Inhibitors of gastric acid secretion increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling.

The use of inhibitors of gastric acid secretion (IGAS), especially proton pump inhibitors (PPI), has been associated with increased cardiovascular risk. While the mechanisms involved are not known, there is evidence supporting increased oxidative stress, a major activator of matrix metalloproteinases (MMP), as an important player in such effect. However, there is no study showing whether other IGAS such as histamine H2-receptor blockers (H2RB) cause similar effects. This study aimed at examining whether treatment with the H2RB ranitidine promotes oxidative stress resulting in vascular MMP activation and corresponding functional and structural alterations in the vasculature, as compared with those found with the PPI omeprazole. Male Wistar rats were treated (4 weeks) with vehicle (2% tween 20), omeprazole (10 mg/Kg/day; i.p.) or ranitidine (100 mg/Kg/day; gavage). Then the aorta was collected to perform functional, biochemical, and morphometric analysis. Both ranitidine and omeprazole increased gastric pH and oxidative stress assessed in situ with the fluorescent dye dihydroethidium (DHE) and with lucigenin chemiluminescence assay. Both IGAS augmented vascular activated MMP-2. These findings were associated with aortic remodeling (increased media/lumen ratio and number of cells/µm2). Both IGAS also impaired the endothelium-dependent relaxation induced by acetylcholine (isolated aortic ring preparation). This study provides evidence that the H2RB ranitidine induces vascular dysfunction, redox alterations, and remodeling similar to those found with the PPI omeprazole. These findings strongly suggest that IGAS increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling, which helps to explain the increased cardiovascular risk associated with the use of those drugs.

Hemodynamic impairment induced by Crotoxin using in vivo and ex vivo approach in a rat model.

Crotalus durissus snakebite represent 10 % of snakebite cases in Brazil, which cardiovascular disorders are associated with severe cases. Considering crotoxin (CTX) as the major venom component, the present study aimed to evaluate the hemodynamic alterations induced by CTX using in vivo and ex vivo approaches in a rat model. In vivo cardiac function parameters were analyzed from anesthetized rats treated with CTX or saline only (Sham), along with serum creatine kinase MB (CK-MB) and lung myeloperoxidase. From the same animals, hearts were isolated and functional parameters evaluated in Langendorff method ex vivo. CTX binding to myoblast cell line in vitro were evaluated using confocal microscopy and flow cytometry. CTX was capable of reducing arterial and diastolic blood pressure, heart rate, along with left ventricle pressure development or decay during systole (LVdP/dtmax and LVdP/dtmin) in vivo, however no differences were found in the ex vivo approach, showing that intrinsic heart function was preserved. In vitro, CTX binding to myoblast cell line was mitigated by hexamethonium, a nicotinic acetylcholine receptor antagonist. The present study has shown that CTX induce hemodynamic failure in rats, which can help improve the clinical management of cardiovascular alterations during Crotalus durissus snakebite.

Quercetin decreases cardiac hypertrophic mediators and maladaptive coronary arterial remodeling in renovascular hypertensive rats without improving cardiac function.

Oxidative stress and MMP activity are found in the hearts and arteries in hypertension and contribute to the resulting hypertrophy and dysfunction. Quercetin is a flavonoid that reduces MMP-2 activity and ameliorates hypertrophic vascular remodeling of hypertension. The hypothesis is that treatment of hypertensive rats with quercetin ameliorates coronary maladaptive remodeling and decreases hypertrophic cardiac dysfunction by decreasing oxidative stress and MMP activity. Male Sprague-Dawley two-kidney, one-clip (2K1C) and Sham rats were treated with quercetin (10 mg/kg/day) or its vehicle for 8 weeks by gavage. Rats were analyzed at 10 weeks of hypertension. Systolic blood pressure (SBP) was examined by tail-cuff plethysmography. Cardiac left ventricles were used to determine MMP activity by in situ zymography and oxidative stress by dihydroethidium. Immunofluorescence was performed to detect transforming growth factor (TGF)-ß and nuclear factor kappa B (NFkB). Morphological analyses of heart and coronary arteries were done by H&E and picrosirius red, and cardiac function was measured by Langendorff. SBP was increased in 2K1C rats, and quercetin did not reduce it. However, quercetin decreased both oxidative stress and TGF-ß in the left ventricles of 2K1C rats. Quercetin also decreased the accentuated MMP activity in left ventricles and coronary arteries of 2K1C rats. Quercetin ameliorated hypertension-induced coronary arterial hypertrophic remodeling, although it did not reduce cardiac hypertrophic remodeling and dysfunction. Quercetin decreases cardiac oxidative stress and TGF-ß and MMP activity in addition to improving coronary remodeling, yet does not ameliorate cardiac dysfunction in 2K1C rats.

On the origin of nitrosylated hemoglobin in COVID-19: Endothelial NO capture or redox conversion of nitrite?: Experimental results and a cautionary note on challenges in translational research.

In blood, the majority of endothelial nitric oxide (NO) is scavenged by oxyhemoglobin, forming nitrate while a small part reacts with dissolved oxygen to nitrite; another fraction may bind to deoxyhemoglobin to generate nitrosylhemoglobin (HbNO) and/or react with a free cysteine to form a nitrosothiol. Circulating nitrite concentrations in healthy individuals are 200-700 nM, and can be even lower in patients with endothelial dysfunction. Those levels are similar to HbNO concentrations ([HbNO]) recently reported, whereby EPR-derived erythrocytic [HbNO] was lower in COVID-19 patients compared to uninfected subjects with similar cardiovascular risk load. We caution the values reported may not reflect true (patho)physiological concentrations but rather originate from complex chemical interactions of endogenous nitrite with hemoglobin and ascorbate/N-acetylcysteine. Using an orthogonal detection method, we find baseline [HbNO] to be in the single-digit nanomolar range; moreover, we find that these antioxidants, added to blood collection tubes to prevent degradation, artificially generate HbNO. Since circulating nitrite also varies with lifestyle, dietary habit and oral bacterial flora, [HbNO] may not reflect endothelial activity alone. Thus, its use as early marker of NO-dependent endothelial dysfunction to stratify COVID-19 patient risk may be premature. Moreover, oxidative stress not only impairs NO formation/bioavailability, but also shifts the chemical landscape into which NO is released, affecting its downstream metabolism. This compromises the endothelium's role as gatekeeper of tissue nutrient supply and modulator of blood cell function, challenging the body's ability to maintain redox balance. Further studies are warranted to clarify whether the nature of vascular dysfunction in COVID-19 is solely of endothelial nature or also includes altered erythrocyte function.

Antiseptic mouthwash inhibits antihypertensive and vascular protective effects of L-arginine.

L-arginine supplementation increases nitric oxide (NO) formation and bioavailability in hypertension. We tested the possibility that many effects of L-arginine are mediated by increased formation of NO and enhanced nitrite, nitrate and nitrosylated species concentrations, thus stimulating the enterosalivary cycle of nitrate. Those effects could be prevented by antiseptic mouthwash. We examined how the derangement of the enterosalivary cycle of nitrate affects the improvement of endothelial dysfunction (assessed with isolated aortic ring preparation), the antihypertensive (assessed by tail-cuff blood pressure measurement) and the antioxidant effects (assessed with the fluorescent dye DHE) of L-arginine in two-kidney, one-clip hypertension model in rats by using chlorhexidine to decrease the number of oral bacteria and to decrease nitrate reductase activity assessed from the tongue (by ozone-based chemiluminiscence assay). Nitrite, nitrate and nitrosylated species concentrations were assessed (ozone-based chemiluminiscence). Chlorhexidine mouthwash reduced the number of oral bacteria and tended to decrease the nitrate reductase activity from the tongue. Antiseptic mouthwash blunted the improvement of the endothelial dysfunction and the antihypertensive effects of L-arginine, impaired L-arginine-induced increases in plasma nitrite and nitrosylated species concentrations, and blunted L-arginine-induced increases in aortic nitrate concentrations and vascular antioxidant effects. Our results show for the first time that the vascular and antihypertensive effects of L-arginine are prevented by antiseptic mouthwash. These findings show an important new mechanism that should be taken into consideration to explain how the use of antibacterial mouth rinse may affect arterial blood pressure and the risk of developing cardiovascular and other diseases.

Omeprazole induces vascular remodeling by mechanisms involving xanthine oxidoreductase and matrix metalloproteinase activation.

Proton pump inhibitors (PPI) are commonly used drugs that may increase the cardiovascular risk by mechanisms not entirely known. We examined whether the PPI omeprazole promotes vascular oxidative stress mediated by xanthine oxidoreductase (XOR) leading to activation of matrix metalloproteinases (MMPs) and vascular remodeling. We studied Wistar rats treated with omeprazole (or vehicle) combined with the XOR inhibitor allopurinol (or vehicle) for four weeks. Systolic blood pressure (SBP) measured by tail-cuff plethysmography was not affected by treatments. Omeprazole treatment increased the aortic cross-sectional area and media/lumen ratio by 25% (P < 0.05). Omeprazole treatment decreased gastric pH and induced vascular remodeling accompanied by impaired endothelium-dependent aortic responses (assessed with isolated aortic ring preparation) to acetylcholine (P < 0.05). Omeprazole increased vascular active MMP-2 expression and activity assessed by gel zymography and in situ zymography, respectively (P < 0.05). Moreover, omeprazole enhanced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay (both P < 0.05). All these biochemical changes caused by omeprazole were associated with increased vascular XOR activity (but not XOR expression assessed by Western blot) and treatment with allopurinol fully prevented them (all P < 0.05). Importantly, treatment with allopurinol prevented the vascular dysfunction and remodeling caused by omeprazole. Our results suggest that the long-term use of omeprazole induces vascular dysfunction and remodeling by promoting XOR-derived reactive oxygen species formation and MMP activation. These findings provide evidence of a new mechanism that may underlie the unfavorable cardiovascular outcomes observed with PPI therapy. Clinical studies are warranted to validate our findings.

Sodium nitrite improves hypertension-induced myocardial dysfunction by mechanisms involving cardiac S-nitrosylation.

Although nitrite improves vascular function and lowers blood pressure, its cardiac effects are not completely known. We investigated whether nitrite improves the cardiac function in normotensive and in hypertensive rats. Two-kidney, one-clip hypertension model (2K1C) was induced in Wistar rats. Blood pressure was evaluated by tail-cuff plethysmography over 6 weeks. By the end of week 2, hypertensive and normotensive rats received nitrite (daily dose of 1 or 15 mg/kg) by gavage for 4 weeks. Cardiac morphology and function were performed by transthoracic echocardiography. Intrinsic heart function was evaluated using the isolated heart model (Langendorff's preparation). Starling curves were generated under nitrite (1 µmol/L) and/or ascorbate (1 mmol/L) or vehicle. Cardiac tissue was collected and snap frozen for biochemical analysis. Nitrite treatment (15 mg/kg) lowered both systolic blood pressure and the increases in left ventricular (LV) mass found in 2K1C rats (P < .05). In addition, nitrite treatment restored the decreased cardiac output in 2K1C rats (P < .05) and improved the cardiac function. These findings were associated with increased nitrite, S-nitrosothiols, and protein S-nitrosylation (all P < .05) assessed in heart tissue. The cardiac effects of nitrite were further investigated in the isolated heart model, and nitrite infusion (1 µmol/L) enhanced cardiac contractility and relaxation. This infusion increased S-nitrosothiols concentrations and protein S-nitrosylation in the heart. Ascorbate completely blunted all nitrite-induced effects. These findings show that treatment with oral nitrite improves cardiac function by mechanisms involving increased S-nitrosothiols generation and S-nitrosylation of cardiac proteins. Pharmacological strategies promoting cardiac S-nitrosylation may be useful to improve myocardial function in heart diseases.

Eye and heart morphogenesis are dependent on melatonin signaling in chick embryos.

Calmodulin is vital for chick embryos morphogenesis in the incubation time 48-66 h when the rudimentary C-shaped heart attains an S-shaped pattern and the optic vesicles develop into optic cups. Melatonin is in the extraembryonic yolk sac of the avian egg; melatonin binds calmodulin. The aim of this study was to investigate the function of melatonin in the formation of the chick embryo optic cups and S-shaped heart, by pharmacological methods and immunoassays. Mel1a melatonin receptor immunofluorescence was distributed in the optic cups and rudimentary hearts. We separated embryonated chicken eggs at 48 h of incubation into basal, control and drug-treated groups, with treatment applied in the egg air sac. At 66 h of incubation, embryos were excised from the eggs and analyzed. Embryos from the basal, control (distilled water), melatonin and 6-chloromelatonin (melatonin receptor agonist) groups had regular optic cups and an S-shaped heart, while those from the calmidazolium (calmodulin inhibitor) group did not. Embryos from the luzindole (melatonin receptor antagonist) and prazosin (Mel1c melatonin receptor antagonist) groups did not have regular optic cups. Embryos from the 4-P-PDOT (Mel1b melatonin receptor antagonist) group did not have an S-shaped heart. Previous application of the melatonin, 6-chloromelatonin or forskolin (adenylate cyclase enhancer) prevented the abnormal appearance of chick embryos from the calmidazolium, luzindole, prazosin and 4-P-PDOT groups. However, 6-chloromelatonin and forskolin only partially prevented the development of defective eye cups in embryos from the calmidazolium group. The results suggested that melatonin modulates chick embryo morphogenesis via calmodulin and membrane receptors.