Endothelial NO Research Articles

Pivotal trial of low-intensity pulsed ultrasound therapy for early Alzheimer's disease: Rationale and design

Background There are lines of evidence suggesting that cerebral microcirculatory dysfunction is involved in the pathogenesis of Alzheimer's disease (AD). We have developed a low-intensity pulsed ultrasound (LIPUS) therapy that upregulates endothelial NO synthase with therapeutic angiogenesis. We demonstrated that the LIPUS therapy ameliorates cognitive declines in mouse models of AD and tended to do so in patients with early AD (mild AD and mild cognitive impairment due to AD) in the pilot trial. Thus, the Japanese government has designated our LIPUS device as the first breakthrough medical device in Japan. Objective We are performing a pivotal clinical trial (LIPUS-AD) to finally address the efficacy and safety of our LIPUS therapy in patients with early AD in Japan. Methods LIPUS-AD is a randomized, double-blind, placebo-controlled trial, in which a total of 220 patients with early AD, who are positive for amyloid-β (Aβ) PET, will be randomized in a 1:1 fashion. The LIPUS therapy is performed for the whole brain for one hour 3 times a week as one session under the special conditions (32 cycles, 0.5 MHz, 0.25 W/cm2). It is performed for 6 sessions with 3-month intervals in the LIPUS group for 72 weeks, while the placebo group receives placebo therapy. Before and at 72 weeks of the trial, all subjects undergo brain Aβ PET and MRI and 9 cognitive functions tests. The primary efficacy endpoint is the changes in ADAS-J-cog-14 scores from baseline to 72 weeks. Conclusions LIPUS-AD addresses efficacy and safety of the LIPUS therapy in patients with early AD. Clinical Trial Gov. No.: NCT05983575

Tobacco cigarette smoking induces cerebrovascular dysfunction followed by oxidative neuronal injury with the onset of cognitive impairment

While chronic smoking triggers cardiovascular disease, controversy remains regarding its effects on the brain and cognition. We investigated the effects of long-term cigarette smoke (CS) exposure (CSE) on cerebrovascular function, neuronal injury, and cognition in a novel mouse exposure model. Longitudinal studies were performed in CS or air-exposed mice, 2 hours/day, for up to 60 weeks. Hypertension and carotid vascular endothelial dysfunction (VED) occurred by 16 weeks of CSE, followed by reduced carotid artery blood flow, with oxidative stress detected in the carotid artery, and subsequently in the brain of CS-exposed mice with generation of reactive oxygen species (ROS) and secondary protein and DNA oxidation, microglial activation and astrocytosis. Brain small vessels exhibited decreased levels of endothelial NO synthase (eNOS), enlarged perivascular spaces with blood brain barrier (BBB) leak and decreased levels of tight-junction proteins. In the brain, amyloid-β deposition and phosphorylated-tau were detected with increases out to 60 weeks, at which time mice exhibited impaired spatial learning and memory. Thus, long-term CSE initiates a cascade of ROS generation and oxidative damage, eNOS dysfunction with cerebral hypoperfusion, as well as cerebrovascular and BBB damage with intracerebral inflammation, and neuronal degeneration, followed by the onset of impaired cognition and memory.

Anti-inflammatory activity and selective inhibition of iNOS gene expression by a polyherbal formulation

Link Samahan® (LS), a product of Link Natural Products (Pvt) Limited, Sri Lanka contains extracts of 14 medicinal plants. It is used as a prophylactic against cold and cold related symptoms. It has immunomodulatory activity, specifically enhancing the humoral immune response. To investigate the anti-inflammatory activity (AIA) of LS and related mechanisms. In vivo AIA was assessed by the inhibition of carrageenan-induced rat paw-edema. Mechanisms of AIA were assessed in vitro by the inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and expression of inducible nitric oxide synthase (iNOS) gene using rat peritoneal cells. LS showed an increasing inhibition in rat paw-edema up to 5h compared to the biphasic pattern exhibited by the reference drug, Indomethacin. Inhibition by LS at the 1st and 3rd-5th hours (44.7% and 73.0-74.6%) was comparable to indomethacin (47.4% and 83.6-76.5%; p>0.05) whereas at the 2nd hour, LS exhibited a significantly higher inhibition compared to indomethacin (66.7% vs 28.6%; p<0.05). LS treatment significantly inhibited the ROS (superoxide; 47.2±0.86%; p<0.05) and RNS (nitrite; 54.0±0.40%; p<0.05) production in rat peritoneal cells. Further, specific inhibition of iNOS gene expression in rat peritoneal cells resulted in a significant reduction in RNS production whereas LS had lesser or no inhibitory effect on endothelial NOS and neuronal NOS gene expression. LS has potent anti-inflammatory activity and selective inhibition of iNOS activity. AIA of LS shown here supports its use as a prophylactic against cold and cold related symptoms.

Intrauterine growth restriction does not lead to pronounced changes in the regulation of arterial contractile responses in rats in the early postnatal period

Intrauterine growth retardation (IUGR) is one of the most common pathologies of pregnancy. As a result of this pathology, the functioning of many systems, including the cardiovascular system, is disrupted. In adult animals who have suffered IUGR, the contribution of procontractile mechanisms regulating vascular tone (for example, the Rho-kinase signaling pathway) increases, and the contribution of anticontractile mechanisms (for example, endothelial NO), on the contrary, decreases, which can lead to vasospasm and impaired blood supply to organs. Since NO and Rho-kinase have a pronounced vasomotor role in early postnatal ontogenesis, the purpose of this work was to assess the influence of IUGR on the contribution of these mechanisms to the regulation of arterial contractile responses in early postnatal ontogenesis. IUGR was modeled by limiting the amount of food consumed by females (by 50%) from the 11th day of pregnancy until birth. In offspring aged 11 - 12 days, the reactions of the isolated saphenous artery were studied in isometric mode, and the content of mRNA and proteins of interest in this artery was also assessed. IUGR did not lead to a change in the reactivity of the arteries of the offspring to the α1-adrenergic receptor agonist methoxamine. The increase in contractile responses to methoxamine in the presence of the NO-synthase inhibitor L-NNA, as well as the expression levels of eNOS (mRNA and protein) and arginase-2 (mRNA) were not changed in the arteries of IUGR rats, while the sensitivity of the arteries to the exogenous NO donor DEA /NO was higher in IUGR compared to control rat pups. Despite the relatively low content of RhoA and Rho-kinase II proteins in the arterial tissue of rat pups from the IUGR group, the decrease in contractile responses under the influence of the Rho-kinase inhibitor Y27632 was equally pronounced in the arteries of rat pups from two experimental groups. Thus, IUGR, caused by maternal nutritional restriction during pregnancy, does not lead to pronounced changes in the regulation of systemic vascular tone in the early postnatal period.

Semaglutide Improves Myocardial Perfusion and Performance in a Large Animal Model of Coronary Artery Disease.

Coronary artery disease is the leading cause of death worldwide. It imposes an enormous symptomatic burden on patients, leaving many with residual disease despite optimal procedural therapy and up to one-thirds with debilitating angina amenable neither to procedures, nor to current pharmacological options. Semaglutide (SEM), a GLP-1 (glucagon-like peptide 1) agonist originally approved for management of diabetes, has garnered substantial attention for its capacity to attenuate cardiovascular risk. Although subgroup analyses in patients indicate promise, studies explicitly designed to isolate the impact of SEM on the sequelae of coronary artery disease, independently of comorbid diabetes or obesity, are lacking. Yorkshire swine (n=17) underwent placement of an ameroid constrictor around the left circumflex coronary artery to induce coronary artery disease. Oral SEM was initiated postoperatively at 1.5 mg and scaled up in 2 weeks to 3 mg in treatment animals (n=8) for a total of 5 weeks, while control animals (n=9) received no drug. All then underwent myocardial harvest with acquisition of perfusion and functional data using microsphere injection and pressure-volume loop catheterization. Immunoblotting, immunohistochemistry, and immunofluorescence were performed on the most ischemic myocardial segments for mechanistic elucidation. SEM animals exhibited improved left ventricular ejection fraction, both at rest and during rapid myocardial pacing (both P<0.03), accompanied by increased perfusion to the most ischemic myocardial region at rest and during rapid pacing (both P<0.03); reduced perivascular and interstitial fibrosis (both P<0.03); and apoptosis (P=0.008). These changes were associated with increased activation of the endothelial-protective AMPK (AMP-activated protein kinase) pathway (P=0.005), coupled with downstream increases in eNOS (endothelial NO synthase; P=0.014). This study reveals the capacity of oral SEM to augment cardiac function in the chronically ischemic heart in a highly translational large animal model, likely through AMPK-mediated improvement in endothelial function and perfusion to the ischemic myocardium.

Polymorphism of genes involved in the regulation of blood pressure in elderly residents of the Arkhangelsk region

BACKGROUND: Living in the northern climate is associated with increased cardiovascular stress, which highlights the necessity for the study of candidate genes associated with the risk of cardiovascular diseases in both the native population and newcomers. Polymorphic loci of the renin-angiotensin system, NO-synthase, and endothelin-1 system genes have been identified as contributors to cardiovascular dysfunction and age-related blood pressure shifts. It is therefore crucial to assess the genetic polymorphism in the elderly population. AIM: To compare frequencies of gene alleles and genotypes involved in blood pressure regulation, including angiotensinogen AGT (rs699 and rs4762), angiotensin 2 type 1 receptor AGTR1 (rs5186), angiotensin converting enzyme ACE (rs4646994), endothelial NO synthase NOS3 and endothelin-1 EDN1 (rs5370) genes, in the native and non-native elderly population of the Arkhangelsk region. MATERIALS AND METHODS: A cross-sectional study was conducted in a random sample of Arkhangelsk residents between the ages of 60 and 74 years (N=604, with 36.4% of males). The molecular genetic analysis was conducted to determine the alleles and genotypes of six genes that are involved in blood pressure regulation. The Stata 18.0 software was used to assess the deviations of empirical genotype distributions from the predicted Hardy–Weinberg equilibrium and to compare the empirical distributions between the study groups. RESULTS: Alleles associated with the risk of cardiovascular diseases were minor in the study population. The genotype frequency distributions for the analyzed genetic variants were consistent with the Hardy–Weinberg principle, with the exception of the T704C variant of the AGT gene (rs699) in the native participants. The allele and genotype frequency distributions in the study sample were found to be similar to those reported worldwide and in European Russia. One exception was AGTR1 gene A1166C frequencies, with their 95% confidence intervals falling below the global level for both native and non-native elderly residents of the Arkhangelsk region. This may suggest that this allele is a selection variant associated with adaptation to the climate of the northern regions. CONCLUSION: The genetic polymorphism in blood pressure regulation was found to be similar between the native and non-native populations of the Arkhangelsk region. However, the AGTR1 gene A1166C frequency among the native population and newcomers was found to be lower than that observed globally.

The endothelial NO synthase content in the blood serum of patients with coronavirus disease (COVID-19) with pneumonia in association with hemostatic parameters depending on the clinical course and its prognostic significance

Aim: to investigate the content of eNOS in the blood serum of patients with coronavirus disease (COVID-19) with pneumonia in association with hemostatic parameters and to determine its prognostic value in assessing the risk of oxygen dependence and death. Material and methods. There were 123 patients with COVID-19 with pneumonia under observation. All patients were examined and treated in accordance with the Order of the Ministry of Health of Ukraine No. 722 dated 28.03.2020. The eNOS content in the patients’ serum was determined by enzyme-linked immunosorbent assay. Results. It was found that the content of eNOS in the blood serum of patients with COVID-19 with pneumonia at the time of hospitalization was 9.0 [7.0; 12.0] days lower (p &lt; 0.001) than in healthy subjects. The development of oxygen dependence in patients with COVID-19 with pneumonia was accompanied by worsening of endothelial dysfunction and procoagulant changes, which was confirmed by a decrease in the content of eNOS in the blood serum (p &lt; 0.001), an increase in the level of fibrinogen (p &lt; 0.05) and D-dimer (p &lt; 0.05). The threshold level of eNOS in the blood serum ≤327.09 pg/ml (AUC = 0.861, p &lt; 0.001) was predictive of the onset of oxygen dependence. In patients with COVID-19 with pneumonia who subsequently died, at the time of hospitalization, eNOS levels were lower (p &lt; 0.001) than in patients who recovered, which was combined with a higher level of D-dimer (p &lt; 0.05) and its more frequent increase (p = 0.04) compared with patients who recovered. The eNOS content in the blood serum of patients with COVID-19 with pneumonia was correlated (p &lt; 0.05) not only with the lethal outcome of the disease, but also with the formation of thrombotic complications, which occurred more often in patients with COVID-19 with pneumonia in the event of an adverse outcome (p = 0.0001). The threshold level of eNOS in the blood serum ≤201.75 pg/ml (AUC = 0.892, p &lt; 0.001) was indicative of a high probability of death. Conclusion. The eNOS content in the blood serum of patients with COVID-19 pneumonia at the time of hospitalization is lower (p &lt; 0.001) than in healthy individuals, and the degree of its decrease depends on the severity of the disease, the development of oxygen dependence and the subsequent outcome of this disease. Limiting levels of eNOS in the blood serum of patients with COVID-19 pneumonia, which are important for predicting the risk of developing oxygen dependence and fatal outcome of the disease, have been established.

INFLUENCE OF SODIUM GLUTAMATE ON REACTIVE OXYGEN AND NITROGEN SPECIES PRODUCTION IN KIDNEY TISSUES OF RATS UNDER ACUTE DESYNCHRONIZATION AND LIPOPOLYSACCHARIDE-INDUCED SYSTEMIC INFLAMMATORY RESPONSE

The study investigated the effect of sodium glutamate on the production of reactive oxygen and nitrogen species (ROS/RNS) in kidney tissues of rats under conditions of acute desynchronization (AD) and lipopolysaccharide (LPS)-induced systemic inflammatory response (SIR) and, thus, contributed to a deeper understanding of the mechanisms involved in the development of renal dysfunction under these conditions. The experiment was conducted on 21 male Wistar rats (210-230 g), randomly divided into three groups: intact animals, a group exposed to AD modeling under LPS-induced systemic inflammatory response (Group II), and a group (Group 3) exposed to AD modeling under LPS-induced systemic inflammatory response, who received sodium glutamate in a dose of 20 mg/kg. The administration of sodium glutamate significantly increased the generation rate of the superoxide anion radical by 13.8% through microsomal monooxygenases, by 8.7% through the mitochondrial respiratory chain, and by 7.7% through leukocyte NADPH oxidase compared to Group 2. However, sodium glutamate did not affect the total NO synthase activity or the activity of its constitutive and inducible isozymes in the kidney homogenate compared to Group 2. The uncoupling index of constitutive NO synthases in kidney tissues also remained unchanged under the administration of sodium glutamate, but the peroxynitrites were 20.5% higher, and S-nitrosothiols were 11.6% higher, than in group 2. The findings indicate that sodium glutamate significantly increases oxidative-nitrosative stress in rat kidney tissues under AD and LPS-induced SIR, resulting in excessive ROS/RNS formation.

Microvascular insulin resistance with enhanced muscle glucose disposal in CD36 deficiency.

Microvascular dysfunction contributes to insulin resistance. CD36, a fatty acid transporter and modulator of insulin signalling, is abundant in microvascular endothelial cells. Humans carrying the minor allele (G) of CD36 coding variant rs3211938 have 50% reduced CD36 expression and show endothelial dysfunction. We aimed to determine whether G allele carriers have microvascular resistance to insulin and, if so, how this affects glucose disposal. Our multi-disciplinary approach included hyperinsulinaemic-euglycaemic clamps in Cd36-/- and wild-type mice, and in individuals with 50% CD36 deficiency, together with control counterparts, in addition to primary human-derived microvascular endothelial cells with/without CD36 depletion. Insulin clamps showed that Cd36-/- mice have enhanced insulin-stimulated glucose disposal but reduced vascular compliance and capillary perfusion. Intravital microscopy of the gastrocnemius showed unaltered transcapillary insulin flux. CD36-deficient humans had better insulin-stimulated glucose disposal but insulin-unresponsive microvascular blood volume (MBV). Human microvascular cells depleted of CD36 showed impaired insulin activation of Akt, endothelial NO synthase and NO generation. Thus, in CD36 deficiency, microvascular insulin resistance paradoxically associated with enhanced insulin sensitivity of glucose disposal. CD36 deficiency was previously shown to reduce muscle/heart fatty acid uptake, whereas here we showed that it reduced vascular compliance and the ability of insulin to increase MBV for optimising glucose and oxygen delivery. The muscle and heart respond to these energy challenges by transcriptional remodelling priming the tissue for insulin-stimulated glycolytic flux. Reduced oxygen delivery activating hypoxia-induced factors, endothelial release of growth factors or small intracellular vesicles might mediate this adaptation. Targeting NO bioavailability in CD36 deficiency could benefit the microvasculature and muscle/heart metabolism. Clinicaltrials.gov NCT03012386 DATA AVAILABILITY: The RNAseq data generated in this study have been deposited in the NCBI Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo/ ) under accession code GSE235988 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235988 ).

The Adhesion GPCR ADGRL2/LPHN2 Can Protect Against Cellular and Organismal Dysfunction.

The most common trigger of sepsis and septic shock is bacterial lipopolysaccharide (LPS). Endothelial cells are among the first to encounter LPS directly. Generally, their function is closely linked to active endothelial NO Synthase (eNOS), which is significantly reduced under septic conditions. LPS treatment of endothelial cells leads to their activation and apoptosis, resulting in loss of integrity and vascular leakage, a hallmark of septic shock. Hence, therapies that prevent endothelial leakage or restore the endothelial barrier would be invaluable for patients. Adhesion GPCRs (aGPCRs) have been largely overlooked in this context, although particularly one of them, ADGRL2/LPHN2, has been implicated in endothelial barrier function. Our study shows that overexpression of ADGRL2 protects endothelial cells from LPS-induced activation, apoptosis, and impaired migration. Mechanistically, ADGRL2 preserves eNOS activity by shifting its binding from Caveolin-1 to Heat Shock Protein 90. Furthermore, ADGRL2 enhances antioxidative responses by increasing NRF2 activity. Notably, we found that this function may be evolutionarily conserved. In the absence of lat-2, a homolog of ADGRL2 in Caenorhabditis elegans, worms show higher ROS levels and altered stress response gene expression. Additionally, lat-2 mutants have a significantly reduced lifespan, altogether indicating a protective role of ADGRL2 against oxidative stress across species.

MODELING OF ENDOTHELIAL DYSFUNCTION AND SEARCH FOR ITS CIRCULATING BIOMARKERS

HighlightsModeling proinflammatory endothelial dysfunction in vitro is achieved by inducing mitochondrial-oxidative stress (alkylating agent mitomycin C), lysosomal-calcium stress (calciprotein particles), cytokine stress (lipopolysaccharide addition), or metabolic stress (palmitic acid addition). In vivo, this can be modeled by comparing aged and young laboratory rodents (specifically hyperlipidemic mice).To model vasospastic endothelial dysfunction in vitro, it is advisable to use inhibitors of endothelial NO synthase. For in vivo modeling, rats with stress-induced hereditary arterial hypertension (SIHAH) should be used, comparing them with normotensive Wistar rats in experiments.For modeling prothrombotic endothelial dysfunction, it is appropriate to use the S1 subunit of the Spike protein of the SARS-CoV-2 virus or its receptor-binding domain (RBD), as well as transgenic K18-hACE2 mice expressing the human ACE2 receptor. AbstractDespite its high clinical relevance to a range of acute (COVID-19, sepsis, multiple organ failure) and chronic (arterial hypertension, frailty syndrome, deep vein thrombosis) conditions and its direct impact on the development of adverse outcomes, the concept of endothelial dysfunction remains rather vague. Considering the various triggers, development mechanisms, molecular markers, and pathological consequences, it is reasonable to classify endothelial dysfunction as a typical pathological process into three types, determined by the leading pathogenetic factor: proinflammatory, vasospastic, and prothrombotic. Except for the hemostatically active high-molecular-weight multimers of von Willebrand factor accompanying the development of prothrombotic endothelial dysfunction in COVID-19, reliable and clinically applicable circulating markers of endothelial dysfunction remain unknown, significantly complicating the study of therapeutic correction methods. This review discusses approaches to modeling these types of endothelial dysfunction in cell cultures and animal models, as well as clinical scenarios for verifying potential markers identified in experiments. For modeling proinflammatory endothelial dysfunction in vitro, it is optimal to use mitomycin C, calciprotein particles, lipopolysaccharide, and palmitic acid; for vasospastic dysfunction – endothelial NO synthase inhibitors; for prothrombotic dysfunction – the S1 subunit of the SARS-CoV-2 Spike protein or its receptor-binding domain. For modeling proinflammatory endothelial dysfunction in vivo, mitomycin C is proposed; for vasospastic dysfunction – rats with hereditary baseline and stress-induced arterial hypertension; for prothrombotic dysfunction – intravenous administration of the S1 subunit or its receptor-binding domain to transgenic mice expressing the human ACE2 receptor (K18-hACE2). The systematic search for markers of endothelial dysfunction is based on step-by-step mass spectrometric profiling and dot-blot profiling (or multiplex immunofluorescent assay based on magnetic microspheres) of the cellular secretome (in serum-free culture medium) and blood serum (after fractionation and removal of high-molecular-weight proteins and supramolecular complexes), followed by verification of the selected markers using enzyme-linked immunosorbent assay.

CURRENT APPROACHES TO THE IDENTIFICATION OF CELLULAR MARKERS OF ENDOTHELIAL DYSFUNCTION

Highlights Potential cellular markers of vasospastic endothelial dysfunction include endothelial NO synthase and its phosphorylated forms, mechanosensitive transcription factors, as well as markers of nitrosative and oxidative stress.Probable cellular markers of proinflammatory endothelial dysfunction may encompass transcription factors of the endothelial-mesenchymal transition, proinflammatory transcription factors, mechanosensitive transcription factors, inducible cell adhesion molecules, components of the basement membrane, and key endothelial integrins.Cellular markers of prothrombotic endothelial dysfunction may include components of the endothelial glycocalyx, as well as membrane and cytoplasmic anti- and prothrombotic molecules responsible for regulating local and systemic hemostasis. AbstractThis review examines several groups of potential cellular markers for various types of endothelial dysfunction: vasospastic, proinflammatory, and prothrombotic. For this purpose, we screened the PubMed database for the respective publications over the past 45 years. Immunohistochemical analysis of the development of vasospastic endothelial dysfunction involves measuring the expression of endothelial NO synthase and its phosphorylated forms (serine-113/117, threonine-495, serine-632, serine-1176/1177), mechanosensitive transcription factors (KLF2, KLF4, and NRF2), markers of nitrosative stress (3-nitrotyrosine and 6-nitrotryptophan), and oxidative stress markers (proteins associated with malondialdehyde or methylglyoxal, xanthine oxidase, and isoforms of NADPH oxidase NOX1, NOX2, NOX4, and NOX5). Potential immunohistochemical markers of proinflammatory endothelial dysfunction include N-cadherin as a mesenchymal cell marker in combination with the loss of endothelial markers (CD31 and VE-cadherin), transcription factors of endothelial-mesenchymal transition (Snail, Slug, Twist1, and Zeb1), proinflammatory transcription factors (NF-κB, IRF1, IRF3, IRF5, IRF7, AP-1, ATF1, ATF2, ATF3, ATF4, ATF6, EGR-1, EGR-3, STAT1, STAT3, and STAT4), NLRP3 as a marker of inflammasomes, the aforementioned mechanosensitive transcription factors, inducible cell adhesion molecules (VCAM1, ICAM1, E-selectin), as well as components of the basement membrane (laminin, type IV collagen, nidogen-1, nidogen-2, perlecan, fibronectin) and endothelial integrins (α2β1, α3β1, α5β1, α9β1, αvβ3, αvβ5). Immunohistochemical analysis of prothrombotic endothelial dysfunction may rely on assessing the expression of components of the endothelial glycocalyx (via immunofluorescent staining with UEA-1, which binds to fucosylated glycans, or through immunohistochemical staining for syndecan-1, heparan sulfate, chondroitin sulfate, hyaluronic acid, perlecan, and glypican-1), as well as evaluating the expression of its antithrombotic (antithrombin III, tissue factor inhibitor, thrombomodulin, CD39, CD73) or prothrombotic proteins (heparanase, hyaluronidase, angiotensin-converting enzyme 2, von Willebrand factor). Verification of immunohistochemical markers differentially expressed in control and model animals should be conducted using electron microscopy of adjacent vascular segments to establish associative and correlative relationships between molecular and pathomorphological markers.

Pathobiological characterization of pulmonary hypertension associated with heart failure with preserved ejection fraction

Abstract Introduction Heart failure with preserved ejection fraction (HFpEF) is an age-related cardiometabolic disease associated with vascular inflammation in cardiac tissue, pulmonary congestion and right ventricular dysfunction. Patients with HFpEF often develop pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. Pulmonary vascular remodeling contributes to combined post- and precapillary PH (CpcPH), which is associated with worse outcome in humans. A recently developed meta-inflammatory murine HFpEF model approximates the characteristics of human HFpEF. Herein, HFpEF is induced by metabolic (high-fat diet (HFD)) and hypertensive stress (inhibition of constitutive NO synthase by Nω-nitro-1-arginomethyl ester (L-NAME)) over a 12-week period of time. With a focus on PH, we have developed a 'three-hit' model based on this model with a supplementary 21- or 42-day hypoxia phase (HFpEF-CpcPH). Methods Adult C57BL/6N mice were fed HFD for 15 or 18 weeks and supplied with L-NAME (0,5g/L or 1g/L) via drinking water, including a final period of 21 or 42 days, respectively, of hypoxia (normobaric, 10 % oxygen) or normoxia. Transthoracic echocardiography was performed using a small animal ultrasound device (Vevo 3100, Visual Sonics) and measurement of right ventricular systolic pressure (RVSP) using a Millar® pressure catheter (Mil-SPR-1000) inserted into the right ventricle. Right ventricular hypertrophy was measured as the ratio of the wet weight of the right ventricle to the left ventricle including the septum (RV/LV+S). Results Administration of HFD and L-NAME had no significant effect on RVSP in either HFpEF-normoxia group (15 weeks (n=8) or 18 weeks (n=5)) compared to the control group (n=8, n=5). 21 days of hypoxia increased RV systolic pressure (RVSP) to 37.05±1.6 (PH, n=8) and 40.33±0.87 mmHg (HFpEF-CpcPH, n=8) respectively, while 42 days of hypoxia caused no further increase (35.1±3.5 mmHg (PH, n=5) and 35.24±3.47 mmgHg (HFpEF-CpcPH, n=4)), respectively. RV hypertrophy (RV/LV+S) was significantly increased after 21 days of hypoxia in HFpEF-CpcPH compared to HFpEF (n=8) (34.42±1.75 vs. 24.93±1.1). Again, 42 days of hypoxia did not lead to a further increase in RV hypertrophy (35.2±3.9 vs. 25.80±4.39) in HFpEF-CpcPH, even though there was a significant difference compared to the HFpEF group. As expected, the heart weight/body weight ratio was significantly increased in HFpEF-CpcPH compared to HFpEF at both time points. However, mice under the prolonged hypoxia phase showed a significantly greater decrease in body weight, which complicates the interpretation of the results. Conclusion In summary, we could show that in the meta-inflammatory HFpEF model no significant PH can be observed within the investigated periods of time. However, a 21 day hypoxia phase was sufficient to induce a HFpEF-CpcPH phenotype. This model provides an important basis to develop new therapeutic approaches.

Lack of AMP-activated protein kinase-α1 reduces nitric oxide synthesis in thoracic aorta perivascular adipose tissue

ObjectivePerivascular adipose tissue (PVAT) releases anti-contractile bioactive molecules including NO. PVAT anti-contractile activity is attenuated in mice lacking AMPKα1 (AMP-activated protein kinase-α1). As AMPK regulates endothelial NO synthase (eNOS) activity in cultured cells, NO synthesis was examined in PVAT from AMPKα1 knockout (KO) mice. Methods and resultsEndothelium-denuded thoracic or abdominal aortic rings were isolated from wild type (WT) and KO mice. NOS inhibition enhanced vasoconstriction in PVAT-intact thoracic aortic rings from mice of either genotype yet had no effect on abdominal rings as assessed by wire myography. Thoracic aorta PVAT exhibited increased NO production, NOS activity and levels of the brown adipose tissue marker uncoupling protein-1 (UCP1) compared to abdominal PVAT. In KO mice, NO production was significantly reduced in thoracic but not abdominal PVAT. Reduced NO production in KO thoracic PVAT was not due to altered levels or phosphorylation of eNOS but was associated with increased caveolin-1:eNOS association and caveolin-1 Tyr14 phosphorylation. A peptide that disrupts eNOS:caveolin-1 association increased NO synthesis and reduced vasoconstriction of PVAT-intact thoracic but not abdominal aortic rings. KO thoracic PVAT also exhibited reduced UCP1 levels. ConclusionsMurine thoracic aorta PVAT exhibits higher NO synthesis and UCP1 levels than abdominal aortic PVAT. Downregulation of AMPK suppresses NO synthesis which may contribute to the reduced anticontractile activity and reduced brown adipose tissue phenotype of KO thoracic PVAT. The mechanism underlying the effect of AMPK downregulation likely results from increased caveolin-1:eNOS association associated with caveolin-1 Tyr14 phosphorylation.

Endoplasmic reticulum stress and expression of nitric oxide synthases in heart failure with preserved and with reduced ejection fraction - pilot study.

Unfolded Protein Response (UPR), endoplasmic reticulum (ER) stress, and inducible nitric oxide synthase (iNOS) overexpression have been found to influence heart failure with preserved ejection fraction (HFpEF) pathogenesis. Their importance in heart failure with reduced ejection fraction (HFrEF) is not entirely established; there is little data involving a detailed comparison between HFpEF and HFrEF from this perspective. This pilot study aimed to compare circulating levels of Glucose-regulated protein 78kDa (GRP78) (ER - stress marker) and all NOS isoforms between both HFpEF and HFrEF and to analyze the correlation between these markers and the clinical characteristics of the patients. Forty-two patients with HFpEF and thirty-eight with HFrEF were involved in this study. Clinical characteristics and echocardiographic data were obtained. Basic laboratory tests were performed and ELISA tests for iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and GRP78. Patients with HFpEF had lower circulating levels of GRP78 and higher iNOS concentrations when compared to HFrEF patients (P = 0.023, P < 0.0001, accordingly). The subgroup of the HFpEF population with eGFR < 60 mL/min/1.73m2 had higher nNOS and eNOS levels than HFpEF patients with normal GFR (P = 0.049, P = 0.035, respectively). In the HFrEF subgroup, patients with coexistent diabetes mellitus had elevated concentrations of nNOS compared to the subpopulation without diabetes mellitus (P = 0.041). There was a positive correlation between eNOS and nNOS concentrations (ρ = 0.86, P < 0.0001). In HFpEF, there is a more intensified iNOS overexpression, while in HFrEF, ER stress is more prominent.

Ziziphus jujuba (Jujube) in Metabolic Syndrome: From Traditional Medicine to Scientific Validation.

This review evaluates the therapeutic potential of Ziziphus jujuba and its main components in managing complications of metabolic syndrome, including diabetes, dyslipidemia, obesity, and hypertension. The reviewed studies provide evidence supporting the use of Z. jujuba and its main components (lupeol and betulinic acid) as natural treatments for complications of metabolic syndrome. These substances enhance glucose uptake through the activation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), reduce hepatic glucose synthesis, and increase glucose uptake by adipocytes and skeletal muscle cells. They also improve insulin sensitivity by modulating AMP-activated protein kinase (AMPK) activity and regulating insulin signaling proteins and glucose transporters. In the field of dyslipidemia, they inhibit triglyceride synthesis, lipid accumulation, and adipogenic enzymes, while influencing key signaling pathways involved in adipogenesis. Z. jujuba and its constituents demonstrate anti-adipogenic effects, inhibiting lipid accumulation and modulating adipogenic enzymes and transcription factors. They also exhibit positive effects on endothelial function and vascular health by enhancing endothelial nitric oxide synthase (eNOS) expression, NO production, and antioxidant enzyme activity. Z. jujuba, lupeol, and betulinic acid hold promise as natural treatments for complications of metabolic syndrome. They improve glucose metabolism, insulin sensitivity, and lipid profiles while exerting anti-adipogenic effects and enhancing endothelial function. However, further research is needed to elucidate the mechanisms and confirm their efficacy in clinical trials. These natural compounds offer potential as alternative therapies for metabolic disorders and contribute to the growing body of evidence supporting the use of natural medicines in their management.

ASSESSMENT OF THE RELATIONSHIP BETWEEN THE CONCENTRATION OF PSGL-1, NO-SYNTHASES AND NITRIC OXIDE METABOLITES IN THE BLOOD SERUM OF PATIENTS WITH ACUTE ARTERIAL THROMBOSIS

Introduction. PSGL-1 is a complex protein, that provides contacts between blood cells and endothelial cells by connecting with cell-adhesion mole-cules selectins, and it is a universal participant in the processes of leukocyte adhesion, inflammation and immune response. The study of the possible regulation of its functioning is an urgent topic, since the process of its interactions with selectins can be a point of therapeutic application. Aim of the study. Evaluation of the relationship between the concentration of universal ligand selectins PSGL-1, inducible (iNOS) and endothelial (eNOS) NO synthases and final metabolites of nitric oxide (II) in the blood serum of patients with acute arterial thrombosis. Material and methods. The concentrations of PSGL-1, iNOS and eNOS in blood serum were determined, using enzyme immunoassay on an enzyme immunoassay analyzer Stat Fax 2100. The total concentration of the final stable metabolites of nitric oxide(II) (nitrates and nitrites) was determined by the spectrophotometric method modified by V.A. Metelskaya, by coloring in the reaction of diazotization with nitrite of sulfonamide present in the com-position of the Griss reagent. The results were processed, using nonparametric statistics methods of Microsoft Office Excel 2016 and IBM SPSS Statistics 26 (StatSoftInc., USA). Results. The study revealed a statistically significant increase in the level of endothelial nitric oxide synthase and the final stable metabolites of nitric oxide in the blood of patients with acute arterial thrombosis, compared with the control group. A direct correlation of moderate severity was also found between the level of eNOS and the concentration of nitrates and nitrites. A low positive correlation was found between the concentration of PSGL-1 and the level of eNOS, and a low negative correlation was found between PSGL-1 and iNOS. Conclusions. According to the results of the study, it can be concluded that nitric oxide, which generated by endothelial NO synthase in patients with arterial thrombosis, may have a regulatory effect on the expression or functioning of the universal glycoprotein ligand of selectins – PSGL-1, which re-quires more in-depth study.

Novel Role of 5-Methyl-(6S)-Tetrahydrofolate in Mediating Endothelial Cell Tetrahydrobiopterin in Pregnancy and Implications for Gestational Hypertension.

Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. We analyzed the maternal blood pressure in pregnant wild-type (Gch1fl/fl) and Gch1fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4.

Abstract 12: Deficiency of Progranulin Decreases Mitochondrial Function and Induces a Whitening Phenotype in Perivascular Adipose Tissue, Suppressing Its Anti-Contractile Effects

Perivascular adipose tissue (PVAT) is recognized as an endocrine organ that secretes a variety of adipokines, which negatively regulate vascular contraction. Progranulin (PGRN), a glycoprotein encoded by the GRN gene, is highly expressed in adipocytes, and is implicated in inflammation, cellular proliferation, and repair mechanisms. The relationship between PGRN and PVAT remains unclear. We hypothesized that the lack of PGRN negatively affects PVAT quality. We used male C57BL/6J wild-type (WT) and global PGRN knockout (KO) mice, aged 10-12 weeks. Energy expenditure was analyzed via the Comprehensive Lab Animal Monitoring System (CLAMS). Vascular function was studied in endothelium-intact aortae with PVAT [PVAT(+)] and without PVAT [PVAT(-)]. Additionally, 3T3-L1 cells were used to evaluate the molecular mechanisms. The KO mice showed a decreased respiratory exchange ratio (RER) and a whitening phenotype of PVAT, which was accompanied by a loss of anti-contractile effects [(mN) WT_PVAT(-): 5.2±0.3 vs PVAT(+): 3.4±0.5*, *P&lt;0.05 and KO_PVAT(-): 6.1±0.7 vs KO_PVAT(+): 5.7±0.4]. Additionally, PVAT from KO displayed a significant reduction in nitric oxide production [(RFU) WT_PVAT: 223.1± 12.3 vs KO_PVAT: 67.3±9.1*, *P&lt;0.05] and endothelial NO synthase phosphorylation. Additionally, the lack of PGRN attenuated the expression of mitochondrial complexes [(AU) WT_PVAT: 1.3± 0.03 vs KO_PVAT: 0.6±0.06*, *P&lt;0.05] measured by the OXPHOS cocktail antibody. Via high-resolution respirometry, we found that isolated mitochondria from KO_PVAT showed impaired succinate-driven respiration or complex II activity. To understand if the whitening phenotype of PVAT was leading to its dysfunction, we induced a browning phenotype of PVAT by treating the mice with a β3-agonist for 7 days (CL 316243, 1mg/kg/day). This treatment increased RER restored the PVAT phenotype, and improved PVAT anti-contractile effect and mitochondrial respiration. Using 3T3-L1 cells, we engineered the overexpression and knockdown of PGRN via lentivirus (LV) infection and siRNA, respectively. Suppression of PGRN increased mRNA of whitening markers (1.5-fold increase vs scramble siRNA), while overexpression elevated mRNA of browning and mitochondrial markers (1.3-fold increase vs scramble LV). In summary, our findings reveal that PGRN is crucial for maintaining the phenotype and anti-contractile function of PVAT and highlight PGRN as a pharmacological target for vascular outcomes in cardiometabolic conditions.

Effects of Neonatal Administration of Non-Opiate Analogues of Leu-Enkephalin on the Delayed Cardiac Consequences of Intrauterine Hypoxia.

Intrauterine hypoxia (gestation days 15-19, pO2 65 mm Hg, duration 4 h) led to an increase in the expression of p53, beclin-1, endothelial NO synthase (eNOS), and caspase-3 proteins in cardiomyocytes and reduced the number of mast cells in the heart of 60-day-old albino rats. Administration of a non-opiate analogue of leu-enkephalin (NALE peptide: Phe-D-Ala-Gly-Phe-Leu-Arg, 100 μg/kg) on days 2-6 of the neonatal period decreased the severity of delayed posthypoxic myocardial reaction. The content of eNOS+ cardiomyocytes and the total number of mast cells of these animals did not differ from the control parameters; the content of p53+ cardiomyocytes was significantly lower than in animals exposed to intrauterine hypoxia. The cardioprotective activity of NALE was partially neutralized by co-administration with the NO synthase inhibitor (L-NAME, 50 mg/kg). Correction of the delayed posthypoxic changes, similar to the effects of NALE peptide, was observed after neonatal administration of its arginine-free analogue, G peptide (Phe-D-Ala-Gly-Phe-Leu-Gly; 100 μg/kg). Non-opiate analogues of leu-enkephalin NALE and G peptides can be considered as promising substances capable of preventing long-term cardiac consequences of intrauterine hypoxia.