eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Anticancer Effect of in Vivo Inhibition of Nitric Oxide Synthase in a Rat Model of Breast Cancer

High expression of nitric oxide (NO)-synthase has been found in different cancers like cervical, breast, and central nervous system. NO-synthase activity inhibition has been suggested as a possible tool to prevent breast cancer. The anti-tumor therapeutic effect of L-nitro arginine methyl ester (L-NAME) in vivo remains understudied. Here we hypothesized that NOS inhibition by L-NAME has some antitumor effects on breast cancer development as it inhibits NO levels, which is a pathophysiological modulator of cell proliferation, cell cycle arrest, apoptosis, and angiogenesis. We utilized a novel anti-cancer treatment model by the administration of NO-synthase inhibitor L-NAME (30 mg/kg in a day, intraperitoneal), injected every third day for five weeks (in parallel to tumors evolution) in opposition to high activity of NOS during 7,12-dimethylbenz[a]anthracene-induced breast tumor in rats in vivo. The blood concentrations of nitrite anions, polyamines, malondialdehyde, NH4+ levels, and arginase activity decreased in DMBA+L-NAME-treated rats compared with DMBA rats. The reduction of these compounds also affects the decrease of the mortality rate of rats, tumor number, weight and volume, and the histopathological grade of breast cancer. Treatment with L-NAME showed increases in time of tumor incidence and body weight compared with DMBA-cancer rats. Therefore, the co-administration of L-NAME influences as a potent anti-cancer agent to treat breast cancer and can lead to the development of therapeutic methods for cancers in the future.

Role of Folic Acid in Type 2 Diabetes

The metabolic condition known as diabetes mellitus is marked by hyperglycemia, a host of symptoms affecting the heart, kidneys, nerves, and other organs. Diabetes nephropathy is one of the leading causes of diabetic impermanence and morbid state. Low parameters of pteroylglutamic acid in the blood are associated with Diabetic Nephropathy, whereas endothelial dysfunction increases the risk for T2D. Endothelial dysfunction is associated with diabetes, which perhaps is caused by the disjunction of the endothelial nitric oxide (NO) synthase enzyme, which reduces NO availability. Because folic acid can repair the disjunction of NO synthase, we sought to see if pteroylglutamic acid supplementation may affect the function of the endothelial layer and inflammatory indicators in type 2 diabetes patients who did not have vascular disease. Recent studies have shown that pteroylglutamic acid also has direct benefits on the function of endo, in addition to its natural function of lowering homocysteine. Folic acid might serve as a "biomarker" for the function of endothelial cells. Many mechanisms have been linked to higher total homocysteine levels and type 2 diabetes risk in diabetic patients. Higher folic acid levels altered endothelial-dependent vasodilation in T2D patients. In patients with coronary heart disease (CAD), folic acid supplementation has been found to reduce homocysteine parameters and improve the function of the endothelial layer. On the other hand, RCTs looking at IR and T2D outcomes have shown mixed results. Several mechanisms link higher total homocysteine levels to increased risk of insulin resistance (IR) and type 2 diabetes mellitus (T2D). Treatment with folate has been shown to bring down homocysteine parameters and improve the endothelium functions in people with coronary heart disease (CAD). Randomized controlled trials (RCTs) on IR and T2D outcomes, on the other hand, have produced a wide range of results.

IMPROVING THE EFFECTIVENESS OF CONSERVATIVE TREATMENT OF ERECTILE DYSFUNCTION IN PATIENTS WITH PEYRONIE'S DISEASE USING THE "BTL 6000" SHOCK WAVE THERAPY APPARATUS AND THE "TIVORTIN" DRUG

Исследовалось эффективность комплексной терапии эректильной дисфункции у пациентов с болезнью Пейрони, с применением препарата "Тивортин", который является субстратом для NO-синтазы - фермента, который катализирует синтез оксида азота в эндотелиоцитах, тем самым вызывая стабилизацию скорости кровотока в артериях полового члена, а также способствует восстановлению морфологической структуры, измененной белочной оболочки; и аппарата ударно-волновой терапий "БТЛ 6000", предназначенный для дезинтеграции бляшек в стадии кальцификации, которая купирует болевые ощущения в 91% случаев и уменьшает деформацию полового члена в 31% случаев, путем ударно-волновой терапии, и улучшения микроциркуляции в кавернозных телах полового члена, тем самым повышает эффективность лечения эректильной дисфункции у больных с болезнью Пейрони. The effectiveness of complex therapy of erectile dysfunction in patients with Peyronie's disease was studied with the use of the drug "Tivortin", which is a substrate for NO-synthase, an enzyme that catalyzes the synthesis of nitric oxide in endotheliocytes, thereby causing stabilization of the blood flow rate in the arteries of the penis, and also contributes to the restoration of the morphological structure, the altered protein envelope; and the shock wave therapy apparatus "BTL 6000", designed for the disintegration of plaques in the calcification stage, which relieves pain in 91% of cases and reduces the deformation of the penis in 31% of cases, by shock wave therapy, and improves microcirculation in the cavernous bodies of the penis, thereby increasing the effectiveness of treatment of erectile dysfunction in patients with Peyronie's disease.

Nitric Oxide Ameliorates Plant Metal Toxicity by Increasing Antioxidant Capacity and Reducing Pb and Cd Translocation

Recently, nitric oxide (NO) has been reported to increase plant resistance to heavy metal stress. In this regard, an in vitro tissue culture experiment was conducted to evaluate the role of the NO donor sodium nitroprusside (SNP) in the alleviation of heavy metal toxicity in a bamboo species (Arundinaria pygmaea) under lead (Pb) and cadmium (Cd) toxicity. The treatment included 200 µmol of heavy metals (Pb and Cd) alone and in combination with 200 µM SNP: NO donor, 0.1% Hb, bovine hemoglobin (NO scavenger), and 50 µM L-NAME, N(G)-nitro-L-arginine methyl ester (NO synthase inhibitor) in four replications in comparison to controls. The results demonstrated that the addition of L-NAME and Hb as an NO synthase inhibitor and NO scavenger significantly increased oxidative stress and injured the cell membrane of the bamboo species. The addition of sodium nitroprusside (SNP) for NO synthesis increased antioxidant activity, protein content, photosynthetic properties, plant biomass, and plant growth under heavy metal (Pb and Cd) toxicity. It was concluded that NO can increase plant tolerance for metal toxicity with some key mechanisms, such as increasing antioxidant activities, limiting metal translocation from roots to shoots, and diminishing metal accumulation in the roots, shoots, and stems of bamboo species under heavy metal toxicity (Pb and Cd).

The role of nitric oxide in the regulation of the electrical activity of the trigeminal nerve in the rat

Nitric oxide (NO) is a gaseous signaling molecule that regulates a number of physiological functions, including its role in the formation of migraine has been established. NO is endogenously produced in the body from L-arginine by NO synthase. The NO donor, nitroglycerin, is a trigger of migraine in humans and is widely used in the modeling of this disease in animals, which suggests the involvement of components of the NO signaling cascade in the pathogenesis of migraine. Based on the results obtained, it was found that an increase in the concentration of both the substrate for the synthesis of NO, L-arginine, and the NO donor, sodium nitroprusside, has a pro-nociceptive effect in the afferents of the trigeminal nerve. In this case, the effect of sodium nitroprusside is associated with the activation of intracellular soluble guanylate cyclase. Key words: nitric oxide, migraine, trigeminal nerve, L-arginine, guanylate cyclase, sodium nitroprusside, nociception.

FUNCTIONING OF NO-CYCLE IN THE SALIVA OF CHILDREN WITH TYPE 1 DIABETES MELLITUS

Introduction. The presence of type 1 diabetes mellitus in children is a major risk factor for periodontal disease. The aim of research work was to determine the activity of NO-synthase and arginase in saliva in children age with insulin-dependent diabetes mellitus. Materials and methods We examined 82 children including 56 children with type 1 diabetes mellitus and 26 children without somatic diseases. NO-synthase (NOS) activity was determined by the difference in nitrite concentration before and after incubation of tissue homogenate. Determining arginase activity was based on analysis the difference in the concentration of L-ornithine before and after incubation in phosphate buffer solution. Statistical processing was performed using Microsoft Office Excel. Research results and their discussion The violation of the indicators’ balance between groups 1 and 3 showed us a decrease in regenerative capacity in the mucous membrane in persons with type 1 diabetes mellitus. Increased ARG activity in group 4 children may lead to rivarly between NOS and ARG for L-arginine. Increased ARG activity in groups 2 and 4 compared with group 1 indicated an adaptive response aimed at repairing gum damage. Based on this, increased NO production from NOS is a consequence of insulin deficiency (systemic factor). Conclusions. The combination of systemic factor (type 1 diabetes mellitus) and local (chronic catarrhal gingivitis) leads to dysregulation of the NO-cycle and increasing of competition between NOS and ARG. ФУНКЦИОНИРОВАНИЕ NO-ЦИКЛА В СЛЮНЕ У ДЕТЕЙ С САХАРНЫМ ДИАБЕТОМ І ТИПА Кузь И.А., Акимов О.Е., Костенко В.А., Шешукова О.В., Максименко А.И., Писаренко Е.А. Полтавский государственный медицинский университет Вступление. Наличие у детей сахарного диабета І типа является основным фактором риска заболеваний пародонта. Целью исследования было определение активности NO-синтазы и аргиназы в слюне у детей с инсулинозависимым сахарным диабетом. Материалы и методы. Обследовано 82 ребенка, в том числе 56 детей с сахарным диабетом І типа и 26 детей без соматических заболеваний. Активность NO-синтазы (NOS) определяли по разнице в концентрации нитрита до и после инкубации гомогената ткани. Определение активности аргиназы основывалось на анализе разницы в концентрации L-орнитина до и после инкубации в фосфатном буферном растворе. Статистическая обработка проводилась с использованием Microsoft Office Excel. Результаты исследований и их обсуждение. Нарушение баланса показателей между 1 и 3 группами свидетельствовало о снижении регенерационной способности слизистой оболочки у лиц с сахарным диабетом І типа. Повышенная активность ARG у детей группы 4 может привести к неравенству между NOS и ARG для L-аргинина. Повышенная активность ARG в группах 2 и 4 по сравнению с группой 1 указала на адаптивный ответ, направленный на восстановление повреждений десен. Исходя из этого, повышенная продукция NO из NOS является следствием дефицита инсулина (системный фактор). Выводы. Сочетание системного фактора (сахарный диабет І типа) и местного (хронический катаральный гингивит) приводит к нарушению регуляции NO-цикла и усилению конкуренции между NOS и ARG.

Vasodilatory Effect of Guanxinning Tablet on Rabbit Thoracic Aorta is Modulated by Both Endothelium-Dependent and -Independent Mechanism

Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of Salvia miltiorrhiza (Danshen) and Ligusticum chuanxiong (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (Kca) and ATP-related potassium channels (KATP) but not voltage-gated potassium channels (kv) and inward rectifying potassium channels (KIR). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca2+/calmodulin (CaM)-Ca2+/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca2+/CaM-Ca2+/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.

ROLE OF ΚB AND AP-1 TRANSCRIPTION FACTORS IN CHANGES IN NITRIC OXIDE PRODUCTION AND UTILIZATION IN THE HEART OF RATS UNDER CHRONIC FLUORIDE INTOXICATION

Fluorides, being hazardous contaminants of soil and drinking water, can get in excessive amounts into human and animal bodies. This is especially true for regions where the fluoride content in soils is very high, for example, Poltava, Dnipropetrovsk, and Kirovohrad regions in Ukraine. Excessive fluoride intake can change the rate of nitric oxide production. The impact of fluorides on changes in nitric oxide production and metabolism in the heart and the role of redox-sensitive transcription factors in these changes are poorly understood. The aim of this study was to determine the effect of activation of κB transcription factors and activator protein 1 on the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, nitrite and nitrate reductase, arginase, concentration of nitrites, peroxynitrite and nitrosothiols in the heart of rats during chronic fluoride intoxication. Materials and methods. The study was performed on 24 adult male Wistar rats weighing 220-260 grams. Animals were randomly divided into 4 groups of 6 animals in each (control, chronic fluoride intoxication group, κB blockade group and activator protein 1 blockade group). The experiment lasted 30 days. We determined the activity of inducible NO-synthase, constitutive isoforms of NO-synthase, the concentration of peroxynitrite alkali and alkaline earth metals, the concentration of nitrites and nitrosothiols, the activity of nitrite reductase, nitrate reductase and arginase. Results. Chronic fluoride intoxication increases the activity of inducible NO-synthase by 1.74 times, does not affect the activity of constitutive isoforms and reduces the activity of arginase by 35.68% compared with the control group of animals. The concentration of nitrites in the heart of rats increases 1.73 times, peroxynitrite 1.43 times, and the concentration of nitrosothiols doubled. The use of κB transcription factor blockers and activator protein 1 reduces nitric oxide production from NO synthases and reduces the concentrations of all nitric oxide metabolites in the heart of rats under conditions of chronic fluoride intoxication. Conclusions. Activation of κB transcription factors and activator protein 1 during chronic excessive intake of fluoride leads to hyperproduction of nitric oxide in the heart of rats due to increased activity of inducible NO-synthase and nitrite reductases. Excess production of nitric oxide under chronic fluoride intoxication leads to the accumulation of nitrites, peroxynitrite and nitrosothiols in the heart of rats.