Purpose of the study. Search for an optimal approach to developing a translational model of endothelial dysfunction (ED).Materials and methods. The experiments were carried out on 58 white male rats, which were randomized into 8 groups: 1st (n = 6) — control for L-methionine (in vitro experiments); 2nd (n = 6) — L-methionine (in vitro experiments); 3rd (n = 6) — control for L-NAME (in vitro experiments); 4th (n = 6) — L-NAME (in vitro experiments); 5th (n = 10) — control for L-methionine (in vivo experiments); 6th (n = 7) — L-methionine (in vivo experiments); 7-a (n = 10) — control for L-NAME (in vivo experiments); 8th (n = 7) — L-NAME (in vivo experiments). In groups 2 and 6, animals were intragastrically administered L-methionine (3 g/kg/day for 7 days), and rats in groups 4 and 8 were administered i.p. with L-NAME (0.025 g/kg/day within 7 days). Control animals received an equivalent volume of solvent. In in vitro experiments, a four-channel myograph (Danish Myo Technology) was used to record the tonic activity of the aortic rings. The contraction strength of the aortic rings caused by norepinephrine (10−7 M) and carbachol (10−5 M) was recorded in isometric mode. In in vivo experiments on anesthetized rats (urethane 1300 mg/kg i.p.), blood microcirculation in the myocardium and skeletal muscle was recorded using laser Doppler flowmetry using a computerized laser analyzer "LAZMA-D" (manufactured by NPP "Lazma", Russia). When assessing microblood flow, the perfusion index (M) was calculated in perfusion units (perf.u.).Results. In vitro experiments have shown that under conditions of hyperhomocysteinemia caused by L-methionine, the vascular response to norepinephrine remains practically unchanged. At the same time, the vasodilating response to carbachol (10−5 M) statistically significantly decreased by 57 % (p = 0.005). In animals treated with L-NAME, not only did the response to carbochol decrease by 55 % (p = 0.009), but also the vasoconstrictor response to norepinephrine increased by 48 % (p = 0.003). Under conditions of ED caused by both hyperhomocysteinemia and L-NAME, blood microcirculation in the myocardium and skeletal muscle is significantly reduced. It has also been shown that in the conditions of the L-NAME-induced ED model, in contrast to the L-methionine-induced ED model, there is no drop in rat body weight and practically no mortality.Conclusion. Thus, ED induced by both hyperhomocysteinemia and L-NAME blockade of endothelial nitric oxide synthase is accompanied by similar changes in blood microcirculation in the myocardium and skeletal muscle. However, taking into account the results of in vitro experiments, we can conclude that the model of ED induced by L-NAME seems more promising, under which, in contrast to ED induced by L-methionine, the vasodilatory response of the vessel to carbachol is not only suppressed, but also the vasoconstrictor reaction to norepinephrine is activated.

Актуальность. Биологическое старение признано наиболее значимым фактором риска развития сердечно-сосудистых заболеваний, которые остаются ведущей причиной смертности в мире. Исследования механизмов старения приобретают особую актуальность, поскольку с каждым годом увеличивается число людей преклонного возраста. До сих пор существуют значительные пробелы в понимании процессов, лежащих в основе старения сосудов и сердца, и практически отсутствуют данные о влиянии половой принадлежности на развитие возраст-ассоциированной патологии. В последние годы активно проводятся исследования роли ионных каналов в развитии различных заболеваний. Значительный прогресс достигнут в понимании вклада классических калиевых каналов внутреннего выпрямления Kir2.x в регуляцию сократимости сосудов и миокарда в норме, но нет четких представлений об их причастности к развитию возрастной патологии. Отсутствуют данные о половых особенностях возрастных изменений свойств этих каналов. Цель исследования – оценка влияния возраста и пола животных на функциональную активность и экспрессию Kir2.1 и Kir2.4 каналов в аорте и сердце крыс. Методика. Исследования проводили на молодых (3 мес) и возрастных (18 мес) крысах самцах и самках породы Вистар. Силу сокращения изолированных фрагментов грудного отдела аорты измеряли в изометрическом режиме. Экспрессию генов Kcnj2 и Kcnj14 соответственно Kir2.1 и Kir2.4 каналов в аорте и в различных отделах сердца оценивали с помощью количественной полимеразной цепной реакции. Результаты. Инкубация сосудов с блокатором Kir2.x каналов BaCl2 (100 мкМ) приводила к усилению сократительной реакции аорты молодых крыс обоего пола в ответ на воздействие возрастающих концентраций серотонина (10-7-10-5 М). Установлено, что в процессе старения у крыс самцов уменьшается вклад Kir2 каналов в формирование базального тонуса аорты, но сохраняется их причастность к реализации механизма отрицательной обратной связи, активируемого воздействием вазоконстрикторов. В отличие от самцов, в аорте самок Kir2 каналы не участвуют в регуляции базального тонуса. В процессе старения у самок утрачивается вовлеченность Kir2 каналов в отрицательную регуляцию сократительных ответов аорты. В аорте старых самцов уровень экспрессии генов Kcnj2 и Kcnj14 оставался неизменным, в то время как у самок наблюдалось двукратное снижение содержания мРНК каналов Kir 2.4. Установлено, что по сравнению с самками сердце самцов наиболее подвержено возрастным изменениям на уровне экспрессии генов Kcnj2 и Kcnj14 каналов Kir2.1 и Kir2.4. Заключение. Полученные данные позволяют предположить, что у особей женского пола в преклонном возрасте большая степень риска развития артериальной гипертензии, в то время как у мужского – патологии сердца. Выявленные половые различия следует учитывать при использовании модуляторов Kir2 каналов в качестве терапевтических средств коррекции сердечно-сосудистых заболеваний в старости. Background. Biological aging is recognized as the most significant risk factor for the development of cardiovascular diseases, which remain the leading cause of death in the world. Research into the mechanisms of aging is becoming particularly relevant as the number of elderly people increases every year. There are still significant gaps in understanding the processes underlying the aging of blood vessels and the heart, and there is virtually no data on the influence of gender on the development of age-associated pathology. In recent years, active research has been carried out on the role of ion channels in the development of various diseases. Significant progress has been made in understanding the contribution of the classical inward rectifying potassium channels Kir2.x to the regulation of vascular and myocardial contractility under normal conditions, but there is no clear understanding of their involvement in the development of age-related pathology. There is no data on gender-related changes in the properties of these channels. The aim of the study is to assess the effect of age and sex of animals on the functional activity and expression of Kir2.1 and Kir2.4 channels in the aorta and heart of rats. Methods. The studies were carried out on young (3 months) and aged (18 months) male and female Wistar rats. The force of contraction of isolated fragments of the thoracic aorta was was measured in isometric mode. The expression of the Kcnj2 and Kcnj14 genes, respectively, Kir2.1 and Kir2.4 channels in the aorta and in various parts of the heart was assessed using quantitative polymerase chain reaction. Results. Incubation of vessels with the Kir2.x channel blocker BaCl2 (100 μM) led to an increase in the contractile response of the aorta of young rats of both sexes in response to exposure to increasing concentrations of serotonin (10-7 – 10-5 M). It has been established that during the aging process in male rats, the contribution of Kir2 channels to the formation of the basal tone of the aorta decreases, but their involvement in the implementation of the negative feedback mechanism activated by the influence of vasoconstrictors remains. Unlike males, in the female aorta Kir2 channels are not involved in the regulation of basal tone. During the aging process in females, the involvement of Kir2 channels in the negative regulation of aortic contractile responses is lost. In the aorta of old males, the level of expression of the Kcnj2 and Kcnj14 genes remained unchanged, while in females a twofold decrease in the mRNA content of Kir 2.4 channels was observed. It was found that, compared with females, the heart of males is most susceptible to age-related changes at the level of expression of the Kcnj2 and Kcnj14 genes of the Kir2.1 and Kir2.4 channels. Conclusion. The data obtained suggest that females in old age have a higher risk of developing arterial hypertension, while males have a higher risk of developing heart disease. The identified sex differences should be taken into account when using Kir2 channel modulators as therapeutic agents for the correction of cardiovascular diseases in old age.

Старение и связанные с ним дисрегуляционные процессы в артериях и сердце являются ведущими предикторами развития сердечно-сосудистых заболеваний. Важную роль в регуляции сократимости сосудов и миокарда играют ионные каналы. Цель исследования – изучение влияния возраста на функциональную активность и экспрессию генов классических калиевых каналов внутреннего выпрямления Kir2.1 и Kir2.4, а также КATP каналов в аорте и сердце крыс. Методика. Эксперименты проводили на крысах самцах Wistar в возрасте 3 и 18 мес. Силу сокращения грудного отдела аорты измеряли в изометрическом режиме, экспрессию генов оценивали при помощи ПЦР анализа. Результаты. Установлено, что у крыс в возрасте 18 мес в аорте снижается функциональная активность Kir2.1 и Kir2.4 каналов, в то время как уровень экспрессии этих каналов остается неизменным. В стареющих сердцах, напротив, выявлен высокий уровень экспрессии генов Kir2.1 и Kir2.4 каналов. Показано, что KATP каналы не влияют на серотонин-индуцированное сокращение аорты молодых крыс (3 мес), но вносят существенный вклад в развитие гиперчувствительности старых сосудов к вазоконстрикторному действию серотонина. Так, блокада KATP каналов глибенкламидом приводила к статистически значимому смещению зависимости «концентрация-эффект» на серотонин вправо только в аорте возрастных крыс. Выявлено возрастное снижение экспрессии генов порообразующей Kir6.2 и регуляторной Sur2 субъединиц KATP канала в аорте и, напротив, значительное повышение в сердце крыс. Заключение. Полученные результаты свидетельствуют о ранних возрастных изменениях функциональной активности калиевых каналов внутреннего выпрямления Kir 2.1 и Kir 2.4, а также KATP каналов в аорте крыс самцов, которые в процессе дальнейшего старения сосудов могут способствовать развитию гипертензии. Предположено, что гиперэкспрессия Kir2.4 и Kir2.1 каналов в стареющем сердце может инициировать нарушения сократительной функции миокарда и возникновение аритмий в старости, в то время как высокий уровень экспрессии субъединиц KATP является показателем компенсаторно-адаптивных процессов, направленных на повышение устойчивости миокарда к гипоксии и стрессу. Aging and related dysregulatory processes in the arteries and heart are the sleading predictors of cardiovascular diseases. Ion channels play an important role in the regulation of vascular and myocardial contractility. The aim of the study was to investigate the effect of age on the functional activity and gene expression of classical inward-rectifying potassium channels Kir2.1 and Kir2.4, as well as KATP channels in the aorta and heart of rats. Methods. Experiments were performed on Wistar male rats at 3 and 18 months of age. The force of thoracic aorta contractions was measured isometrically, and the gene expression was assessed by PCR analysis. Results. In the aorta of 18-month-old rats, the functional activity of Kir2.1 and Kir2.4 channels was reduced while the expression of these channels remained unchanged. In the aging hearts, on the contrary, the expression level of Kir2.1 and Kir2.4 channel genes was high. The KATP channels had no effect on serotonin-induced aortic contractions in young, 3-month-old rats, but contributed significantly to the development of hypersensitivity of aging blood vessels to serotonin-induced vasoconstriction. Thus, blockade of KATP channels with glibenclamide led to a significant shift of the concentration-effect curve of serotonin to the right only for the aorta from old rats. Age-related decreases in the gene expression of pore-forming Kir6.2 and regulatory Sur2 subunits of the KATP channel were observed in the aorta, and, vice versa, significant increases in their expression were observed in the rat heart. Conclusion. The study results indicated early age-related changes in the functional activity of Kir 2.1 and Kir 2.4 inward-rectifying potassium channels, as well as of KATP channels in the aorta of male rat. During further vascular aging, these changes may contribute to the development of hypertension. It was suggested that Kir2.4 and Kir2.1 channel overexpression in the aging heart may initiate disorders of myocardial contractility and arrhythmias in older age. At the same time, a high level of KATP subunit expression indicates compensatory-adaptive processes aimed at increasing myocardial resistance to hypoxia and stress.

The sex differences of age-related changes in the functional activity and expression of receptor and regulatory proteins of the renin-angiotensin system (RAS) in the aorta and myocardium of rats is evaluated. It was found that with age in females, the sensitivity of the aorta to the vasoconstriction action of angiotensin II increases, while in older males it remains unchanged. In the aorta of old females, a decrease in the mRNA levels of ACE, MasR and ACE2 was revealed by 1.6, 1.8 and 4.4 times, respectively. It has been shown that during aging in the left ventricle of females, the level of MasR and ACE2 mRNA decreases, in males, on the contrary, the level of ACE and MasR mRNA increases. The results obtained indicate the sexual characteristics of age-related changes in RAS activity in the vessels and heart of rats.

Введение. Гипергомоцистеинемия (Ггц) - повышение уровня гомоцистеина в плазме крови выше 12 мкмоль/л - независимый фактор риска сердечно-сосудистых заболеваний. Гипергомоцистеинемия, являясь сравнительно распространенным состоянием, ассоциирована также с рядом других патологий, таких как болезнь Альцгеймера, шизофрения, заболевания почек, остеопороз, инсулиннезависимый диабет. Повышенные концентрации гомоцистеина (Гц) способны вызывать клеточный стресс (окислительный, стресс эндоплазматического ретикулума), что может приводить к эндотелиальной дисфункции и клеточной гибели. Ггц может отягощать течение инфекционных заболеваний, но механизмы такой модуляции изучены недостаточно. Предполагается, что Ггц является одним из факторов неблагоприятного прогноза SARSCoV-2 инфекции. Цель исследования - изучение в эндотелиальных клетках механизмов действия гомоцистеина, липополисаридов, их комбинации, а также модулирующего действия фолиевой кислоты и витамина В12. Методика. Эндотелиоцитоподобные клетки EA.hy926 подвергали действию дитиотрейтола, гомоцистеина, липополисаридов, гомоцистеина в комбинации с липополисахаридами, с дополнительной инкубацией с витамином В12 и фолиевой кислотой или только фолиевой кислотой. Использовали ДНК-цитометрию для подсчета процента гиподиплоидных клеток, а также МТТ-тест для оценки цитотоксических эффектов. Методом ПЦР-РВ оценивали уровни экспрессии мРНК генов, вовлеченных в ответ клетки на стресс эндоплазматического ретикулума: BiP, XBP1 (общего и сплайсированной формы), IRE1, ATF6 в двух временных точках после воздействия - 6 ч и 24 ч. Результаты. Впервые показано, что совместное действие липополисахаридов и гомоцистеина приводит к увеличению активности пути IRE1-XBP1 - сигнальной ветви ответа на стресс эндоплазматического ретикулума. Являясь слабыми индукторами стресса ЭПР, липополисариды значимо потенцируют активированный гомоцистеином сигнальный путь IRE1-XBP1, что мы связываем с взаимодействием сигнальных путей врожденного иммунитета и ответа на стресс. Обнаруженное потенцирование может объяснять небольшое увеличение цитотоксичности при сочетанном действии Гц и ЛПС в связи с тем, что пролонгированная активность IRE1 обладает проапоптотическим действием. Впервые показано, что фолиевая кислота и витамин В12 снижают активность пути IRE1-XBP1 при действии гомоцистеина и ЛПС, а также дитиотрейтола. Заключение. В ходе исследования установлено значимое стимулирование сигнального пути IRE1-XBP1 комбинацией ЛПС и Гц, а также атеннюация этого пути фолиевой кислотой и витамином В12. Однако, потенцирование гомоцистеином цитотоксического эффекта ЛПС невелико. Хотя полученные в ходе данного in vitro исследования результаты не позволяют в полной мере рассматривать Ггц как фактор, усложняющий течение инфекционных заболеваний, состояние сосудистой системы и возрастающий риск ее дисфункции должны приниматься во внимание при выборе методов профилактики, терапии и прогноза течения инфекционных заболеваний на фоне гипергомоцистеинемии. Introduction. Hyperhomocysteinemia (HHcy) is a pathological condition described with high levels in plasma of homocysteine (Hcy), a nonprotein α-amino acid homologue of cysteine. It might cause oxidative and endoplasmic reticulum stress with resultant endothelial dysfunction and cell death. HHcy, that is a widespread condition, is associated with a number of other pathologies, such as Alzheimer’s disease, schizophrenia, kidney disease, osteoporosis, and diabetes. Early studies emphasize HHcy as one of the crucial and independent risk factors for development of cardiovascular disorders. Moreover, hyperhomocysteinemia can escalate the severity of infectious diseases and even appears as a potential marker for forecasting the course of SARSCoV-2 infection, as many patients are currently at risk for major clotting events once the infection is established. However, the mechanisms of these processes were poorly studied previously. Therefore, the major focus of this work is to study the mechanisms of actions of homocysteine and lipopolysaccharides (LPS) separately and combined together, and modulating actions of them by folic acid and vitamin B12 in endothelial cells. Methods. Endotheliocyte-like EA.hy926 cells were exposed to dithiothreitol, homocysteine, LPS, and Hcy in combination with LPS, with additional incubation with vitamin B12 and folic acid or folic acid alone.The DNA cytometry was performed to calculate the percentage of hypodiploid cells, and the MTT test was conducted to assess cytotoxic effects. RT-qPCR was done in order to evaluate the mRNA expression of the genes involved in the cell response to endoplasmic reticulum stress: BiP, XBP1 (common and spliced form), IRE1, ATF6 at two time points after exposure, 6 h and 24 h. Results. During our research, it was discovered that the coaction of LPS and Hcy increased the activity of the IRE1-XBP1 pathway, a signaling branch of ER stress. LPS, as weak inducers of ER stress, significantly potentiates the Hcy-activated IRE1-XBP1 signaling pathway. We interpret this event as the interaction of signaling pathways of innate immunity and stress response. The observed potentiation may explain the elevated cytotoxicity under the coaction of Hcy and LPS due to the proapoptotic effect of IRE1prolonged activity. In this paper, our research demonstrated that folic acid and vitamin B12 restrict the activity of the IRE1-XBP1 pathway under the action of Hcy and LPS as well as of dithiothreitol. Conclusion. A significant stimulation of the IRE1-XBP1 signaling pathway by a combination of LPS and Hcy, as well as attenuation of this pathway by folic acid and vitamin B12 have been shown. However, the potentiation of the cytotoxic effect of LPS by homocysteine is relatively small. Although the results obtained during this in vitro study do not let us to fully consider HHcy as a factor aggravating the course of infectious diseases, the state of the vascular system and the increasing risk of its dysfunction should be taken into account while choosing methods for the prevention, treatment and forecasting of the infectious diseases course against the background of hyperhomocysteinemia.

В современном обществе социальные и демографические изменения привели к увеличению распространённости одиночества и социальной изоляции, особенно среди лиц пожилого возраста. Преклонный возраст, как и психосоциальный хронический стресс, являются независимыми факторами риска развития сердечно-сосудистых заболеваний. Цель исследования - изучение влияния социально-изоляционного стресса на половые различия возрастных изменений чувствительности сосудов к аргинин-вазопрессину (AVP) и экспрессию вазопрессиновых и глюкокортикоидных рецепторов в аорте и миокарде крыс. Методы исследования. Социально-изоляционный стресс моделировали путем изоляции крыс в отдельные клетки с 22-го по 24-й месяцы жизни включительно. Силу сокращения изолированных сосудов оценивали в изометрическом режиме на 4-канальном миографе (Danish Myo Technology, модель Multi Myograph System - 620M). Экспрессию генов оценивали при помощи ПЦР-анализа. Результаты. Установлено, что у старых (24 мес) крыс наблюдается значительный рост силы сократительного ответа на воздействие AVP, причем, в отличие от самцов, у самок увеличивается не только сила, но и длительность индуцированной тонической реакции. Отмечено, что чувствительность аорты, как молодых, так и старых самок по отношению к действию AVP была в 2,7 и 2,2 раза выше, чем у самцов соответственно. В сосудах выявлены половые различия возрастных изменений профиля экспрессии вазопрессиновых рецепторов 1-го типа (V1AR): уровень мРНК у старых самок снижается в 1,4 раза, у старых самцов, напротив, возрастает в 2 раза по сравнению с показателями молодых крыс. Установлено, что независимо от пола у старых крыс в правом и левом предсердиях значительно возрастает экспрессия гена глюкокортикоидных рецепторов (GR), где локализованы важнейшие регуляторы прохождения электрического импульса между предсердиями и желудочками. Социально-изоляционный стресс не оказывал существенного влияния на возрастные изменения реактивности сосудов в ответ на действие AVP и уровень экспрессии генов V1AR и GR. Установлено, что длительный хронический стресс приводил к значительному повышению экспрессии V1AR в сердце старых самцов и, напротив, к снижению у старых самок крыс. Изоляционный стресс старых крыс у самок приводил к снижению, а у самцов - к повышению экспрессии гена GR в левом желудочке. Заключение. Одним из механизмов агонист-индуцированного усиления вазоконстрикторной реакции сосудов старых самцов крыс является высокий уровень экспрессии гена AV1AR. Выраженная по силе и продолжительности сократительная реакция сосудов старых самок на фоне снижения экспрессии гена AV1AR, позволяет предположить, что у них важную роль в регуляции тонуса сосудов играет Rho-киназный каскад, активация которого может влиять не только на силу, но и на кинетику сократительного ответа. Связанное с возрастом увеличение экспрессии гена GR в предсердиях старых крыс самцов и самок может быть одним из механизмов нарушения сердечного ритма и гемодинамики. Негативное влияние социально-изоляционного стресса проявляется в большей мере на сердце старых крыс. In the modern society, social and demographic changes have increased the prevalence of loneliness and social isolation, especially among senior people. Old age and chronic psychosocial stress are independent risk factors for cardiovascular disease. The aim was to study the effect of social isolation stress on sex differences in age-related changes of vascular sensitivity to arginine-vasopressin (AVP) and the expression of vasopressin and glucocorticoid receptors in the rat aorta and myocardium. Methods. Social isolation stress was modeled by isolating rats in individual cages from the age of 22 months through 24 months. The contraction force of isolated blood vessels was measured in the isometric mode with a four-channel myograph (Danish Myo Technology, Multi Myograph System - 620M). Gene expression was assessed by the PCR analysis. Results. In old (24 months) rats, the strength of the contractile response to AVP was significantly increased. As distinct from males, in females, not only the contraction force but also the duration of AVP-induced tonic reaction was increased. In both young and old females, the aorta sensitivity to AVP was 2.7 and 2.2 times higher than in males, respectively. Blood vessels showed sex differences in the age-related changes of the vasopressin receptor V1 (V1AR) expression profile: in old females, the mRNA level was decreased 1.4 times, while in old males, in contrast, mRNA was twofold increased compared to young rats. In old rats, regardless of the gender, the glucocorticoid receptor (GR) gene expression was significantly increased in right and left atria, the area of the most important regulators of the electrical pulse passage between the atria and the ventricles. Social isolation stress had no significant effect on the age-related changes in vascular reactivity to AVP and the V1AR and GR gene expression. Prolonged, chronic stress induced a significant increase in the cardiac V1AR expression in old male rats and, on the contrary, a decrease in old females. Isolation stress in old females induced a decrease, while in old males, it induced an increase in the left ventricular GR gene expression. Conclusion. One of the mechanisms for the increased agonist-induced vasoconstrictor response in old male rats is a high level of AV1AR gene expression. The stronger and more lengthy vasoconstrictor reaction of old female rats associated with the decreased AV1AR gene expression suggests that the Rho-kinase cascade plays a significant role in their regulation of vascular tone, since the cascade activation can influence not only the contraction force but also its kinetics. The age-related increase in the atrial GR gene expression in old male and female rats may be a mechanism underlying disorders of both the heart rhythm and hemodynamics. The adverse impact of social isolation stress is more pronounced in the heart of old rats.

Social isolation stress was modeled by long-term isolation of 12-month-old rats in individual cages over 28 weeks. It was found that sensitization of blood vessels to the vasoconstrictor action of serotonin due to overexpression of 5HT2A-type receptor genes, as well as an imbalance in the expression level of endothelin ETA- and ETB-receptors (55 and 153%, respectively) are the early signs of vascular aging. A significant contribution to the development of age-related changes in the contractile properties of blood vessels is made by the stress component, which is manifested at the level of glucocorticoid-dependent mechanisms of regulation of gene expression. The decrease in the expression of glucocorticoid receptors caused by isolation stress leads to a decrease in the expression of the genes responsible for the synthesis of V1A-R and ATII-R and to the development of vascular hyporeactivity to the vasoconstrictor action of ATII and AVP. In the aorta of stressed rats, the α1-AR mRNA level increases by 3 times. At the same time, stress did not affect the dynamics of age-related changes in the expression of genes encoding 5HT2A-R and ETA/ETB-R.

Наиболее тяжелым проявлением SARS-CoV-2 инфекции является развитие острого респираторного дистресс-синдрома. Высокий уровень гомоцистеина в крови пациентов - прогностически неблагоприятный фактор течения COVID-19 инфекции. Также развитие полиорганной недостаточности при COVID-19 инфекции часто утяжеляется эндотоксемией. Цель исследования. Изучение влияния гипергомоцистеинемии и липополисахарида (ЛПС) Salmonella Typhimurium на сократимость аорты и почечной артерии крыс и экспрессию генов рецепторных и регуляторных белков. Методикы. Моделирование гипергомоцистеинемии проводили путем добавления в воду L-метионина в концентрации 5 г/л (0,5%) в течение 14 дней. Эндотоксемию вызывали введением ЛПС Salmonella Typhimurium (Sigma, 5 мг/кг, в/б). Силу сокращения изолированных сосудов измеряли в изометрическом режиме. Экспрессию генов оценивали при помощи ПЦР-анализа. Результаты. Установлено, что через 24 часа после однократного введения животным ЛПС сила сокращения аорты возрастает в ответ на воздействие ангиотензина II (ATII, в 1,6 раза), аргнинвазопрессина (AVP, в 1,5 раза), и снижается после введения эндтелина-1 (ET-1, в 1,6 раза), по сравнению с аналогичными показателями контрольных крыс. При повторном введении ЛПС сосуды теряют чувствительность к вазоконстрикторам. У крыс после метиониновой нагрузки в плазме крови более чем в 3 раза повышается уровень общего гомоцистеина. Гомоцистеинемия приводит к выраженным нарушениям нейроэндокринной регуляции сократимости сосудов: снижается чувствительность аорты и почечной артерии к вазоконстрикторному действию ATII (в 1,7 и 2,6 раза соответственно), AVP (в 1,5 и 1,7 раза), серотонина (5HT, в 1,2 и 1,8 раза) и ET1 (в 1,5 и 1,6 раза). Установлено, что введение ЛПС крысам на фоне высокого уровня гомоцистеина в плазме ещё в большей степени усиливает выявленные нарушения сократительной активности сосудов. У крыс после введения ЛПС выявлен низкий уровень экспрессии вазоконстрикторных адренорецепторов α1A-AR, α1D-AR и вазодилататорных β1- и β2-адренорецепторов и высокий - ETA и ETB рецепторов. Как ЛПС, так и гипергомоцистеинемия приводят к значительному снижению содержания мРНК ангиотензинпревращающих ферментов ACE, ACE2 и рецепторов MasR, V1AR. У крыс после метиониновой нагрузки выявлено снижение экспрессии генов инозитолтрисфосфатных рецепторов IP3R2, IP3R3, что свидетельствует о нарушении кальциевого гомеостаза в сосудах. Метиониновая нагрузка не приводила к летальным исходам. В группе животных после однократного введения ЛПС смертность составляла 7%, а в группе с введением ЛПС после метиониновой нагрузки - 50%. Заключение. Таким образом, гипергомоцистеинемия усиливает ЛПС-индуцированные нарушения нейроэндокринной регуляции тонуса сосудов, отягощает течение и увеличивает смертность при эндотоксемии. The most severe manifestation of SARS-CoV-2 infection is the development of acute respiratory distress syndrome. A high blood level of homocysteine in patients is a prognostically unfavorable factor for the course of COVID-19 infection. The development of multiple organ failure in COVID-19 infection is aggravated by endotoxemia. The aim of the study was to investigate the effect of hyperhomocysteinemia and Salmonella Typhimurium lipopolysaccharide (LPS) on the rat aorta and renal artery contractility and the gene expression of receptor and regulatory proteins. Methods. Hyperhomocysteinemia was modeled in rats by adding L-methionine to drinking water at a concentration of 5 g/L (0.5%) for 14 days. Endotoxemia was induced with a single injection of Salmonella Typhimurium LPS (Sigma, 5 mg/kg, intraperitoneally). The contractile force of isolated blood vessels was measured in the isometric mode. Gene expression was assessed with the PCR analysis. Results. At 24 h after a single injection of LPS, the force of aortic contractile response to angiotensin II (ATII) increased 1.6 times and to arginine vasopressin (AVP) 1.5 times whereas the response to endothelin-1 (ET1) decreased by 37.5% compared to the respective values for control rats. Upon repeated administration of LPS, the blood vessels lost the sensitivity to vasoconstrictors. After methionine loading, the plasma level of total homocysteine increased more than three times. Homocysteinemia resulted in severe disorders in the neuroendocrine regulation of vascular contractility, including decreased sensitivity of the aorta and the renal artery to vasoconstrictor effects of ATII (41.2% and 61.5%, respectively), AVP (33.3% and 41.2%, respectively), serotonin (5HT, 16.7% and 44.4%, respectively) and ET1 (33.3% and 37.5%, respectively). The administration of LPS to rats on the background of a high plasma level of homocysteine further aggravated the disorders of vascular contractile activity. Administration of LPS was associated with lower expression of vasoconstrictor α1A- and α1D-adrenoceptors, and of vasodilator β1- and β2-adrenoceptors and with a higher expression of ETA and ETB receptors. Both LPS and hyperhomocysteinemia led to significant decreases in angiotensin-converting enzymes ACE and ACE2 mRNA, and in MasR and V1AR receptor mRNA. The methionine loading induced a decrease in the gene expression of inositol trisphosphate receptors IP3R2 and IP3R3, which indicated a disorder of calcium homeostasis in the blood vessels. The methionine loading was not fatal. In the group of animals after a single administration of LPS, the mortality rate was 7%, and in the group after the administration of LPS and methionine loading, the mortality rate was 50%. Conclusion. Thus, hyperhomocysteinemia enhances LPS-induced disorders in the neuroendocrine regulation of vascular tone, aggravates the course of endotoxemia, and increases the mortality rate.

The study was aimed at the assessment of age-related changes in the transcriptional activity of genes involved in the regulation of Ca2+ signaling, specifically of the genes of IP3R and RyR and of their activity modulators CaM and Epac, in the vessels and myocardium of rats of both sexes. Expression was evaluated by the content of the mRNA of the studied protein related to the content of beta-actin mRNA. It was shown that upon aging, unidirectional changes in the expression of genes encoding IP(3)Rs and RyR2 develop in the aorta of rats of both sexes, as well as various, sex-specific changes in the expression of genes of proteins CaM and Epac that modulate the activity of IP3R and RyR. In the vessels of old rats (24 months) of both sexes, relative levels of mRNA for IP3R of types 2 and 3 were reduced but remained unchanged for IP(3)R1 and significantly increased for RyR2 as compared with these indicators in young rats (4 months). In the aorta of old females, the relative mRNA content was reduced for CaM and Epac1 but remained unchanged for Epac2; in males, on the contrary, the expression of Epacl and Epac2 was increased by 67% and 50%, respectively, as compared with corresponding parameters in young rats (4 months), but was not changed for CaM, which indicates gender differences in the violation of the subtle mechanisms of modulation of RyR2 and IP(3)Rs activity in blood vessels. Significant changes were revealed in the myocardium of old rats. In older males, an elevated expression was demonstrated for RyR2, IP(3)R1,2,3 in the left ventricle, for RyR2 and IP(3)R1 in the left atrium, and for RyR2 and IP(3)R3 in the right atrium. In contrast to males, in the left ventricle of females the mRNA content for RyR2 and IP(3)R1,2,3 was significantly lower than in young animals. In old females, the right atrium exhibited high expression levels of IP(3)R1 and IP(3)R3, and in the left atrium the expression level of IP(3)R3 was increased. In all parts of the heart of females, the expression of IP(3)R2 was unchanged. In the myocardium of old rats of both sexes, the expression of proteins CaM and Epac2 increased significantly. The revealed age-related differences in the transcriptional activity of genes involved in the regulation of intracellular Ca2+ signaling at the level of IP(3)Rs and RyR2-mediated mechanisms suggest that with increasing life expectancy in males, the likelihood of developing myocardial hypertrophy and heart rhythm disturbance is significantly higher than in females.

The expression of Epac proteins (exchange protein directly activated by cAMP) and calmodulin (CaM) was assessed by the content of the corresponding mRNA in biopsy specimens of cardiac atrium, left ventricle, and thoracic aorta of rats with alcoholic cardiomyopathy. In the myocardium, overexpression of Еpac1 , Ерас2 , and СаМ mRNA was found. The content of Epac2 mRNA in the left ventricle was elevated by 2.9 times ( p =0.000001), in the left atrium by 3.2 times ( p =0.00001), in the right atrium by 3 times ( p =0.00001). In contrast to the myocardial tissue, the content of CaM mRNA in the thoracic aorta was not increased, but showed a tendency to decrease, when compared to the control values, while the level of Epac1 and Epac2 mRNA was increased. The assumption is made that regulatory proteins Epac and CaM can play a key role in arrhythmogenesis development under conditions of alcoholic cardiomyopathy.

Original translational rat model of chronic heart failure provoked by experimental anterior transmural myocardium infarction was employed to examine the preventive action of anxiolytic Afobazole (15 mg/kg/day administered intraperitoneally during the first 15 days after coronary occlusion) on the development of the heart failure assessed in 3 months after infarction. Afobazole prevented the development of pathologic remodeling of the myocardium, maintained its inotropic function, and decreased the plasma level of brain natriuretic peptide known as a biochemical marker of chronic heart failure. In the myocardium, Afobazole down-regulated overexpression of the genes induced in chronic heart failure and assessed by corresponding RNA levels, which code angiotensin (AT1A-R), vasopressin (V1A-R), and glucocorticoid (GR) receptors as well as Epac2 protein. The revealed biochemical changes are consistent with the data on cardioprotective action of Afobazole.

Возраст является основным фактором риска развития сердечно-сосудистых заболеваний. Однако механизмы влияния старения на развитие заболеваний до сих пор не понятны. Цель исследования - изучение влияния старения на функциональную активность и экспрессию рецепторов эндогенных вазоконстрикторов в аорте крысы. Методы. Исследования проводили на беспородных крысах-самцах в возрасте 4 и 12 мес. Силу сокращения изолированных фрагментов грудного отдела аорты измеряли в изометрическом режиме по методу Мульвани. Выделение РНК из аорты проводили с помощью набора GeneJET, синтез кДНК - используя набор RevertAid H Minus First Strand cDNA Synthesis Kit («Thermo Fisher Scientific», США), ПЦР-РВ проводили с помощью набора qPCRmix-HS («Евроген», Россия), используя праймеры и флуоресцентные зонды («ДНК-синтез», Россия), согласно протоколам производителей. Результаты. Ранними признаками старения сосудов является их сенситизация к вазоконстрикторному действию серотонина (5НТ), обусловленная гиперэкспрессией гена рецепторов 5HT2A-типа (194% от уровня в сосудах 4-мес. крыс). Чувствительность аорты по отношению к воздействию норадреналина, ангиотензина II, аргининвазопрессина и эндотелина-1 у крыс в возрасте 12 мес. остается такой же, как у молодых животных. Установлено, что в аорте стареющих крыс значительно снижается уровень мРНК эндотелиновых рецепторов ETA-типа (55%). Сохранение чувствительности сосудов к вазоконстрикторному действию ET1 при двукратном снижении экспрессии гена ETA-R косвенно свидетельствует об участии данных рецепторов не только в поддержании тонуса сосудов, но и в реализации других эффектов ET1. Заключение. Таким образом, повышение функциональной активности и экспрессии серотониновых 5HT2A-R, а также гипоэкспрессия эндотелиновых ETA-R являются ранними проявлениями старения аорты крыс и могут рассматриваться как маркеры возрастных изменений сосудов. Age is the major risk factor for cardiovascular disease. However, mechanisms of the effect of aging on the disease development are still unclear. The aim of this study was to investigate the effect of aging on functional activity and expression of endogenous vasoconstrictor receptors in the rat aorta. Methods. The study was performed on mongrel male rats aged 4 and 12 months. Contraction force of fragments isolated from the thoracic aorta i was measured in an isometric mode using the Mulvani method. RNA was isolation from the aorta using the GeneJET kit; cDNA synthesis was measured using the RevertAidTM H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA); PCR-RT was performed using the qPCRmix-HS kit (Evrogen, Russia) with primers and fluorescent probes (DNA synthesis, Russia) according to manufacturer’s protocols. Results. Early signs of vascular aging included blood vessel sensitization to the vasoconstrictor effect of serotonin (5HT) due to overexpression of 5HT2A-type receptors (194% of the level in blood vessels of 4-month old rats). The aorta sensitivity to norepinephrine, angiotensin II, arginine vasopressin, and endothelin-1 in 12-month old rats remained the same as in younger animals. The level of ETA-type endothelin receptor mRNA was significantly reduced in the aorta of aging rats (55%). Preservation of vascular sensitivity to the vasoconstrictor action of ET1 with a twofold decrease in the expression of the ETA-R gene indirectly indicated an involvement of these receptors not only in maintaining the vascular tone but also in mediating other ET1 effects. Conclusion. Therefore, increases in the functional activity and expression of serotonin 5HT2A-R along with the hypoexpression of endothelin ETA-R are early manifestations of rat aorta aging and can be considered as markers for age-related changes in blood vessels.

It has been shown that the Epac1 and Epac2 protein inhibitor ESI-09 has no effect on the amplitude of contraction of aortic rings caused by the influence of serotonin, noradrenaline, or KCl depolarizing solution, but changes the kinetics of the contractile response. It was noted that in the presence of ESI-09 the curve of the relaxation phase in intact and deendothelized vessels moved to the left under the impact of serotonin or KCl and the phase of prolonged tonic contraction, which developed after the exposure to noradrenalin, was canceled. It was found that ESI-09 exerted different effects on the induced growth in the concentration of cytoplasmic Ca2+ in the aortic smooth muscle cells of rats depending on the agonist, whereas the selective inhibitor Epac2 ESI-05 has no effect on vascular contractility and calcium metabolism in the aortic smooth muscle of rats. The cAMP-independent participation of Epac1 in the formation of the contractile response to the influence of vasoconstrictor compounds was revealed.

We found that the inhibitors of the serotonin (5HT) transporter fluoxetine and clomipramine significantly inhibit 5HT-induced constriction of isolated rings of the aorta. The most prominent inhibitory effect was observed for clomipramine, which at a concentration of 2 μM, prevented aorta constriction in response to low and moderate doses of 5HT and multiply attenuated it in response to high doses (10 μM). The inhibitors of the 5HT-transporter attenuated the strength of norepinephrine-induced aorta constriction by 40–60% and eliminated long-term tonic constriction. Application of clomipramine or fluoxetine on the vessels preliminarily constricted by norepinephrine resulted in 100% relaxation, which was maintained in the presence of the NO-synthase inhibitor L-NAME. The inhibitors of the 5HT-transporter decreased but did not prevent 5HT-induced [Ca2+]cyt increase in smooth muscle cells (SMCs) of the aorta even at high concentrations. Clomipramine and fluoxetine did not affect the vasopressin-induced [Ca2+]cyt increase in SMCs and the strength of constriction of isolated aorta rings. We found that the sensitivity of the rat aorta to the vasoconstrictor effect of 5HT and the role of the 5HT-transporter in regulation of the vascular tone increased with aging.

On the model of alcohol cardiomyopathy studied the effect of chronic ethanol consumption and the insulation stress on the reactivity of isolated rat aorta and the expression of the endogenous vasoconstrictor receptors in the aorta. Pushing alcoholization outbred rats was carried out for 24-28 weeks, using as the sole source of liquid 10% ethanol solution. In assessing the results of the study took into account the age of the animals. It is found that the reactivity of isolated aortic rings dissected from the body of old (40-45 weeks) nonstressed rats in response to endothelin-1 (ET1), noradrenaline (NA), arginine vasopressin (AVP) or angiotensin II (ATII) is not different from such reactivity for young animals. However, with the increase in life expectancy increases the sensitivity of vessels to vasoconstrictor action of serotonin (5HT). Prolonged stress insulation and the consumption of high doses of ethanol the stress lead to increased ET1- and NA-induced contraction of the aortic rings and a significant decrease in contractile response of the aorta to the impact ATII and AVP. Stress and alco- hol in combination with stress causing reduction mRNA ETA-R, AT1A-R. and V1A-R and increased mRNA α₁-AR in rat aorta. It is found that in the vessels of stressed and alcoholized animals reduced level of expression of cytosolic glucocorticoid receptors (GR), which is a transcription factor for genes ETA-R, AT1A-R V1A-R. It is propoused that the development of vascular hyporesponsiveness of stressed and alcoholized rats to action ATII and AVP is the result of reducing the expression of their receptors on the GR-dependent mechanism. It is shown that under the influence of ethanol vessels become hyporeactivity selectively with respect to the action of 5HT. The mechanism of this process is unclear. Importantly, the changes in the contractile properties vessels recovered from the rat at 1 month after the abolition of the reception of ethanol (step abstinence) were similar to changes found at the alcohohzed animals. Thus, the importance of breaking the neuroendocrine regulation of vascular tone during long-term consumption of ethanol has a stressor components. Furthermore, in this experimental model we not received data in favor ethanol direct impact on the development of hypertension.

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

It has been shown that the agonist of 5HT1A-receptors 8-OH-DPAT induces contraction of aortic rings in the presence of angiotensin II. This effect is not associated with activation of α1-adrenoceptors by 8-OH-DPAT as it is reproduced in the presence of prazosin which completely suppresses the nonspecific vasoconstrictive effect of 8-OH-DPAT via α1-adrenoceptors on the aorta incubated without angiotensin II. Synergism in the action of angiotensin II and 8-OH-DPAT is completely preserved after partial desensitization of the receptors of angiotensin II. It has been found that 8-OH-DPAT increases the free cytoplasmic calcium concentration in cultured smooth muscle cells from the rat aorta. The data obtained support the hypothesis about the existence of “silent” vasoconstrictive 5HT1A-receptors. It has been suggested that activation of these receptors underlies synergism in vasoconstrictive action of serotonin and angiotensin II.