Anna Zemancikova’s research while affiliated with Slovak Academy of Sciences and other places

Background: Angiotensin converting enzyme 2 (ACE2) plays a crucial role in the infection cycle of SARS-CoV-2 responsible for formation of COVID-19 pandemic. In the cardiovascular system, the virus enters the cells by binding to the transmembrane form of ACE2 causing detrimental effects especially in individuals with developed hypertension or heart disease. Zofenopril, a H2S-releasing angiotensin-converting enzyme inhibitor (ACEI), has been shown to be effective in the treatment of patients with essential hypertension; however, in conditions of ACE2 inhibition its potential beneficial effect has not been investigated yet. Therefore, the aim of the study was to determine the effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats, an animal model of human essential hypertension and heart failure, under conditions of ACE2 inhibition induced by the administration of the specific inhibitor MLN-4760 (MLN). Results: Zofenopril reduced MLN-increased visceral fat to body weight ratio although no changes in systolic blood pressure were recorded. Zofenopril administration resulted in a favorable increase in left ventricle ejection fraction and improvement of diastolic function regardless of ACE2 inhibition, which was associated with increased H2S levels in plasma and heart tissue. Similarly, the acute hypotensive responses induced by acetylcholine, L-NAME (NOsynthase inhibitor) and captopril (ACEI) were comparable after zofenopril administration independently from ACE2 inhibition. Although simultaneous treatment with zofenopril and MLN led to increased thoracic aorta vasorelaxation, zofenopril increased the NO component equally regardless of MLN treatment, which was associated with increased NO-synthase activity in aorta and left ventricle. Moreover, unlike in control rats, the endogenous H2S participated in maintaining of aortic endothelial function in MLN-treated rats and the treatment with zofenopril had no impact on this effect. Conclusions: Zofenopril treatment reduced MLN-induced adiposity and improved cardiac function regardless of ACE2 inhibition. Although the concomitant MLN and zofenopril treatment increased thoracic aorta vasorelaxation capacity, zofenopril increased the participation of H2S and NO in the maintenance of endothelial function independently from ACE2 inhibition. Our results confirmed that the beneficial effects of zofenopril were not affected by ACE2 inhibition, moreover, we assume that ACE2 inhibition itself can lead to the activation of cardiovascular compensatory mechanisms associated with Mas receptor, nitrous and sulfide signaling.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects host cells through angiotensin-converting enzyme 2 (ACE2). Concurrently, the product of ACE2 action, angiotensin 1–7 (Ang 1–7), binds to Mas receptors within the cardiovascular system and provides protective effects. Therefore, it is crucial to reveal the role of ACE2 inhibition, especially within pre-existing cardiovascular pathologies. In our study, we imitated the action of SARS-CoV-2 in organisms using the low dose of the ACE2 inhibitor MLN-4760 with the aim of investigating to what degree ACE2 inhibition is detrimental to the cardiovascular system of spontaneously hypertensive rats (SHRs), which represent a model of human essential hypertension. Our study revealed the complex action of MLN-4760 in SHRs. On the one hand, we found that MLN-4760 had 1) (pro)obesogenic effects that negatively correlated with alternative renin-angiotensin system activity and Ang 1–7 in plasma, 2) negative effects on ACE1 inhibitor (captopril) action, 3) detrimental effects on the small arteries function and 4) anti-angiogenic effect in the model of chick chorioallantoic membrane. On the other hand, MLN-4760 induced compensatory mechanisms involving strengthened Mas receptor-, nitric oxide- and hydrogen sulfide-mediated signal transduction in the aorta, which was associated with unchanged blood pressure, suggesting beneficial action of MLN-4760 when administered at a low dose.

The aim of the current study was to evaluate the influence of a high-fat diet and its combination with high-fructose intake on young normotensive rats, with focus on the modulatory effect of perivascular adipose tissue (PVAT) on the reactivity of isolated arteries. Six-week-old Wistar–Kyoto rats were treated for 8 weeks with a control diet (10% fat), a high-fat diet (HFD; 45% fat), or a combination of the HFD with a 10% solution of fructose. Contractile and relaxant responses of isolated rat arteries, with preserved and removed PVAT for selected vasoactive stimuli, were recorded isometrically by a force displacement transducer. The results demonstrated that, in young rats, eight weeks of the HFD might lead to body fat accumulation and early excitation of the cardiovascular sympathetic nervous system, as shown by increased heart rate and enhanced arterial contractile responses induced by endogenous noradrenaline released from perivascular sympathetic nerves. The addition of high-fructose intake deteriorated this state by impairment of arterial relaxation and resulted in mild elevation of systolic blood pressure; however, the increase in arterial neurogenic contractions was not detected. The diet-induced alterations in isolated arteries were observed only in the presence of PVAT, indicating that this structure is important in initiation of early vascular changes during the development of metabolic syndrome.

Melatonin is released by the pineal gland and can modulate cardiovascular system function via the G protein-coupled melatonin receptors MT1 and MT2. Most vessels are surrounded by perivascular adipose tissue (PVAT), which affects their contractility. The aim of our study was to evaluate mRNA and protein expression of MT1 and MT2 in the mesenteric artery (MA) and associated PVAT of male rats by RT-PCR and Western blot. Receptor localization was further studied by immunofluorescence microscopy. Effects of melatonin on neurogenic contractions were explored in isolated superior MA ex vivo by measurement of isometric contractile tension. MT1, but not MT2, was present in MA, and MT1 was localized mainly in vascular smooth muscle. Moreover, we proved the presence of MT1, but not MT2 receptors, in MA-associated PVAT. In isolated superior MA with intact PVAT, neuro-adrenergic contractile responses were significantly smaller when compared to arteries with removed PVAT. Pre-treatment with melatonin of PVAT-stripped arterial rings enhanced neurogenic contractions, while the potentiating effect of melatonin was not detected in preparations with preserved PVAT. We hypothesize that melatonin can stimulate the release of PVAT-derived relaxing factor(s) via MT1, which can override the direct pro-contractile effect of melatonin on vascular smooth muscle. Our results suggest that melatonin is involved in the control of vascular tone in a complex way, which is vessel specific and can reflect a sum of action on different layers of the vessel wall and surrounding PVAT.

The aim of this study was to evaluate the mutual relationship among perivascular adipose tissue (PVAT) and endogenous and exogenous H 2 S in vasoactive responses of isolated arteries from adult normotensive (Wistar) rats and hypertriglyceridemic (HTG) rats, which are a nonobese model of metabolic syndrome. In HTG rats, mild hypertension was associated with glucose intolerance, dyslipidemia, increased amount of retroperitoneal fat, increased arterial contractility, and endothelial dysfunction associated with arterial wall injury, which was accompanied by decreased nitric oxide (NO)-synthase activity, increased expression of H 2 S producing enzyme, and an altered oxidative state. In HTG, endogenous H 2 S participated in the inhibition of endothelium-dependent vasorelaxation regardless of PVAT presence; on the other hand, aortas with preserved PVAT revealed a stronger anticontractile effect mediated at least partially by H 2 S. Although we observed a higher vasorelax-ation induced by exogenous H 2 S donor in HTG rats than in Wistar rats, intact PVAT subtilized this effect. We demonstrate that, in HTG rats, endogenous H 2 S could manifest a dual effect depending on the type of triggered signaling pathway. H 2 S within the arterial wall contributes to endothelial dys-function. On the other hand, PVAT of HTG is endowed with compensatory vasoactive mechanisms, which include stronger anti-contractile action of H 2 S. Nevertheless, the possible negative impact of PVAT during hypertriglyceridemia on the activity of exogenous H 2 S donors needs to be taken into consideration.

Background: Quercetin (QCT) was shown to exert beneficial cardiovascular effects in young healthy animals. The aim of the present study was to determine cardiovascular benefits of QCT in older, 6-month and 1-year-old Zucker diabetic fatty (ZDF) rats (model of type 2 diabetes). Methods: Lean (fa/+) and obese (fa/fa) ZDF rats of both ages were treated with QCT for 6 weeks (20 mg/kg/day). Isolated hearts were exposed to ischemia-reperfusion (I/R) injury (30 min/2 h). Endothelium-dependent vascular relaxation was measured in isolated aortas. Expression of selected proteins in heart tissue was detected by Western blotting. Results: QCT reduced systolic blood pressure in both lean and obese 6-month-old rats but had no effect in 1-year-old rats. Diabetes worsened vascular relaxation in both ages. QCT improved vascular relaxation in 6-month-old but worsened in 1-year-old obese rats and had no impact in lean controls of both ages. QCT did not exert cardioprotective effects against I/R injury and even worsened post-ischemic recovery in 1-year-old hearts. QCT up-regulated expression of eNOS in younger and PKCε expression in older rats but did not activate whole PI3K/Akt pathway. Conclusions: QCT might be beneficial for vascular function in diabetes type 2; however, increasing age and/or progression of diabetes may confound its vasculoprotective effects. QCT seems to be inefficient in preventing myocardial I/R injury in type 2 diabetes and/or higher age. Impaired activation of PI3K/Akt kinase pathway might be, at least in part, responsible for failing cardioprotection in these subjects.

Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase-dependent reactive oxygen species (ROS) overproduction and decreased nitric oxide (NO) bioavailability lead to vascular dysfunction and development of hypertension. The goal of our study was to analyze an effect of salt diet and NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure (BP), arterial reactivity, NO production, as well as ROS level in adult rats pretreated with low dose of L-NAME (2 mg/kg/day) for three weeks. Higher dose of L-NAME (40 mg/kg/day), or salt diet (8% NaCl), or combination of both were applied for the following four weeks. The administration of L-NAME in low dose had no effect on BP but enhanced the expression of eNOS. Both higher dose of L-NAME and salt diet elevated BP, decreased NOS activity, and impaired the endothelium-dependent arterial relaxation. However, salt diet did not increase ROS production and sympathoadrenergic arterial contractions in low dose L-NAME-pretreated rats. Combination of salt diet with higher dose of L-NAME did not evoke additive decrease of NOS activity, but it caused elevation of conjugated dienes (CD) concentration and NADPH oxidase 2 (Nox-2) protein expression. In conclusion, these findings indicate that chronic low dose of L-NAME treatment has a potential to trigger adapting mechanisms to attenuate some cardiovascular disorders.

Aliskiren, a renin inhibitor, has been shown to have cardioprotective and blood pressure (BP) lowering effects. We aimed to determine the effects of nanoparticle-loaded aliskiren on BP, nitric oxide synthase activity (NOS) and structural alterations of the heart and aorta developed due to spontaneous hypertension in rats. Twelve week-old male spontaneously hypertensive rats (SHR) were divided into the untreated group, group treated with powdered or nanoparticle-loaded aliskiren (25 mg/kg/day) and group treated with nanoparticles only for 3 weeks by gavage. BP was measured by tail-cuff plethysmography. NOS activity, eNOS and nNOS protein expressions, and collagen content were determined in both the heart and aorta. Vasoactivity of the mesenteric artery and wall thickness, inner diameter, and cross-sectional area (CSA) of the aorta were analyzed. After 3 weeks, BP was lower in both powdered and nanoparticle-loaded aliskiren groups with a more pronounced effect in the latter case. Only nanoparticle-loaded aliskiren increased the expression of nNOS along with increased NOS activity in the heart (by 30%). Moreover, nanoparticle-loaded aliskiren decreased vasoconstriction of the mesenteric artery and collagen content (by 11%), and CSA (by 25%) in the aorta compared to the powdered aliskiren group. In conclusion, nanoparticle-loaded aliskiren represents a promising drug with antihypertensive and cardioprotective effects.

Perivascular adipose tissue (PVAT) and its vasomodulatory effects play an important role in the physiology and pathophysiology of blood vessels. Alterations in PVAT associated with reduction in its anticontractile influence are proven to contribute to vascular dysfunction in hypertension. The aim of this study was to examine whether the changes in PVAT properties could participate in progression of vascular abnormalities in developing spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) rats and SHR, both in 5th and in 12th week of age, were used. Systolic blood pressure was similar between WKY rats and SHR in 5th week of age; however, in 12th week, it was significantly increased in SHR comparing to WKY rats. The amount of retroperitoneal fat was higher in WKY rats in both age groups, whereas body weight was higher in WKY rats only in 12th week, when compared to age-matched SHR. From isolated superior mesenteric arteries, two ring preparations were prepared for isometric tension recording, one with PVAT intact and other with PVAT removed. In WKY rats as well as in SHR, arterial contractile responses to noradrenaline, applied cumulatively on rings, were significantly inhibited in the presence of intact PVAT. In both age groups, anticontractile effect of PVAT was higher in WKY rats than in SHR. Neurogenic contractions, induced by electrical stimulation of perivascular sympathoadrenergic nerves, were significantly attenuated in the presence of PVAT in WKY mesenteric arteries from both age groups; however, in arteries from SHR, intact PVAT had no influence on this type of contractile responses. The results suggest that in SHR impairment of anticontractile effect of PVAT precedes hypertension and might contribute to its development.