Regulation Of Vascular Tone Research Articles

Analysis of the condition of the kallikrein-kinin system in the comorbid course of chronic pancreatitis and type 2 diabetes

Background. According to evidence, within approximately 8 years, 50% of patients with chronic pancreatitis (CP) develop endocrine and exocrine insufficiency, which manifests itself as steatorrhea, weight loss, diabetes mellitus (DM), and nutritional disorders. Diabetes mellitus aggravates these phenomena, which deepens the disorder of metabolic processes in CP, including protein metabolism. Protein structures include components of the kallikrein-kinin system (KKS), which plays a role in the regulation of vascular tone, diuresis, inflammation, coagulation, and pain reception. Due to repeated exacerbations of pancreatitis, there is fibrosis of the pancreas, which increases the risk of insulin resistance and the formation of type 2 diabetes. Thus, disorders of the kallikrein-kinin system and the progression of CP are interrelated, which makes it important to study the state of KKS in CP, especially in combination with type 2 diabetes mellitus. The purpose is to investigate the state of the kallikrein-kinin system in chronic pancreatitis in outpatients, depending on the presence of concomitant type 2 diabetes. Material and methods. 137 outpatients with CP with concomitant diabetes mellitus and without diabetes were studied: the main group— 112 patients with CP without exacerbation in combination with diabetes mellitus in a state of complete or subcompensation, and the comparison group— 25 patients with isolated CP. Evaluation of general and specific proteolysis (α1-proteinase inhibitor, α2-macroglobulin, kallikrein, kininase II activity, prekallikrein, plasma proteolytic activity) was performed by the method analysis using standard kits from BIOSERV ELISA. Results. There was a 20.8% increase in the level of the total proteolytic activity of plasma in the group of patients with comorbidity of CP and diabetes mellitus compared with the group with isolated CP, 51.9% increase in specific proteolysis (or kininogenesis)— the level of kallikrein (proteolysis enzyme), p<0.05. At the same time, a decrease in prekallikrein (inactive precursor of kallikrein) was found in the group of patients with comorbidity by 19.4% compared to that in isolated CP (p < 0.05). The presence of dissociation of protective parameters of kallikrein-kinin system is proved; increase by 9.4% of α1-proteinase inhibitor content at comorbidity of CP and DM2 in relation to that in isolated CP; decrease in the content of α2-macroglobulin in CP relative to the control group (p<0.05), which showed a decrease in the body’s compensatory capacity in CP, at the same time found an increase in α2-macroglobulin in the comorbidity of CP with DM2 by 49.5% relative to isolated CP; reduction of kininase-II activity in CP and diabetes mellitus by 14.5% relative to that in CP. Conclusions. Activation of KKS with multidirectional changes in KKS parameters and general and specific proteolysis in CP was noted. In CP and in the comorbid course with diabetes mellitus, further activation of proteolysis took place with the simultaneous inclusion of protective mechanisms for resolving inflammation and detoxification. Excessive production of kinins in concomitant DM2 has been proven, which weakened the body’s protective response.

High-efficient serum-free differentiation of endothelial cells from human iPS cells

IntroductionEndothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs’ physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications.MethodHuman induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h.ResultThis combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4).ConclusionThe differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45− cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45- cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cellsfrom arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

Hydrogen Sulfide-Induced Vasodilation: The Involvement of Vascular Potassium Channels.

Hydrogen sulfide (H2S) has been highlighted as an important gasotransmitter in mammals. A growing number of studies have indicated that H2S plays a key role in the pathophysiology of vascular diseases and physiological vascular homeostasis. Alteration in H2S biogenesis has been reported in a variety of vascular diseases and H2S supplementation exerts effects of vasodilation. Accumulating evidence has shown vascular potassium channels activation is involved in H2S-induced vasodilation. This review aimed to summarize and discuss the role of H2S in the regulation of vascular tone, especially by interaction with different vascular potassium channels and the underlying mechanisms.

MET235THR POLYMORPHISM IN THE AGT GENE AS THE DEVELOPMENT FACTOR OF SEVERE PREECLAMPSIA

Objective: Every year, as a prediction risk of developing preeclampsia, experts are attracted by polymorphisms of some genes involved in the regulation of vascular tone and endothelial function. The purpose - to establish the features of Met235Thr polymorphism in the AGT gene in the development of severe preeclampsia. Design and method: We examined 139 pregnant women in the third trimester of gestation which were divided into 2 groups: The main group - 56 pregnant women with severe preeclampsia. The control group is 83 conditionally healthy women. The age of the pregnant women was 19–37 years. The material for the study was DNA samples from pregnant women. Isolation of DNA from blood and PCR analysis were performed with kits of reagents and test systems from Ampli Prime Ribot-prep. Results: The shares of the Thr allele, being at very close levels in values, were insignificantly less in the control group, where they were 36.9%, versus 38.4% among patients with severe preeclampsia. The detection rate of the homozygous wild Met / Met genotype of the Met235Thr polymorphism of the AGT gene was also insignificantly higher in the control group, being at the level of 43.0% (x2 = 0.15; p = 0.70; RR = 0.92; 95% CI: 0.33–2.54; OR = 0.87; 95% CI: 0.42–1.79), which was insignificantly higher than among patients with severe preeclampsia 39.5%. The heterozygous Met / Thr genotype, also relatively evenly distributed between the groups, was insignificantly less frequently detected in the control group, amounting to 40.2% (x2 = 0.20; p = 0.67; RR = 1.1; 95% CI: 0.41–2.97; OR = 1.18; 95 % CI: 0.58–2.41), versus 44.2% among patients with severe preeclampsia. The homozygous mutant Thr / Thr genotype, also being at a comparable level, prevailed statistically insignificantly in the control group (16.8%), relative to the group of patients (16.3%) (x2 = 0.01; p = 0.94; RR = 0.97; 95% CI: 0.25–3.72; OR = 0.96; 95% CI: 0.38–2.44) Conclusions: We can conclude that the Met235Thr polymorphism in the AGT gene in women with severe preeclampsia has clinical prognostic significance.

RED BLOOD CELL COLOUR VARIATION AS A MARKER OF CALCIUM DISTRIBUTION: WHAT ROLE DOES IT HAVE IN HEART FAILURE PATIENTS WITH HYPERTENSION AND LONG COVID?

Objective: Red blood cell (RBC) role is both passive action, oxygen delivery to the tissues as well as carbon dioxide to the lungs and active action involvement in the regulation of vascular tone. The aim was to investigate pathophysiological and ultrastructural changes of RBC in heart failure (HF) patients with hypertension (HT) and long Covid. Design and method: In total 12 patients with HF of Coronary Artery Disease origin, HT, and long Covid were examined. Mean age of patients was 62 ± 5.8 years. The control group consisted of 10 apparently healthy people. The functional state and ultrastructure of RBC were studied using electron microscopy. Results: During ultrastructure examination, structural pathologies of RBC in HF patients with HT and long Covid were revealed. RBC anisocytosis and poikilocytosis as structural damage variations in size and shape were found respectively. Reticulocytes were found much more often in HF patients with HT and long Covid than in the control group. In healthy control group, RBC had a typical discoid shape. In the presence of long Covid, both calcification as a marker of RBC apoptosis and destruction was also detected (Fig.1). Neutrophil extracellular traps (NETs) were found in RBC surrounding (Fig.1). Conclusions: Altered RBC function has important implications for HF patients with HT and long Covid. RBC has been shown to induce endothelial cell dysfunction and to increase cardiac injury as well as increased inflammatory processes in long Covid. The presence of HF, HT and long Covid leads to RBC calcification and activation of blood cell apoptosis. Prognostic role of RBC calcium distribution in combination with other important prognostic measures, such as biomarkers like Thrombospondin - 1, NT-proBNP and ST2 is subject of interest and requires further research.

HYPERTENSIVE PATIENTS WITH ANGINA AND NON-OBSTRUCTIVE CORONARY ARTERY DISEASE ARE CHARACTERIZED BY INCREASED SYMPATHETIC NERVOUS SYSTEM ACTIVATION

Objective: There are data supporting that sympathetic nervous system (SNS) is implicated in the regulation of endothelium function and vascular tone in different cardiovascular disease states. Patients with hypertension and angina frequently present non-obstructive coronary artery disease (CAD) in angiography. The aim of our study was to assess the level of SNS activation in hypertensive patients with angina and non-obstructive CAD, in hypertensives without angina and normotensive controls. Design and method: Our population consisted of 10 treated hypertensive patients with angina who had non-obstructive CAD in coronary angiography [7 females, mean age: 60.5 ± 9.6 years, office blood pressure (BP): 157/93 ± 13/11 mmHg], 16 treated hypertensive patients without angina (9 females, mean age: 59.5 ± 7.5 years, office BP:142/88 mmHg) and 11 normotensive individuals without angina (8 females, mean age: 51.8 ± 11 years, BP:125/87 mmHg). In all participants, muscle sympathetic nerve activity (MSNA) was assessed. Results: Hypertensives with angina and non-obstructive CAD (n = 10) compared to hypertensive patients without angina (n = 16) and normotensives without angina (n = 11) exhibited higher levels of MSNA (57.6 ± 6.1 vs 43 ± 8.4 vs 45 ± 9.5 bursts per minute, respectively; p < 0.01 for all) and 24-h ambulatory BP (144/83 ± 20/14 vs 141/83 ± 7/10 vs to normotensives 125/81 ± 7/6 mmHg, respectively; p < 0.001 for all). There were no differences in renal function between groups and no gender related effects on MSNA values (p = NS). Conclusions: Hypertensive patients with angina and non-obstructive CAD are characterized by higher SNS drive compared to hypertensives without angina and normotensive controls, as reflected by MSNA levels. These preliminary results indicate that therapies targeting SNS tone could be of potential clinical benefit in this high-risk hypertension phenotype.

Involvement of β-adrenoceptors in the cardiovascular responses induced by selective adenosine A2A and A2B receptor agonists.

A2A and A2B adenosine receptors produce regionally selective regulation of vascular tone and elicit differing effects on mean arterial pressure (MAP), whilst inducing tachycardia. The tachycardia induced by the stimulation of A2A or A2B receptors has been suggested to be mediated by a reflex increase in sympathetic activity. Here, we have investigated the role of β1‐ and β2‐adrenoceptors in mediating the different cardiovascular responses to selective A2A and A2B receptor stimulation. Hemodynamic variables were measured in conscious male Sprague‐Dawley rats (350–450 g) via pulsed Doppler flowmetry. The effect of intravenous infusion (3 min per dose) of the A2A‐selective agonist CGS 21680 (0.1, 0.3, 1.0 µg.kg−1.min−1) or the A2B‐selective agonist BAY 60–6583 (4.0, 13.3, 40.0 µg.kg−1.min−1) in the absence or following pre‐treatment with the non‐selective β‐antagonist propranolol (1.0 mg.kg−1), the selective β1‐antagonist CGP 20712A (200 µg.kg−1), or the selective β2‐antagonist ICI 118,551 (2.0 mg.kg−1) was investigated (maintenance doses also administered). CGP 20712A and propranolol significantly reduced the tachycardic response to CGS 21680, with no change in the effect on MAP. ICI 118,551 increased BAY 60–6583‐mediated renal and mesenteric flows, but did not affect the heart rate response. CGP 20712A attenuated the BAY 60–6583‐induced tachycardia. These data imply a direct stimulation of the sympathetic activity via cardiac β1‐adrenoceptors as a mechanism for the A2A‐ and A2B‐induced tachycardia. However, the regionally selective effects of A2B agonists on vascular conductance were independent of sympathetic activity and may be exploitable for the treatment of acute kidney injury and mesenteric ischemia.

The Effects of Acidosis on eNOS in the Systemic Vasculature: A Focus on Early Postnatal Ontogenesis.

The activity of many vasomotor signaling pathways strongly depends on extracellular/intracellular pH. Nitric oxide (NO) is one of the most important vasodilators produced by the endothelium. In this review, we present evidence that in most vascular beds of mature mammalian organisms metabolic or respiratory acidosis increases functional endothelial NO-synthase (eNOS) activity, despite the observation that direct effects of low pH on eNOS enzymatic activity are inhibitory. This can be explained by the fact that acidosis increases the activity of signaling pathways that positively regulate eNOS activity. The role of NO in the regulation of vascular tone is greater in early postnatal ontogenesis compared to adulthood. Importantly, in early postnatal ontogenesis acidosis also augments functional eNOS activity and its contribution to the regulation of arterial contractility. Therefore, the effect of acidosis on total peripheral resistance in neonates may be stronger than in adults and can be one of the reasons for an undesirable decrease in blood pressure during neonatal asphyxia. The latter, however, should be proven in future studies.

TWIK-Related Acid-Sensitive Potassium Channels (TASK-1) Emerge as Contributors to Tone Regulation in Renal Arteries at Alkaline pH.

Aim: TASK-1 channels are established regulators of pulmonary artery tone but their contribution to the regulation of vascular tone in systemic arteries is poorly understood. We tested the hypothesis that TASK-1 channel functional impact differs among systemic vascular beds, that this is associated with differences in their expression and may increase with alkalization of the extracellular environment. Therefore, we evaluated the expression level of TASK-1 channels and their vasomotor role in mesenteric and renal arteries. Methods: Pulmonary, mesenteric and renal arteries from male Wistar rats were used for TASK-1 channel mRNA (qPCR) and protein content (Western blotting) measurements. The functional role of TASK-1 channels was studied by wire myography using the TASK-1 channel blocker AVE1231. In some experiments, the endothelium was removed with a rat whisker. Results: Expression levels of both mRNA and protein of the TASK-1 channel pore-forming subunit were highest in pulmonary arteries, lowest in mesenteric arteries and had an intermediate value in renal arteries. Blockade of TASK-1 channels by 1 µM AVE1231 increased U46619-induced contractile responses of pulmonary arteries but did not affect basal tone and contractile responses to methoxamine of mesenteric and renal arteries at physiological extracellular pH (pHo = 7.41). At alkaline extracellular pH = 7.75 (increase of NaHCO3 to 52 mM) AVE1231 evoked the development of basal tone and increased contractile responses to low concentrations of methoxamine in renal but not mesenteric arteries. This effect was independent of the endothelium. Conclusion: In the rat systemic circulation, TASK-1 channels are abundant in renal arteries and have an anticontractile function under conditions of extracellular alkalosis.

Ex vivo dual perfusion of an isolated human placenta cotyledon: Towards protocol standardization and improved inter-centre comparability

Since the full development of the ex vivo dual perfusion model of the human placenta cotyledon, the technique has provided essential insight into how nutrients, lipids, gases, immunoglobulins, endocrine agents, pharmaceuticals, chemicals, nanoparticles, micro-organisms and parasites might traverse the maternofetal barrier. Additionally, the model has been instrumental in gaining a better understanding of the regulation of vascular tone, endocrinology and metabolism within this organ. The human placenta is unique amongst species in its anatomy and transfer modalities. This orthologous diversity therefore requires an appropriate consideration of placental transfer rates of compounds, particles and micro-organisms specific to humans.Different research centres have adapted this model with a wide variation in perfusion parameters, including in the establishment of perfusion, perfusate composition, gassing regime, cannulation method, flow rates, perfused tissue mass, and also in the application of quality control measures. The requirement to harmonise and standardise perfusion practice between centres is largely driven by the need to obtain consistency in our understanding of placental function, but also in the qualification of the model for acceptance by regulatory agencies in drug and toxicology testing.A pilot study is proposed, aiming to describe how existing inter-centre variation in perfusion methodology affects placental metabolism, protein synthesis, oxygen consumption, the materno-fetal transfer of key molecular markers, and placental structure.

Features of vascular motor endothelial function in hypertensive patients with chronic heart failure

Abstract Funding Acknowledgements Type of funding sources: None. The main part of the pathogenesis of arterial hypertension (AH) is a remodeling of the vascular system and the state of the vascular endothelium, which functions as a trigger mechanism for the implementation vasoregulating mechanisms. Vascular endothelium is involved in the provision of local processes of hemostasis, proliferation, migration of blood cells in the vessel wall, which is also associated with the regulation of vascular tone and the development of atherosclerosis. Purpose To study the vascular motor endothelial function in hypertensive patients with chronic heart failure (CHF). Methods Vascular mechanisms of pathogenesis have been studied in 172 patients aged (54,2±6,2) years, men 84 and women 88, with uncomplicated hypertension II-III degree. Echographic system Acuson Sequoia 512 was used for ultrasound imaging of the heart. The diameter of the brachial artery (D, mm), flow velocity (V, m/s), the ratio D/V, Δd (%) - an increase in the diameter of the artery decompression, ΔdN (%) - an increase in the diameter of the artery sample with nitroglycerin and the ratio Δd/ΔdN were measured. Results Due to the reduction in brachial artery diameter (with AH stage II 55, l% and AH stage III 48.7%, p <0.01) decreased blood flow velocity in the whole group is 36.4%, which is as much depended on the degree of hypertension (p <0.01). Conducting tests for decompression and nitroglycerin showed that reduced growth in brachial artery diameter in the total group on the decompression was compared with the normal 51.1%: in AH II degree - by 48.8% and III level by 52.2% (p<0.01). The test with nitroglycerin: the larger group of hypertensive patients reduced vasodilation was 21.1%, II degree - 13.8%, and III degree - 30.7% (p<0.01). Conducting tests for decompression showed that normal type vasodilatory response occurred only in 27.4% of patients, with II degree in 33.8% and AH III in 22% of patients. Reduced response to decompression was 60% of patients with AH II - in 43.2%, and III degree - at 56.8% and constrictor - in 12.6% of them in AH II degree at - 6.2% and 17 % at AH III. The test with nitroglycerin: normal vascular reaction occurred in 33.5% of patients, decreased in 59.9% and constrictor in 9.6% of patients. Reduced vasoconstrictor response to nitroglycerin was more common in patients with AH II degree. Cunclusion These results define the nature of the preferred choice of antihypertensive drugs such as normalizing pressure and have a positive effect on the endothelium, which is also associated with a reduction of myocardial hypertrophy and vascular wall.

Sphingolipids Imbalanced in Brain Microvessel of Predisposed Reduced Uterine Perfusion Mice

Preeclampsia (PE), a hypertension disorder, is one of the most serious complications that can occur during pregnancy. It affects at least 2‐8% of pregnancies, causing up to 76,000 maternal deaths and 500,000 infant fatalities. PE negatively impacts the vasculature of multiple body systems and has significant neurovascular implications including reversible cerebral vasoconstriction syndrome and ischemic stroke. Although PE resolves after delivery, long term cerebrovascular effects remain. Women who have experienced PE are estimated to have an 80% increased risk of future stroke. Although the consequences of PE are extensive and prevalent, its full etiology is not yet known. It is suspected that cerebrovascular dysfunction plays a significant role in these critical ramifications of PE. Interestingly, the ceramide and sphingosine‐1‐phosphate (S1P) pathway is known to regulate many of the systems that are altered in PE. Specifically, ceramide and S1P are involved in regulation of blood pressure, vascular tone, and vessel permeability in addition to other cell and tissue processes. Because of the significant involvement of ceramide, S1P, and their signaling constituents in endothelial cell and vascular integrity, variations within this pathway may explain the link between PE and cerebrovascular disease. We hypothesize that the ceramide and S1P signaling pathway is linked to dysfunctional brain vasculature in mice that had a PE‐induced pregnancy. This study aims to prove the ceramide and S1P signaling pathway is altered in the cerebral microvasculature of PE murine models. C57BL female mice underwent reduced uterine perfusion pressure (RUPP) surgery at 10 weeks old to simulate PE or sham surgery. Brain microvessels (BMVs) were isolated from these mice dams nine months following delivery. Two step reverse transcription qPCR was performed to assess the mRNA expression of the various ceramide and S1P signaling components. It was found that RUPP mice BMVs express significantly less sphingosine kinase 1 (SphK1) in comparison to sham BMVs. RUPP mice BMVs also demonstrated significantly increased sphingosine‐1‐phosphate lyase 1 (Sgpl1) and phospholipid phosphatase 1 (Plpp1). RUPP mice BMVs exhibited decreased expression of ceramide kinase and sphingosine kinase 2 (Sphk2) as well. SphK1 and Sphk2 are key enzymes in producing S1P, an active signaling lipid responsible for maintaining the blood brain barrier, cerebrovascular integrity, and angiogenesis. Sgpl1 is responsible for the degradation of S1P into its inactive form, therefore contributing to endothelial apoptosis and diminished vessel integrity. Plpp1 catalyzes the production of pro‐apoptotic ceramide and was discovered to promote the metabolism of S1P. Ceramide kinase is a crucial enzyme in producing anti‐apoptotic ceramide‐1‐phosphate. These discrepancies in ceramide and S1P signaling pathway expression may contribute to impaired cerebrovascular health, providing insight into the increased incidence of stroke with PE.

Smooth Muscle‐Specific Deletion of Neuropilin‐1 Increases Vascular Contractility and Blood Pressure

IntroductionNeuropilin‐1 (NRP1) is a transmembrane glycoprotein that acts as a co‐receptor for the Class 3 Semaphorin (SEMA) ligand SEMA3A and mediates the inhibition of RhoA signaling. NRP1 is expressed in vascular smooth muscle cells (VSMCs), however, its role in regulating VSMC contractility and blood pressure in vivo is unknown.HypothesisWe hypothesize that SEMA3A‐NRP1 signaling is essential in mitigating RhoA activity in VSMCs and that loss of NRP1 increases vascular tone and blood pressure in vivo.MethodsTo study the role of NRP1 in SMCs, mice with inducible, smooth muscle cell‐specific deletion of NRP1 (SM22a‐CreERT2; Nrp1flox/flox) were generated. Following recombination using 4‐hydroxy tamoxifen (SM‐NRP1 KO) in male and female adult mice (8‐12 weeks), systolic blood pressure (SBP) was measured using a tail cuff and compared to age‐ and sex‐matched mice that did not receive tamoxifen (control). Vascular reactivity to various contractile agonists was measured using ex vivo tension myography.ResultsSBP was significantly increased in SM‐NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Aortas of SM‐NRP1 KO mice displayed significantly enhanced contractile response to phenylephrine, KCl, and the thromboxane agonist U44619, and reduced relaxation to the endothelium‐independent vasodilator sodium nitroprusside. In vitro, treatment of murine primary aortic VSMC with SEMA3A decreased angiotensin II‐induced Rho‐GTP activation. Gene expression of Sema3a in the aorta was downregulated in two models of hypertension, angiotensin II‐dependent and genetic model of hypertension, as identified by qPCR and transcriptomic analysis respectively, further supporting the role of SEMA3A‐NRP1 signaling in basal blood pressure regulation. Additionally, control and SM‐NRP1 KO mice (starting at 2 weeks post‐recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM‐NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, gene expression of atrial natriuretic peptide was increased (p=0.06) in hearts of angiotensin II‐infused SM‐NRP1 KO mice. Preliminary analyses indicated an increase in aortic medial thickness at baseline and an increase in aortic adventitial collagen following angiotensin II infusion in SM‐NRP1 KO mice compared to controls.ConclusionTogether, our data point to the role of NRP1 as a novel regulator of basal vascular tone and blood pressure, and the loss of NRP1 leads to the onset of hypertension and exacerbates pathological vascular remodeling and cardiac dysfunction.

Calcium‐activated Chloride Channel TMEM16A/ANO1 Does Not Mediate the Regulation of Sweating and Cutaneous Vasodilation in Humans In Vivo

The regulation of human body temperature during a heat stress is dependent on the activation of the heat loss responses of cutaneous vasodilation and eccrine sweating. While significant advancements have been made in our understanding of the mechanisms governing their regulation, new potential modifiers such as transmembrane member 16A (TMEM16A), a Ca2+‐activated Cl‐ channel also known as anoctamin 1 (ANO1), continue to be identified. In vitro and animal studies suggest a role for TMEM16A in the regulation of sweat secretion and vascular tone. For example, Ca2+ induced Cl‐current was abolished in TMEM16A deficit, but not BEST2 deficit, NCL‐SG3 human eccrine sweat gland cell lines. Further, animal models showed the activation of TMEM16A on vascular smooth muscle cells blunts vasodilation. However, direct evidence supporting a role of TMEM16 in mediating cutaneous vasodilation as well as eccrine sweating in humans in vivo remains to be established. Therefore, we evaluated the hypothesis that TMEM16A mediates the regulation of sweating and cutaneous vasodilation during a whole‐body heat stress in young adults.Twelve young (24 ± 2 years) adults (6 females) underwent a whole‐body heat stress induced by a water‐perfused suit to raise core temperature 1.1±0.1ºC above baseline resting levels. Sweat rate (ventilated capsule) and cutaneous vascular conductance (assessed as skin blood flow/mean arterial pressure and normalised to maximal conductance achieved via simultaneous local skin heating to 44°C and administration of sodium nitroprusside) were assessed at four skin sites on the forearm treated with 1) lactated Ringers (control), 2) 5 % dimethyl sulfoxide serving as a vehicle control, 3) 1 mM T16Ainh‐A01 (a TMEM16A blocker) or 4) 2 mM benzbromarone (another TMEM16A blocker). All drugs were administered continuously via intradermal microdialysis.Sweat rate increased as heating continued at all skin sites (P < 0.001 for a main effect of heating stage). Neither the main effect of treatment site nor the interaction between treatment site and heating stage was significant (both P ≥ 0.318). Cutaneous vascular conductance increased in parallel to elevations in core temperature (P < 0.001 for a main effect of heating stage). Although no main effect of treatment site was observed (P = 0.109), a significant interaction between heating stage and treatment site was measured (P < 0.001). Post‐hoc analysis revealed that cutaneous vascular conductance at the DMSO‐treated site was higher than the control site at baseline (P = 0.047). By contrast, cutaneous vascular conductance at moderate elevations in core temperature (i.e., ≥0.4‐0.8 °C) was greater at the control site relative to the DMSO‐treated site (all P ≤ 0.013). Importantly, no differences were measured in cutaneous vascular conductance between the DMSO‐treated site and TMEM16A blocker‐treated sites (all P ≥ 0.073).Taken together, our findings demonstrate for the first time that TMEM16A does not contribute to the regulation of sweating and cutaneous vasodilation during whole‐body heating in young adults in vivo.

Does Dietary Methionine Alter Perivascular Adipose Tissue Function?

Perivascular adipose tissue (PVAT) is an adipose depot surrounding blood vessels and is a paracrine regulator of vascular tone. Obesity is associated with the loss of PVAT‐mediated anti‐contractile properties, thus contributing to obesity‐associated hypertension. Hydrogen sulfide (H2S) is a gaseous signaling molecule that is produced in the vasculature and is important for regulating vascular tone. In PVAT, cystathionine gamma lyase (Cse) is the primary source of H2S which acts as an important adipose‐derived relaxing factor. Dietary methionine restriction recapitulates many of the metabolic effects of caloric restriction and leads to preferential expression of activating transcription factor 4 (ATF4) which facilitates Cse transcription. We hypothesized that dietary methionine restriction will prevent high fat diet‐induced loss of PVAT anti‐contractile properties, through activation of the integrated stress response (ISR) and increased ATF4‐mediated transcription of Cse. Male Wistar rats were fed either a control (C, 10% calories from fat) or a high fat diet (HFD) (H, 60% calories from fat) that was either methionine replete (R, 0.86% methionine) or methionine depleted (D, 0.12% methionine) for 12 weeks such that there were four groups: CR, CD, HR, and HD. Dietary methionine restriction prevented body weight gain, while rats fed a methionine replete diet gained weight over the 12 week feeding period. There was no difference in weight gain between control or HFD fed rats. Arterial blood pressure, measured in conscious rats using tail cuff plethysmography, was not different between the four groups over the 12 week feeding period. After 12 weeks on diets, arterial contractility was assessed using wire myography in 2nd order mesenteric arteries (MAs) with and without attached PVAT. In our hands, the presence of PVAT was associated with reduced contraction to norepinephrine (NE, 10 µM) and greater relaxation to acetylcholine (ACh,10 µM) in MAs from all groups, and HFD was not associated with a loss of PVAT anti‐contractile properties. In the presence of PVAT, dietary methionine restriction increased relaxation to ACh in NE‐precontracted MAs, while HFD had no effect on ACh‐induced relaxation. In conclusion, in male Wistar rats, while HFD had no effect on weight gain, blood pressure, or PVAT modulation of MA contractility, dietary methionine restriction for 12 weeks had profound effects on body weight gain, no effect on arterial blood pressure, and a small effect on MA ACh‐induced relaxation in the presence of PVAT. Future studies include examining gene and protein expression of Cse and other ATF4 responsive genes using real time qPCR, Western blot analysis, and immunohistochemical staining of arterial cross‐sections. These studies will provide further insights into dietary interventions to minimize the cardiovascular co‐morbidities associated with obesity.

Perivascular adipose tissue modulates the effects of flavonoids on rat aorta rings: Role of superoxide anion and β3 receptors

Several studies demonstrate the beneficial effects of dietary flavonoids on the cardiovascular system. Since perivascular adipose tissue (PVAT) plays an active role in the regulation of vascular tone in both health and diseases, the present study aimed to assess the functional interaction between PVAT and flavonoids in vitro on rat aorta rings. Several flavonoids proved to display both antispasmodic and spasmolytic activities towards noradrenaline-induced contraction of rings deprived of PVAT (-PVAT). However, on PVAT-intact (+PVAT) rings, both actions of some flavonoids were lost and/or much decreased. In rings-PVAT, the superoxide donor pyrogallol mimicked the effect of PVAT, while in rings+PVAT the antioxidant mito-tempol restored both activities of the two most representative flavonoids, namely apigenin and chrysin. The Rho-kinase inhibitor fasudil, or apigenin and chrysin concentration-dependently relaxed the vessel active tone induced by the Rho-kinase activator NaF; the presence of PVAT counteracted apigenin spasmolytic activity, though only in the absence of mito-tempol. Similar results were obtained in rings pre-contracted by phenylephrine. Finally, when β3 receptors were blocked by SR59230A, vasorelaxation caused by both flavonoids was unaffected by PVAT. These data are consistent with the hypothesis that both noradrenaline and apigenin activated adipocyte β3 receptors with the ensuing release of mitochondrial superoxide anion, which once diffused toward myocytes, counteracted flavonoid vasorelaxant activity. This phenomenon might limit the beneficial health effects of dietary flavonoids in patients affected by either obesity and/or other pathological conditions characterized by sympathetic nerve overactivity.

The combined effect of low-level laser therapy and dietary supplements at the microcirculation system in athletes

The problem of athletes’ body resistance to effects of extreme loadings which lead to decreased adaptive capabilities of the body and over training is particularly relevant in modern sports. In order to improve the physical performance of athletes, there is a constant search for new tools and methods. Aim: to study the effect of dietary supplements at the microcirculation system, both in isolated use and in combination with low-level laser therapy. Material and methods. The study included 28 highly qualififi ed male all-around athletes of the CMC and MS levels. To study the parameters of skin microcirculation, laser Doppler flfl owmetry (LDF) was used, device “LAKK-02” (NPP “LAZMA”). The following parameter was calculated: microcirculation index (MI) proportional to the number of red blood cells in the diagnosed volume and their average velocity. The received signal give the information about endothelial, neurogenic and myogenic active mechanisms of vascular tone regulation and passive mechanisms – respiratory and cardiac flfl uctuations in blood flfl ow; flfl ax index, specififi c oxygen consumption index by tissues, erythrocyte concentration index in the probed blood volume and the ratio of coenzymes of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flfl avinadenine dinucleotide (FAD) (NADH/FAD). Results. After isolated use of dietary supplements, the level of perfusion increases signififi cantly by 48 % (p < 0.05), and the value of the flfl ax increases by 78 % (p < 0.05). More red blood cells by 22 % (p < 0.05) are released from blood depots into the microcirculatory bed. The tone of arterioles decreases by 48 % (p < 0.05), of precapillary sphincters – by 59 % (p < 0.05). In mitochondria, ratio of NADH/FAD coenzymes increases by 55 % (p < 0.05). Conclusion. Laser radiation in combination with dietary supplements optimizes recovery processes in the body of athletes who are subjected to heavy physical exertion. Under the combined effect, the intensity of microcirculation increases, oxygen output of erythrocytes by hemoglobin improves as well as its active diffusion from blood into tissues and participation in the ATP synthesis at the level of cell mitochondrial. It is worth noting that there is similarity in the isolated use of dietary supplements and in combination with low-energy laser radiation which enhances the mechanism of action of dietary supplements.

The Vasoactive Effect of Perivascular Adipose Tissue and Hydrogen Sulfide in Thoracic Aortas of Normotensive and Spontaneously Hypertensive Rats.

The objective of this study was to investigate the vasoregulatory role of perivascular adipose tissue (PVAT) and its mutual interaction with endogenous and exogenous H2S in the thoracic aorta (TA) of adult normotensive Wistar rats and spontaneously hypertensive rats (SHRs). In SHRs, hypertension was associated with cardiac hypertrophy and increased contractility. Regardless of the strain, PVAT revealed an anticontractile effect; however, PVAT worsened endothelial-dependent vasorelaxation. Since H2S produced by both the vascular wall and PVAT had a pro-contractile effect in SHRs, H2S decreased the sensitivity of adrenergic receptors to noradrenaline in Wistar rats. While H2S had no contribution to endothelium-dependent relaxation in Wistar rats, in SHRs, H2S produced by the vascular wall had a pro-relaxant effect. We observed a larger vasorelaxation induced by exogenous H2S donor in SHRs than in Wistar rats. Additionally, in the presence of PVAT, this effect was potentiated. We demonstrated that PVAT of the TA aggravated endothelial function in SHRs. However, H2S produced by the TA vascular wall had a pro-relaxation effect, and PVAT revealed anti-contractile activity mediated by the release of an unknown factor and potentiated the vasorelaxation induced by exogenous H2S. All these actions could represent a form of compensatory mechanism to balance impaired vascular tone regulation.