Endothelin-1 Production Research Articles

Differences in Endothelial Activation and Dysfunction Induced by Antiphospholipid Antibodies Among Groups of Patients With Thrombotic, Refractory, and Non-refractory Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by pregnancy morbidity or thrombosis and persistent antiphospholipid antibodies (aPL) that bind to the endothelium and induce endothelial activation, which is evidenced by the expression of adhesion molecules and the production of reactive oxygen species (ROS) and subsequent endothelial dysfunction marked by a decrease in the synthesis and release of nitric oxide (NO). These endothelial alterations are the key components for the development of severe pathological processes in APS. Patients with APS can be grouped according to the presence of other autoimmune diseases (secondary APS), thrombosis alone (thrombotic APS), pregnancy morbidity (obstetric APS), and refractoriness to conventional treatment regimens (refractory APS). Typically, patients with severe and refractory obstetric APS exhibit thrombosis and are classified as those having primary or secondary APS. The elucidation of the mechanisms underlying these alterations according to the different groups of patients with APS could help establish new therapies, particularly necessary for severe and refractory cases. Therefore, this study aimed to evaluate the differences in endothelial activation and dysfunction induced by aPL between patients with refractory obstetric APS and other APS clinical manifestations. Human umbilical vein endothelial cells (HUVECs) were stimulated with polyclonal immunoglobulin-G (IgG) from different groups of patients n = 21), including those with primary (VTI) and secondary thrombotic APS (VTII) and refractory primary (RI+), refractory secondary (RII+), and non-refractory primary (NR+) obstetric APS. All of them with thrombosis. The expression of adhesion molecules; the production of ROS, NO, vascular endothelial growth factor (VEGF), and endothelin-1; and the generation of microparticles were used to evaluate endothelial activation and dysfunction. VTI IgG induced the expression of adhesion molecules and the generation of microparticles and VEGF. RI+ IgG induced the expression of adhesion molecules and decreased NO production. RII+ IgG increased the production of microparticles, ROS, and endothelin-1 and reduced NO release. NR+ IgG increased the production of microparticles and endothelin-1 and decreased the production of VEGF and NO. These findings reveal differences in endothelial activation and dysfunction among groups of patients with APS, which should be considered in future studies to evaluate new therapies, especially in refractory cases.

Ovarian Cancer-Driven Mesothelial-to-Mesenchymal Transition is Triggered by the Endothelin-1/β-arr1 Axis

Transcoelomic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and host components. Tumor-derived factors might allow the conversion of mesothelial cells (MCs) into tumor-associated MCs, providing a favorable environment for SOC cell dissemination. However, factors and molecular mechanisms involved in this process are largely unexplored. Here we investigated the tumor-related endothelin-1 (ET-1) as an inducer of changes in MCs supporting SOC progression. Here, we report a significant production of ET-1 from MCs associated with the expression of its cognate receptors, ETA and ETB, along with the protein β-arrestin1. ET-1 triggers MC proliferation via β-arrestin1-dependent MAPK and NF-kB pathways and increases the release of cancer-related factors. The ETA/ETB receptor activation supports the genetic reprogramming of mesothelial-to-mesenchymal transition (MMT), with upregulation of mesenchymal markers, as fibronectin, α-SMA, N-cadherin and vimentin, NF-kB-dependent Snail transcriptional activity and downregulation of E-cadherin and ZO-1, allowing to enhanced MC migration and invasion, and SOC transmesothelial migration. These effects are impaired by either blockade of ETAR and ETBR or by β-arrestin1 silencing. Notably, in peritoneal metastases both ETAR and ETBR are co-expressed with MMT markers compared to normal control peritoneum. Collectively, our report shows that the ET-1 axis may contribute to the early stage of SOC progression by modulating MC pro-metastatic behaviour via MMT.

Abstract 11265: Sex-Specific Response to Endothelin-1 Overexpression Mediates Thoracic Aortic Aneurysm Development

Introduction: Thoracic aortic aneurysms (TAAs) are common diseases associated with high morbidity and mortality. Although TAAs are more common in men, women have worse outcomes and dissect at smaller aortic diameters. Endothelin-1 (ET-1) is the most potent vasoconstrictor in humans, and an important mediator of vascular stiffness and tone. ET-1 levels are increased in patients with TAA, and genetic variants that affect ET-1 production and receptor expression are associated with vascular diseases. Though ET-1 levels do not differ between men and women with TAA, it is unknown if response to ET-1 mediates sex-specific differences. Hypothesis: ET-1 has different effects on vascular stiffness and aortic dissection risk in men and women. Methods: Angiotensin-II (AngII) was delivered subcutaneously at 1 ug/kg/min dose and rate using osmotic pumps for 28 days in endothelial cell-specific ET-1 transgenic (eET-1) and matched WT control mice. We measured blood pressure, aortic stiffness, and aortic size at the end of the study. Results: We observed marked sex-specific ET-1 effects on TAA. Despite elevated blood pressure in all animals that received AngII (Fig1A), we observed increased aortic stiffness in male eET-1 mice (+1.5 m/s eET-1 vs. +0.74 m/s WT) but decreased aortic stiffness in female eET-1 mice (-1.9 m/s eET-1 vs. +0.69 m/s WT), measured by pulse wave velocity (Fig1B). Consistent with these results, aortic diameter was only increased in male eET-1 mice (+0.61 mm eET-1 vs. +0.11 mm WT) but not in female eET-1 mice (+0.06 mm eET-1 vs. +0.11 mm WT) (Fig1C). Significantly, 2 of 3 male eET-1 animals died before 28 days from dissected TAAs, whereas all females survived the length of the study. Conclusions: In the AngII-TAA model, there is a sex dimorphic effect of ET-1 overexpression. Male mice have greater vascular stiffness and develop TAA in response to the same level of ET-1 as in matched female mice. This difference may explain the increased incidence of TAA in men.

The Causal Relationship between Endothelin-1 and Hypertension: Focusing on Endothelial Dysfunction, Arterial Stiffness, Vascular Remodeling, and Blood Pressure Regulation.

Hypertension (HTN) is one of the most prevalent diseases worldwide and is among the most important risk factors for cardiovascular and cerebrovascular complications. It is currently thought to be the result of disturbances in a number of neural, renal, hormonal, and vascular mechanisms regulating blood pressure (BP), so crucial importance is given to the imbalance of a number of vasoactive factors produced by the endothelium. Decreased nitric oxide production and increased production of endothelin-1 (ET-1) in the vascular wall may promote oxidative stress and low-grade inflammation, with the development of endothelial dysfunction (ED) and increased vasoconstrictor activity. Increased ET-1 production can contribute to arterial aging and the development of atherosclerotic changes, which are associated with increased arterial stiffness and manifestation of isolated systolic HTN. In addition, ET-1 is involved in the complex regulation of BP through synergistic interactions with angiotensin II, regulates the production of catecholamines and sympathetic activity, affects renal hemodynamics and water–salt balance, and regulates baroreceptor activity and myocardial contractility. This review focuses on the relationship between ET-1 and HTN and in particular on the key role of ET-1 in the pathogenesis of ED, arterial structural changes, and impaired vascular regulation of BP. The information presented includes basic concepts on the role of ET-1 in the pathogenesis of HTN without going into detailed analyses, which allows it to be used by a wide range of specialists. Also, the main pathological processes and mechanisms are richly illustrated for better understanding.

Pathophysiological Association between Diabetes Mellitus and Endothelial Dysfunction.

Endothelial dysfunction plays a critical role in atherosclerosis progression, leading to cardiovascular complications. There are significant associations between diabetes mellitus, oxidative stress, and endothelial dysfunction. Oxidative stress is increased by chronic hyperglycemia and acute glucose fluctuations induced by postprandial hyperglycemia in patients with diabetes mellitus. In addition, selective insulin resistance in the phosphoinositide 3-kinase/Akt/endothelial nitric oxide (NO) synthase pathway in endothelial cells is involved in decreased NO production and increased endothelin-1 production from the endothelium, resulting in endothelial dysfunction. In a clinical setting, selecting an appropriate therapeutic intervention that improves or augments endothelial function is important for preventing diabetic vascular complications. Hypoglycemic drugs that reduce glucose fluctuations by decreasing the postprandial rise in blood glucose levels, such as glinides, α-glucosidase inhibitors and dipeptidyl peptidase 4 inhibitors, and hypoglycemic drugs that ameliorate insulin sensitivity, such as thiazolidinediones and metformin, are expected to improve or augment endothelial function in patients with diabetes. Glucagon-like peptide 1 receptor agonists, metformin, and sodium-glucose cotransporter 2 inhibitors may improve endothelial function through multiple mechanisms, some of which are independent of glucose control or insulin signaling. Oral administration of antioxidants is not recommended in patients with diabetes due to the lack of evidence for the efficacy against diabetic complications.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Relationship between endothelin and nitric oxide pathways in the onset and maintenance of hypertension in children and adolescents

The mechanisms that regulate blood pressure are numerous and complex; one mechanism that plays an important role in this scenario is represented by the balance between the vasoconstrictor effect of endothelin-1 and the vasodilator effect of nitric oxide. While there is agreement on the fact that increased endothelin-1 activity and decreased nitric oxide bioavailability are present in hypertensive adults, the situation is less clear in children and adolescents. Not all studies agree on the finding of an increase in plasma endothelin-1 levels in hypertensive children and adolescents; in addition, the picture is often confused by the concomitant presence of obesity, a condition that stimulates the production of endothelin-1. Furthermore, there is recent evidence that, in younger obese and hypertensive subjects, there is an overproduction of nitric oxide, rather than a reduction. This condition may change over time, causing endothelial dysfunction due to a reduced availability of nitric oxide in hypertensive adolescents. The purpose of this review is to address the main biochemical and pathophysiological aspects of endothelin and nitric oxide involvement in hypertension and to summarize the available scientific evidence on their role in the onset and maintenance of high blood pressure in children and adolescents.

Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

Invitro effect of pine bark extract on melanin synthesis, tyrosinase activity, production of endothelin-1, and PPAR in cultured melanocytes exposed to Ultraviolet, Infrared, and Visible light radiation.

French maritime pine bark (Pinus pinaster) extract (PBE), the registered trade name of which is Pycnogenol® , has been studied for its depigmenting action due to its antioxidant, anti-inflammatory, and anti-melanogenic activity. However, the mechanisms through which PBE are still not fully clear. Evaluate the impact of PBE on four in vitro parameters closely associated with cutaneous pigmentation, including melanin synthesis, tyrosinase activity, endothelin-1 (ED1), and production of peroxisome proliferator-activated receptor α, δ, and γ (PPAR α, δ, and γ), by studying the modulation of action of ultraviolet radiation A (UVA)/ultraviolet radiation B (UVB), infrared-A (IR-A), visible light (VL), and association of UVA/UVB, IR-A, and VL (ASS). Human melanocytes were incubated in a dry extract solution of PBE, exposed to UVA/UVB, IR-A, VL, and ASS for subsequent quantification of melanin, ED1, and PPAR α, δ, and γ. The effects of PBE on inhibition of tyrosinase activity were also performed by monophenolase activity assay. UVA/UVB, IR-A, VL, and ASS radiation caused significant increases in the synthesis of melanin, ED1, and PPAR α, δ, and γ when compared to baseline control. However, PBE significantly reduced the production of melanin, ED1, and PPAR α, δ, and γ, as well as reducing about 66.5% of the tyrosinase activity. PBE reduces in vitro melanin production by downregulating tyrosinase and reducing pigmentation-related mediators, such as ED1 and PPAR α, δ, and γ, therefore contributing to the inhibition of pathways associated with skin hyperpigmentation.

Calcification of aortic valvular interstitial cells induced by endothelin receptor blockers

Calcified aortic stenosis (AS) is the most common acquired valvulopathy for which there is still no pharmacological treatment. Endothelin-1 (ET-1) is not only a powerful vasoconstrictor but also a pro-inflammatory and pro-fibrotic peptide whose role in AS remains unclear. The aim of this study was to characterize the role of ET-1 in the aortic valve calcification. Valvular endothelial cells (VECs), isolated from human aortic valves, were cultured in a cell perfusion system to assess ET-1 production in different fluid flow shear stress conditions. In addition, valvular interstitial cells (VICs) were cultured in a pro-calcifying culture medium (inorganic phosphate: 2 mM) with or without small-molecule and monoclonal antibody ET A and ET B receptor antagonists during 10 days. Calcium content was assessed using an o-cresolphtalein-based assay and red alizarin staining. VEC prepro-ET-1 and VIC osteogenic mRNA expression levels were evaluated by RTqPCR. Visualization of VIC ET A and ET B receptor was performed by immunohistochemistry. Turbulent shear stress, mimicking the flow conditions suffered by the valve at the aortic side that is the most prone to calcify, increased VEC prepro-ET-1 mRNA expression level and ET-1 release compared to laminar shear stress. Unexpectedly, calcium content and alizarin red staining of VICs were increased after blockade of ET B receptor and this effect was potentiated by concomitant blockade of ET A receptor. The mRNA expression of osteopontin, RUNX2, and BMP2 was similarly increased by ET B blockade. A perinuclear localization of ET A and ET B receptors in VICS was observed, suggesting a direct implication in the regulation of calcification genes. These results support a protective role of the endothelin system against the development of AS. Further studies are warranted to characterize the intracellular pathways and to confirm these results in ex vivo and in vivo models.

Endothelin ETB Receptor-Mediated Astrocytic Activation: Pathological Roles in Brain Disorders.

In brain disorders, reactive astrocytes, which are characterized by hypertrophy of the cell body and proliferative properties, are commonly observed. As reactive astrocytes are involved in the pathogenesis of several brain disorders, the control of astrocytic function has been proposed as a therapeutic strategy, and target molecules to effectively control astrocytic functions have been investigated. The production of brain endothelin-1 (ET-1), which increases in brain disorders, is involved in the pathophysiological response of the nervous system. Endothelin B (ETB) receptors are highly expressed in reactive astrocytes and are upregulated by brain injury. Activation of astrocyte ETB receptors promotes the induction of reactive astrocytes. In addition, the production of various astrocyte-derived factors, including neurotrophic factors and vascular permeability regulators, is regulated by ETB receptors. In animal models of Alzheimer’s disease, brain ischemia, neuropathic pain, and traumatic brain injury, ETB-receptor-mediated regulation of astrocytic activation has been reported to improve brain disorders. Therefore, the astrocytic ETB receptor is expected to be a promising drug target to improve several brain disorders. This article reviews the roles of ETB receptors in astrocytic activation and discusses its possible applications in the treatment of brain disorders.

Angiotensin I-Converting enzyme (ACE) inhibitory and dipeptidyl Peptidase-4 (DPP-Ⅳ) inhibitory activity of umami peptides from Ruditapes philippinarum

The functional activity of umami peptides after digestion is poorly known. We studied the dipeptidyl peptidase-4 (DPP- IV) and angiotensin I-converting enzyme (ACE) inhibition activity of 14 umami peptides from Ruditapes philippinarum in vivo and in vitro. GRVSNCAA (IC50 = 57.93 μmol/L) and TYLPVH (IC50 = 1.37 μmol/L) exhibited noncompetitive ACE inhibitory activity in vitro, and GPAGPAGPR showed DPP-IV inhibitory activity (IC50 = 160.5 μmol/L). The peptides also decreased systolic blood pressure and reduced the blood sugar level in the oral glucose tolerance test and the single blood pressure test. To understand the hypoglycemic and hypotensive mechanism of peptides, we conducted cell experiments with HepG2 and human umbilical vein endothelial cells. GRVSNCAA and TYLPVH significantly (p < 0.01) promoted nitric oxide secretion and reduced endothelin-1 production. GPAGPAGPR exerted hypoglycemic activity by increasing the content of hexokinase and pyruvate kinase.These results suggest that some umami peptides have the potential to provide human health benefits.

Abstract MP15: Hypoxia and Endothelin-1 in Microglial Cell Activation

Oxidative stress and inflammatory responses play critical roles in hypoxic-ischemic brain injury. Microglial cells are rapidly activated in response to injury and stressful stimuli, including hypoxia. Endothelin-1 (ET-1) is a potent vasoconstrictor that has been associated with cerebrovascular diseases. Hypoxia stimulates endothelial ET-1 production. However, the role of ET-1 in microglia under hypoxia is not clear. The aim of this project was to characterize the effect of hypoxia in a human microglial cell line, HMC3. We induced hypoxia using a chamber (1% O2, 5% CO2, and 92 % N2) at 37 °C for 4h, 24h, and 48h. MUSE Oxidative Stress Assay was performed to measure reactive oxygen species (ROS) formation, ELISA to determine TNF, IL-6, and ET-1 levels, qPCR to measure gene expression of ET-1, and immunofluorescence staining to visualize and compare the presence of ET-1 in hypoxic and control group cells. It was observed that, when compared to normoxic HMC3 cells, hypoxic HMC3 exposure significantly increased the ROS by a factor of 2.5 (p&lt;0.001, n=3), the pro-inflammatory cytokine TNF-α increased 3.8 times (p&lt;0.01, n=4), and IL-6 increased by a factor of 1.6 (p&lt;0.01, n=4). In addition, hypoxia stimulates ET-1 gene expression 5.0-fold (p&lt;0.001, n=4) and increased protein production 1.3 times (p&lt;0.01, n=4). Consequently, treatment with ET-1 increased the amount of ROS, TNF-α, and IL-6 in HMC3 cells by a factor of 1.4 (p&lt;0.05, n=4), 1.6 (p&lt;0.001, n=4), and 1.9 (p&lt;0.05, n=4), respectively. All these events were blocked by ET-1 receptor A (ETRA) antagonist, BQ123. Our results suggest that hypoxic conditions create a cycle of microglial cell activation leading to increased ROS and ET-1 production that further stimulate microglial cells. Thus, we posit that the ET-1 receptor blockade represents a promising therapeutic approach to regulate microglial cell responses in hypoxic-ischemic brain injury.

From Green Technology to Functional Olive Oils: Assessing the Best Combination of Olive Tree-Related Extracts with Complementary Bioactivities

Our aim was to assess the combination of olive tree-related extracts with the most favorable profile of in vitro bioactive properties. We tested the antioxidant (increment of low-density lipoprotein resistance against oxidation), vasoactive (promotion of nitric oxide release and decrease of endothelin-1 production in human umbilical vein endothelial cells), anti-inflammatory (decrease of the endothelial production of vascular cell adhesion molecule-1), and antithrombotic (reduction of the endothelial release of plasminogen activator inhibitor-1) capacities of six phenolic extracts and three triterpenic acid solutions (Ps and Ts, respectively). We tested extracts alone and in combination, at nutritional (Ps: 0.05–0.5 μmol/L; Ts: 0.001–0.1 μmol/L) and nutraceutical doses (Ps: 1–10 μmol/L; Ts: 0.25–10 μmol/L). The combination of Ps rich in 3,4-dihydroxyphenylglycol (76%, P2), hydroxytyrosol (95%, P3), and oleuropein (70%, P4) (final nutritional concentration: 0.15 μmol/L; final nutraceutical concentration: 3 μmol/L) was the best in order to prepare functional products and nutraceuticals with cardioprotective properties, despite the fact that the isolated extract with the greatest in vitro properties was P5 (75% oleocanthal), suggesting a potential synergistic effect among different olive components.

Obesity-related changes in the vascular actions of insulin

Over the past 2 to 3 decades, research has focused on the changes in the vascular effects of insulin occurring in insulin resistant states like obesity. Consistent evidence indicates that obesity results in reduced endothelial release of nitric oxide in response to insulin, associated with concomitant enhancement in the production of endothelin-1. More recent work has pointed toward reduced vascular permeability and changes in the physical-chemical characteristics of the perivascular extracellular matrix as additional mechanisms of impaired insulin sensitivity in obesity. All these perturbations are important, because they contribute to impaired delivery of insulin itself and metabolic substrates to the target tissues and may play a role in the development of both diabetes and vascular damage. This review will describe the physiological vascular actions of insulin and their changes in obesity, focusing on some established pathophysiological determinants of the derangement of vascular insulin signaling, such as the lipid overflow from expanded fat depots and signals originating from inflamed obese adipose tissue (both distant and perivascular). Also, it will outline novel evidence underscoring the contribution of dysregulated adipokine secretion and changes in intestinal permeability and gut microbiome. Finally, it will touch upon some unresolved issues, such as the potential role of vascular insulin resistance in obesity-driven adipose tissue remodeling, and will discuss perspectives for future studies, regarding in particular possible therapeutic strategies with translational implications for the patients care.

Disorders of microcirculation in a comorbid patient

Background. Diabetes mellitus (DM) is a global epidemic. According to the experts, by 2045 the number of patients with diabetes will rise by 48 %. Patients with diabetes are characterized by the high cardiovascular mortality and a significant reduction in life expectancy. Almost half of the deaths associated with diabetes are due to cardiovascular diseases (CVD). Patients with diabetes often have concomitant pathology (hypertension, dyslipidemia, obesity), which increases the cardiovascular risk.&#x0D; Objective. To describe microcirculation disorders in patients with comorbid conditions.&#x0D; Materials and methods. Analysis of literature sources on this topic.&#x0D; Results and discussion. Type 2 diabetes (DM2) is a multifaceted disease that has a number of cardiovascular, metabolic and renal complications. The links of the pathogenesis of cardiovascular complications of DM2 include dyslipidemia, systemic inflammation, insulin resistance, autonomic imbalance, and endothelial dysfunction (ED). ED leads to vasoconstriction, increased chronic inflammation, increased vascular permeability and hypercoagulation, which ultimately causes micro- and macroangiopathy. Neurological complications of diabetes are also mediated by ED and microangiopathies, which lead to nerve hypoxia with a decrease in conductivity velocity. In recent years, the literature has been actively discussing the syndrome of early vascular aging – premature and accelerated development of structural and functional age-related changes in blood vessels. Microcirculation disorders that accompany diabetes, CVD and their complications are mediated by nitric oxide (NO) imbalance. NO reduces the contractility and proliferation of smooth muscle cells, platelet aggregation, endothelin production, adhesion of monocytes and platelets, and oxidation of low-density lipoproteins. In humans, NO is produced from L-arginine. Increased L-arginase activity and decreased NO-synthase activity lead to a decrease in NO content and to the development of ED, atherosclerosis, and decreased insulin sensitivity. L-arginine as a substrate of NO improves the functional state of the endothelium, reduces the manifestations of oxidative stress, reduces the level of pro-inflammatory cytokines and adhesion molecules, inhibits platelet aggregation, reduces insulin resistance. It should be noted that the administration of 4.2 g of L-arginine in DM is not enough. In clinical studies, doses of 6.4-9 g per day were found to be effective in diabetes and obesity. Therefore, it is advisable to use Tivortin-200 (“Yuria-Pharm”), which contains 8.4 g of L-arginine in one vial.&#x0D; Conclusions. 1. Patients with diabetes are characterized by the high cardiovascular mortality and a significant reduction in life expectancy. 2. The links of the pathogenesis of cardiovascular complications of DM2 include dyslipidemia, systemic inflammation, insulin resistance, autonomic imbalance, and ED. 3. Disorders of microcirculation that accompany diabetes, CVD and their complications, are mediated by NO imbalance. 4. L-arginine as a substrate of NO improves the functional state of the endothelium, reduces the manifestations of oxidative stress, reduces the level of pro-inflammatory cytokines and adhesion molecules, inhibits platelet aggregation, reduces insulin resistance. 5. In diabetes and obesity, it is advisable to use Tivortin-200, which contains 8.4 g of L-arginine in one vial.

Рівні ендотеліну-1 та тестостерону в крові хворих на цукровий діабет 1-го та 2-го типів

Із цукровим діабетом (ЦД) пов’язана ендотеліальна дисфункція (ЕД) — одна з ключових патологічних подій у розвитку хронічних діабетичних ускладнень. Важливим ефектом ЕД є збільшення продукції та біологічної активності ендотеліну (ЕТ). ET‑1, який виробляється переважно в ендотелії, є основною серцево-судинною ізоформою ендотелінової системи. ЦД — одне із захворювань, пов’язаних із патологічно підвищеним рівнем ЕТ. Отримані докази того, що андрогени можуть грати певну роль у визначенні специфічного для статі артеріального тиску. Чоловіки мають більш високий ризик розвитку ішемічної хвороби серця або гіпертонічної хвороби порівняно з жінками в пременопаузі. Мета — визначення концентрації ЕТ‑1 та тестостерону (Т) в крові хворих на цукровий діабет першого (ЦД1) та другого (ЦД2) типів із різним індексом маси тіла (ІМТ) та різних вікових категорій. Матеріал і методи. Рівень ET‑1 оцінювали методом ІФА в 103 осіб: 17 здорових осіб та 86 пацієнтів із ЦД1 і ЦД2. Для визначення концентрації ET‑1 використовували набір Endotelin (1-21) EIA. Загальний Т визначали за допомогою набору EIA‑1559. Результати. У всіх пацієнтів із діабетом рівень ЕТ‑1 у крові був значно вищим, ніж в осіб контрольної групи. Різниця в кількості ЕТ‑1 у крові пацієнтів із ЦД1 і ЦД2 була невірогідною. Рівень ЕТ‑1 у чоловіків був вищим, ніж у жінок у середньому на 37%. У групі з вищим індексом маси тіла кількість ET‑1 була вищою на 71%, а Т — нижчою на 25%. Рівень ЕТ‑1 у віковій групі до 50 років був нижчим, ніж у старшій групі для обох типів ЦД (123% для ЦД1 і 148% для ЦД2), а кількість Т у хворих на ЦД2 — вища майже на 20%. У групі хворих на ЦД2 з рівнем Т&lt;3 нг/мл концентрація ЕТ‑1 у крові була вищою, ніж у хворих із рівнем Т&gt;3 нг/мл (відповідно 0,550 фмоль/мл і 0,392 фмоль/мл). Висновки. У чоловіків, хворих на ЦД2 з ожирінням спостерігається підвищення концентрації ЕТ‑1 і зниження рівня Т. Ці показники, напевно, безпосередньо не пов’язані з вмістом глікованого гемоглобіну (Нв1Ас). Із віком рівень ЕТ‑1 у крові хворихна ЦД зростає, а рівень Т знижується. Ці процеси можуть бути взаємопов’язаними. Концентрація ЕТ‑1 у крові в чоловіків, хворих на ЦД обох типів вірогідно вища, ніж у жінок.

POLYMORPHISM OF AGT, AGT2R1 AND NOS3 GENES AS A RISK FACTOR FOR IMBALANCE IN VASOACTIVE FACTORS

The aim of the study was to identify associations between allelic variants of AGT, AGT2R1 and NOS3 genes with production of endo-thelin-1 (ET-1) and nitric oxide (NO) in young residents of the Russian North without a history of cardiovascular diseases. Methods. The sample consisted of 286 young adults (116 females and 170 males) born in the Arkhangelsk region. Single nucleotide polymorphisms in the AGT, AGT2R1 and NOS3 genes were determined by polymerase chain reaction (PCR). Plasma NO oxide and ET-1 levels were measured. Results. T704C polymorphism in AGT gene was not associated with the studied vasoactive endothelial factors. However, vasoactive factors production in homozygotes with mutant T/T alleles of C521T polymorphism in the same gene is shifted towards vasoconstrictive factors. The level of ET-1 in the group with T/T genotype was higher than in the groups with C/T and C/C genotypes (p = 0.006 and p = 0.04, respectively). Homozygotes with mutant C alleles of AGT2R1 gene had higher concentration of ET-1 as compared to homozygotes with wild-type A alleles (p = 0.012). Young men with 786 C/C of NOS3 gene had higher plasma concentration of ET-1 than individuals with C/T (p = 0.01) and T/T (p = 0.07). ET-1 was higher in the group of girls homozygotes with mutant C alleles, however no statistically significant differences were found. Conclusions. Our results suggest that the risk of imbalance in vasoactive factors in young healthy residents of the Russian North is associated with the increase in ET-1 production level, but not with NO level. The presence of 521 T/T genotype in AGT gene, 1666C/C in AGT2R1 gene and -786C/C in NOS3 gene increases the risk of vasoactive factors imbalance development towards vasoconstriction.

Biological effects of inhaled hydraulic fracturing sand dust. III. Cytotoxicity and pro-inflammatory responses in cultured murine macrophage cells

Cultured murine macrophages (RAW 264.7) were used to investigate the effects of fracking sand dust (FSD) for its pro-inflammatory activity, in order to gain insight into the potential toxicity to workers associated with inhalation of FSD during hydraulic fracturing. While the role of respirable crystalline silica in the development of silicosis is well documented, nothing is known about the toxicity of inhaled FSD. The FSD (FSD 8) used in these studies was from an unconventional gas well drilling site. FSD 8was prepared as a 10 mg/ml stock solution in sterile PBS, vortexed for 15 s, and allowed to sit at room temperature for 30 min before applying the suspension to RAW 264.7cells. Compared to PBS controls, cellular viability was significantly decreased after a 24 h exposure to FSD. Intracellular reactive oxygen species (ROS) production and the production of IL-6, TNFα, and endothelin-1 (ET-1) were up-regulated as a result of the exposure, whereas the hydroxyl radical (.OH) was only detected in an acellular system. Immunofluorescent staining of cells against TNFα revealed that FSD 8 caused cellular blebbing, and engulfment of FSD 8 by macrophages was observed with enhanced dark-field microscopy. The observed changes in cellular viability, cellular morphology, free radical generation and cytokine production all confirm that FSD 8 is cytotoxic to RAW 264.7 cells and warrants future studies into the specific pathways and mechanisms by which these toxicities occur.

Mitigative Effect of Sipjeondaebo-tang on RhoA Activation in Cold-Exposed Vascular Cells

Objective To investigate the vasodilatory effect of Sipjeondaebo-tang by inhibiting RhoA activity in vascular cells during cold exposure. Methods Human vascular endothelial cells and pericytes were pretreated with Sipjeondaebo-tang for 30 min, followed by incubation at 37 °C (control) or 25 °C (cold exposure) for 30 min. Activation of endothelin-1-mediated RhoA in pericytes was assessed by pretreating the cells with Sipjeondaebo-tang for 30 min, followed by incubation with endothelin-1 at 37 °C for 30 min. Western blotting was performed to measure the expression of active RhoA. Endothelin-1 and nitric oxide release from endothelial cells was examined with enzyme-linked immunosorbent assay kits. The formation of stress fibers and focal adhesion complexes was analyzed by immunocytochemistry. Results Cold treatment activated RhoA in both pericytes and vascular endothelial cells, whereas Sipjeondaebo-tang treatment inhibited this activation. Sipjeondaebo-tang treatment also reversed the cold-mediated production of endothelin-1 and nitric oxide. Cold exposure promoted the formation of stress fibers and focal adhesion complexes by increasing the expression of phospho-focal adhesion complex kinase, whereas Sipjeondaebo-tang treatment suppressed this response. Conclusions These findings suggested that Sipjeondaebo-tang inhibits cold-induced RhoA activation and its related pathway components, including endothelin-1 and nitric oxide, in vascular cells. Therefore, Sipjeondaebo-tang could be beneficial for the treatment of Raynaud’s phenomenon. Keywords: Raynaud’s phenomenon, cold, rhoA, endothelin-1, nitric oxide, Sipjeondaebo-tang