. MCTp caused moderate pulmonary inflammation, remodelling of small distal vessels (percentage of muscularized arteries: 33,5 vs 20,6%, p≤0,0006) and a right ventricular dysfunction (RVSP 27,8mmHg vs 16,4mmHg, p≤0,0001; Fulton index 0,35 vs 0,26, p≤0,0007). These vascular effects were associated with a decrease in eNOS protein expression in lung tissues and resolved after 45 days. In conclusion, we developed a model of endothelial dysfunction and transient pulmonary hypertension in mice.

The Regulatory Role of Nuclear Factor Kappa B in the Heart of Hereditary Hypertriglyceridemic Rat

Activation of nuclear factor-κB (NF-κB) by increased production of reactive oxygen species (ROS) might induce transcription and expression of different antioxidant enzymes and also of nitric oxide synthase (NOS) isoforms. Thus, we aimed at studying the effect of NF-κB inhibition, caused by JSH-23 (4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine) injection, on ROS and NO generation in hereditary hypertriglyceridemic (HTG) rats. 12-week-old, male Wistar and HTG rats were treated with JSH-23 (bolus, 10μmol, i.v.). After one week, blood pressure (BP), superoxide dismutase (SOD) activity, SOD1, endothelial NOS (eNOS), and NF-κB (p65) protein expressions were higher in the heart of HTG rats compared to control rats. On the other hand, NOS activity was decreased. In HTG rats, JSH-23 treatment increased BP and heart conjugated dienes (CD) concentration (measured as the marker of tissue oxidative damage). Concomitantly, SOD activity together with SOD1 expression was decreased, while NOS activity and eNOS protein expression were increased significantly. In conclusion, NF-κB inhibition in HTG rats led to decreased ROS degradation by SOD followed by increased oxidative damage in the heart and BP elevation. In these conditions, increased NO generation may represent rather a counterregulatory mechanism activated by ROS. Nevertheless, this mechanism was not sufficient enough to compensate BP increase in HTG rats.

Abstract P038: Dietary Capsaicin May Decrease Blood Pressure Through Enhancing NO With eNOS Activation in 2-Kidney, 1-Clip Hypertensive Rats

Objective: Capsaicin, a component of chili peppers, is reported to have beneficial effects on cardiovascular system through the vasodilative effects. We recently demonstrated the alleviation of blood pressure (BP) elevation by consuming a low concentration of capsaicin diet in 2-kidney, 1-clip (2K1C) hypertensive rats. Since the alleviation was diminished when 2K1C rats took NG-nitro-L-arginine methyl ester, a NO synthase (NOS) inhibitor, during the protocol, we hypothesized that NO has a key role in the effect of capsaicin in 2K1C rats. In this study, we observed eNOS mRNA expression and protein expressions of eNOS and phosphorylated eNOS in 2K1C rats fed a diet containing capsaicin. Methods: Six-week old male Sprague-Dawley rats were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). One week after the surgery, each group of rats were further divided into 2 groups randomly, which received either a control diet (CTL) or a diet containing 0.006% capsaicin (CAP) for 6 weeks. The systolic BP was measured by a tail-cuff method once per week throughout the protocol. At the end of the protocol, rats were euthanized and the abdominal aortas were collected for extracting mRNA and protein. Then, the expression of eNOS mRNA and protein in aorta was evaluated in each group of rats by real time RT-PCR and Western blotting. Results: As shown in Table, capsaicin diet alleviated BP elevation in 2K1C rats. After the dietary protocol, eNOS mRNA expression in 2K1C-CAP was significantly higher than in 2K1C-CTL. Although there were no significant differences in eNOS protein expression among four groups, phosphorylated eNOS protein expression in 2K1C-CAP was marginally significantly higher than in 2K1C-CTL. The expression was also significantly higher in 2K1C rats than in SHAM. Discussion: The present data suggested that dietary capsaicin decreases BP through enhancing NO with activation of eNOS in 2K1C hypertensive rats. It may be a clue for developing a dietary therapy for prevention of hypertension.

PI3K-Akt/eNOS in remote postconditioning induced by brief pulmonary ischemia

Purpose: Postconditioning, a series of brief ischemia-reperfusion sequences given before an ischemic heart undergoes sustained reperfusion, has been shown to lessen ischemia/reperfusion injury. The current study establishes a rabbit model of myocardial ischemia-reperfusion and studied the effects of pulmonary remote postconditioning in this model. Methods: Serum levels of creatine kinase (CK), superoxide dismutase (SOD), and malondialdehyde (MDA), protein expression of endothelial nitric oxide synthase (eNOS), Rho kinase (ROCK- 2), and protein kinase B (Akt) in myocardial cells and the apoptosis index of myocardial cells were examined. Results: Pulmonary remote postconditioning decreased CK, significantly decreased MDA, and increased SOD. Postconditioning significantly increased eNOS protein expression. Administration of eNOS inhibitor, L-NAME, dramatically suppressed the postconditioning-induced eNOS protein expression and serum SOD level, but significantly increased MDA level. The two longer sessions of postconditioning increased Akt, although this increase was not accompanied by changes in levels of the Akt inhibitor, ROCK-2. Blocking eNOS activity with L-NAME had no visible effect on either Akt or ROCK-2. Conclusion: Our results suggest a role for Akt in remote postconditioning-induced myocardial protection, but do not support an involvement of eNOS in Akt-mediated action.

Prolonged AMP-activated protein kinase induction impairs vascular functions

AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism and is involved in the pathogenesis of several diseases, including type 2 diabetes and cardiovascular diseases. Data showing the effects of AMPK on vasculature are controversial. Therefore, the aim of this study was to determine the impact of prolonged AMPK activation on vascular functions. For this purpose we have examined the role of AMPK in endothelium-dependent and -independent relaxation and vascular contractions. For this, we incubated thoracic aortic rings, from rats, with AMPK activator 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR, 500 μmol/L or 2 mmol/L) in the presence or absence of AMPK inhibitor compound C (10 μmol/L). Next, cumulative dose–response curves to acetylcholine (ACh) (10−9−10−4 mol/L), nitroglycerine (NG) (10−9–3 × 10−5 mol/L), and noradrenaline (NA) (10−9−10−4 mol/L) were obtained. Endothelial nitric oxide synthase (eNOS) protein expression was determined. Our results show that endothelium-dependent relaxation was inhibited after AICAR treatment, and that this effect was reversed by AMPK inhibition. Moreover, AICAR enhanced the contractile response to NA and caused a decrease in eNOS protein expression. In conclusion, prolonged AMPK induction causes endothelial impairment, possibly via increased degradation and (or) reduced expression of eNOS.

eNOS phosphorylation and translocation are altered in male but not female mice by increased activation of the Gαq protein

Little is known about sex-dependent physiological and pathophysiological differences in cardiac endothelial nitric oxide synthase (eNOS) expression and activation. Therefore, we investigated cardiac morphology and eNOS protein expression, including its translocation-dependent activation and phosphorylation, in cardiac tissue of male and female wild-type mice and transgenic heart-failure mice having a cardiac-specific, 5-fold overexpression of the Gαq protein. In addition, we measured calcineurin protein expression. Heart-to-body weight ratio was increased in Gαq mice. Female wild-type mice showed higher eNOS protein expression and activation (translocation and phosphorylation) than did wild-type males. In cardiac tissue of Gαq mice, these sex-dependent differences remained or were enhanced. Protein expression of the catalytic subunit calcineurin A, which has been shown to dephosphorylate eNOS, was higher in wild-type males than in wild-type females. These differences were increased in the Gαq mice model. We conclude that sex differences exist in cardiac eNOS protein expression and phosphorylation. Increased activation of the Gαq protein appears to alter eNOS protein expression and phosphorylation only in males.

Endothelial and vascular dysfunctions and insulin resistance in rats fed a high-fat, high-sucrose diet

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis.