Experimental Hypertension Research Articles

Disease severity-related alterations of cardiac microRNAs in experimental pulmonary hypertension.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart‐relevant microRNAs, that is myomiRs (miR‐1, miR‐133a, miR‐208, miR‐499) and miR‐214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT‐qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR‐1/miR‐133a/miR‐208a/miR‐499) were decreased and the expression of miR‐214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR‐214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.

Melatonin inhibits Th17 differentiation

Abstract Hypertension is a leading risk factor for cardiovascular disease, which is a major cause of death worldwide. T cells are required for the development of hypertension. Specifically, we showed that T helper 17 (Th17) cells produce the pro-inflammatory cytokine, IL-17A, which leads to sustained elevations of blood pressure and renal and vascular dysfunction in experimental hypertension. T cell differentiation and trafficking is regulated by circadian clocks. High fat and high salt intake, typical of Western diets, disrupt circadian rhythms and are associated with increased risk for developing cardiovascular disease. These diets are also associated with an increase in Th17 cell differentiation. Melatonin is a major circadian hormone with anti-inflammatory and anti-oxidant actions. To determine the interplay between circadian rhythms and Th17 cell differentiation, we tested the hypothesis that in vitro treatment of Th17 cells with melatonin would inhibit IL-17A cytokine production. Splenic naïve CD4+ T cells from wild type C57Bl/6J male mice were cultured for 72 hours on anti-CD3/anti-CD28 antibody coated plates in the presence or absence of Th17-polarizing cytokines and treated with melatonin (2ng/ml; Sigma Aldrich) or vehicle (0.02% DMSO). IL-17A cytokine levels were analyzed by ELISA. In cells treated with Th17 polarizing cytokines, IL-17A cytokine production was significantly reduced by melatonin. Thus, this study highlights the involvement of circadian control of Th17 cells via the actions of melatonin to suppress Th17 differentiation and IL-17A production. Future studies will determine the effect of melatonin treatment in vivo on development of hypertension and renal/vascular dysfunction.

Sex differences in blood pressure of aged Ren-2 transgenic rats.

Sex-related differences were observed not only in human but also in experimental hypertension. The aim of our study was to compare blood pressure (BP) of aged male and female heterozygous transgenic rats (TGR) harboring Ren-2 mouse gene, with their normotensive Hannover Sprague-Dawley (HanSD) controls. At the age of 9 months, systolic (SBP) and diastolic blood pressure (DBP) were measured by a direct puncture of carotid artery in rats awaking from isoflurane anesthesia. Thiobarbituric acid-reactive species (TBARS) formation was monitored as indicator of lipid peroxidation damage in heart, kidney and liver, whereas intracellular content of reduced glutathione was determined in the same organs as the main intracellular antioxidant. Furthermore, plasma triglycerides and total cholesterol as well as high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions of cholesterol were measured. As compared to HanSD rats, we found significantly elevated BP only in male TGR (MAP: 123±1 vs. 171±5, SBP: 150±2 vs. 208±7, and DBP: 99±3 vs. 140±4 mm Hg), but not between TGR and HanSD females, which were both normotensive. We also did not find any significant differences in TBARS and reduced glutathione in the three above mentioned organs as well as in plasma cholesterol or its HDL and LDL fractions between transgene-negative HanSD and TGR animals of either sex. However, we found significant sex differences in TBARS, glutathione and plasma lipids in both rat strains. Our results confirmed that aged TGR exhibit a marked sexual BP dimorphism, which does not seem to be dependent on oxidative stress or abnormal cholesterol metabolism.

MicroRNA‐483 amelioration of experimental pulmonary hypertension

Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR‐483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA‐seq and bioinformatics analyses showed that miR‐483 targets several PAH‐related genes, including transforming growth factor‐β (TGF‐β), TGF‐β receptor 2 (TGFBR2), β‐catenin, connective tissue growth factor (CTGF), interleukin‐1β (IL‐1β), and endothelin‐1 (ET‐1). Overexpression of miR‐483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF‐β, TGFBR2, β‐catenin, CTGF, IL‐1β, and ET‐1. In contrast, inhibition of miR‐483 increased these genes in ECs. Rats with EC‐specific miR‐483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR‐483. These results indicate that PAH is associated with a reduced level of miR‐483 and that miR‐483 might reduce experimental PH by inhibition of multiple adverse responses.

Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension

Arterial hypertension is the most important risk factor for the development of cardiovascular disease. Recently, aircraft noise has been shown to be associated with elevated blood pressure, endothelial dysfunction, and oxidative stress. Here, we investigated the potential exacerbated cardiovascular effects of aircraft noise in combination with experimental arterial hypertension. C57BL/6J mice were infused with 0.5 mg/kg/d of angiotensin II for 7 days, exposed to aircraft noise for 7 days at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A), or subjected to both stressors. Noise and angiotensin II increased blood pressure, endothelial dysfunction, oxidative stress and inflammation in aortic, cardiac and/or cerebral tissues in single exposure models. In mice subjected to both stressors, most of these risk factors showed potentiated adverse changes. We also found that mice exposed to both noise and ATII had increased phagocytic NADPH oxidase (NOX-2)-mediated superoxide formation, immune cell infiltration (monocytes, neutrophils and T cells) in the aortic wall, astrocyte activation in the brain, enhanced cytokine signaling, and subsequent vascular and cerebral oxidative stress. Exaggerated renal stress response was also observed. In summary, our results show an enhanced adverse cardiovascular effect between environmental noise exposure and arterial hypertension, which is mainly triggered by vascular inflammation and oxidative stress. Mechanistically, noise potentiates neuroinflammation and cerebral oxidative stress, which may be a potential link between both risk factors. The results indicate that a combination of classical (arterial hypertension) and novel (noise exposure) risk factors may be deleterious for cardiovascular health.

Implication of in vivo circulating fibrocytes ablation in experimental pulmonary hypertension murine model.

Recruitment and involvement of bone-/blood-derived circulating fibrocytes (CF) in the promotion of fibrotic tissue remodelling processes have been shown. However, their direct contribution to pathological changes is not clear. The present study investigates the causal role of CF in the pathogenesis of pulmonary hypertension (PH). For selective ablation of CF, we applied the suicidal gene strategy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir. The transgenic mice were generated, having HSV-TK-GFP transgene under the collagen 1 promoter. To selectively target CF, HSV-TK-GFP+ bone marrow transplanted into irradiated wild type mice. These chimera mice were subjected to hypoxia for PH induction and ganciclovir for CF ablation. In vivo CF ablation reduced right ventricular hypertrophy and vascular remodelling with reduced total collagen content. We quantified the CF recruited in the perivascular area and arterial wall of small pulmonary arteries. There was significant recruitment of CF in the lung in response to hypoxia. The characterization of CF showed the expression of CD45 and collagen1 (GFP) along with α-smooth muscle actin (αSMA). Our data demonstrated that CF ablation has a potential impact on right ventricular hypertrophy and vascular remodelling in the setting of experimental pulmonary hypertension induced by hypoxia. The beneficial effects may be related to the direct contribution of fibrocytes or its paracrine effect on other resident cell types. Thus, clinical manipulation of CF may represent a novel therapeutic approach to ameliorate the disease state in pulmonary hypertension.

Effects of short‐term therapy with high dose nutritional inorganic nitrite and nitrate on vascular dysfunction, oxidative damage and epigenetic regulation in experimental arterial hypertension

BackgroundArterial hypertension is one of the major risk factors leading to highly relevant diseases like coronary artery disease, stroke or peripheral artery disease. Even short‐term application of angiotensin‐II (AT‐II) in animal models of arterial hypertension is leading to vascular dysfunction, enhanced production of reactive oxygen species (ROS) and modification of epigenetic processes. Nitric oxide (NO) represents an important opponent to AT‐II induced vasoconstriction and high blood pressure. Dietary uptake (e.g. vegetables) of inorganic nitrite (NO2−) and nitrate (NO3−) leads to enhanced NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2− and NO3− co‐therapy in hypertensive mice.Methods and resultsHigh‐dose AT‐II (1mg/kg/d for 1 week) was used to induce arterial hypertension in male C57BL/6 mice. In parallel high dose inorganic nitrite (7.5mg/kg/d) and inorganic nitrate (150mg/kg/d) was administered via drinking water. Hemodynamic effects were evaluated by blood pressure measurements and isometric tension studies. To elucidate underlying epigenetic regulation processes gene expression analyses and quantification of ROS formation were performed.Administration of AT‐II induced hypertension, cardiac hypertrophy and impaired endothelial function. Co‐therapy with high dose inorganic nitrite, but not with nitrate, normalised vascular function and arterial hypertension. In addition, oxidative stress markers and inflammatory pathways were enhanced in hypertensive mice and mostly normalized by nitrite therapy. We observed hypertension induced changes, at least by trend, of epigenetic proteins like histone deacetylase 2 or lysine demethylase 3A in cardiac tissue, which were mostly normalized by nitrite therapy.ConclusionNutritional nitric oxide precursors represent complementary therapy to classical antihypertensive drugs, representing a dietary treatment applicable to the general population (e.g. by eating certain vegetables). Short‐term therapy with inorganic nitrite showed beneficial effects on vascular function, oxidative stress and epigenetic regulation in experimental hypertension, which were less pronounced with inorganic nitrate. The observed differences may rely on superior bioconversion of nitrite to NO or intoxication through methaemoglobinaemia because of high dose inorganic nitrate therapy. Therefore, in particular inorganic nitrite seems to represent a cost‐efficient, drug‐free, alternative in antihypertensive therapy. Further clinical research is necessary to further support these preclinical findings and to demonstrate whether the incidence and severity of hypertension in particular and cardiovascular outcome in general can be influenced by inorganic nitrite therapy.Support or Funding InformationWe gratefully acknowledge the financial support by Stiftung Mainzer Herz (to M.O. and A.D.).

Biventricular diastolic dysfunction, thrombocytopenia, and red blood cell macrocytosis in experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension is a fatal disease, where death is associated with right heart failure and reduced cardiorespiratory reserve. The Sugen 5416, hypoxia and normoxia Fischer rat model mimics human pulmonary arterial hypertension, although the cause(s) of death remains incompletely understood. Here, we hypothesized that these animals develop biventricular diastolic dysfunction that contributes to tissue hypoperfusion coincident with severe pulmonary arterial hypertension. We performed comprehensive echocardiographic and hematologic assessments. Serial echocardiogram at 3–5 weeks was performed followed by blood sampling via aortic or cardiac puncture. Echocardiogram revealed pulmonary arterial hypertension in pulmonary artery Doppler waves, including notched wave envelopes, and decreased pulmonary artery acceleration time/pulmonary artery ejection time ratio and right ventricular outflow tract velocity time integral. Impaired right ventricular systolic function, assessed by decreased tricuspid annular plane systolic excursion and tricuspid tissue Doppler systolic positive wave velocity, was observed in pulmonary arterial hypertension. Tricuspid and mitral pulsed wave and tissue Doppler findings suggested biventricular diastolic dysfunction, with dynamic changes in early and late diastolic filling waves, their fusion patterns, and a decrease in e' velocity. Heart rate and ejection fraction did not change, but cardiac output, stroke volume, and end-diastolic volume were decreased, and inferior vena cava respiratory variation was decreased. Blood electrolyte values were suggestive of intravascular volume expansion early in the disease followed by volume contraction and tissue hypoperfusion in the latter stages of disease. Complete blood count showed thrombocytopenia and non-anemic macrocytosis with reticulocytosis and an increase in red blood cell distribution width. Thus, pulmonary, cardiac, and hematological findings in Fischer animals with pulmonary arterial hypertension are characteristic of humans and provide an insightful experimental platform to resolve mechanisms of disease progression.

Using Pulmonary Angiogram to Estimate Vascular Remodeling in Mice

BackgroundPulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH) and animals with experimental pulmonary hypertension (PH). Utilizing pulmonary angiography technique, we are able to visualize vascular blockade and structural changes in the lung vasculature of patients with PAH and animals with experimental PH. Given the differences in ventilation (V), perfusion (Q) and V/Q relationship in different regions of the lung, it is important to know whether vascular structure and density are different in various areas of the lung. In this study, we aimed to examine whether pulmonary vascular structure and density are different in the upper (apical), middle and lower (basal) regions of the lung in mice and whether pulmonary hemodynamic measurement via right heart catheterization affects the angiographic measurement.MethodsA polyethylene tube was cannulated into the pulmonary artery (PA) via the right ventricle in anesthetized mice to perfuse microfilm polymer (yellow) into the lung vasculature using an automated pump. The lungs filled with microfil were dehydrated using ethanol and placed in methyl salicylate for overnight to show only the vasculature. Lungs were then photographed with a digital camera. The peripheral lung vascular images from the upper, middle and lower regions were selected and converted to binary images for quantitative analysis.ResultsIn the skeletonized images (showing only the vasculature), the length of branches, the number of branches, and the number of junctions were first measured and compared among the upper, middle and lower regions of normal mouse lungs. The data showed that there was no significant difference among different regions in terms of the length of branches as well as the numbers of branches and junctions. Furthermore, we compared these three parameters in different regions between the lungs isolated from mice in which we measured right ventricular pressure using right heart catheterization and the lungs isolated from control mice. The data indicated that the length of branches (22±3, 20±3 and 21±1 vs. 21±1, 22±1 and 19±2 mm/mm2, respectively), the number of branches (420±70, 364±83 and 411±44 vs. 388±50, 426±26 and 353±27 per mm2, respectively, and the number of junctions (186±34, 186±14 and 212±38 vs. 183±35, 191±13 and 155±18 per mm2, respectively) in the upper, middle and lower regions of the lungs from mice with right heart catheterization were not significantly different from the lungs of control mice.ConclusionPeripheral vascular structure and density in the mouse lung, determined by the length of branches and the numbers of branches and junctions, are not significantly different among the apical, middle and basal regions of the mouse lung. Lung hemodynamic measurement or invasive right heart catheterization has little effect on the peripheral vascular density. These results suggest that lung angiogram on the peripheral vasculature is a reliable experimental tool to evaluate lung vascular structural changes in small animals.Support or Funding InformationNational Natural Science Foundation of China (81800035)

Tie-2 Cre-Mediated Deficiency of Extracellular Signal-Regulated Kinase 2 Potentiates Experimental Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension in Neonatal Mice.

Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is a significant lung morbidity of infants, and disrupted lung angiogenesis is a hallmark of this disease. We observed that extracellular signal-regulated kinases (ERK) 1/2 support angiogenesis in vitro, and hyperoxia activates ERK1/2 in fetal human pulmonary microvascular endothelial cells (HPMECs) and in neonatal murine lungs; however, their role in experimental BPD and PH is unknown. Therefore, we hypothesized that Tie2 Cre-mediated deficiency of ERK2 in the endothelial cells of neonatal murine lungs would potentiate hyperoxia-induced BPD and PH. We initially determined the role of ERK2 in in vitro angiogenesis using fetal HPMECs. To disrupt endothelial ERK2 signaling in the lungs, we decreased ERK2 expression by breeding ERK2flox/flox mice with Tie-Cre mice. One-day-old endothelial ERK2-sufficient (eERK2+/+) or –deficient (eERK2+/−) mice were exposed to normoxia or hyperoxia (FiO2 70%) for 14 d. We then performed lung morphometry, gene and protein expression studies, and echocardiography to determine the extent of inflammation, oxidative stress, and development of lungs and PH. The knockdown of ERK2 in HPMECs decreased in vitro angiogenesis. Hyperoxia increased lung inflammation and oxidative stress, decreased lung angiogenesis and alveolarization, and induced PH in neonatal mice; however, these effects were augmented in the presence of Tie2-Cre mediated endothelial ERK2 deficiency. Therefore, we conclude that endothelial ERK2 signaling is necessary to mitigate hyperoxia-induced experimental BPD and PH in neonatal mice. Our results indicate that endothelial ERK2 is a potential therapeutic target for the management of BPD and PH in infants.

Epigenetic Metabolic Reprogramming of Right Ventricular Fibroblasts in Pulmonary Arterial Hypertension: A Pyruvate Dehydrogenase Kinase-Dependent Shift in Mitochondrial Metabolism Promotes Right Ventricular Fibrosis.

Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-β1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

Paneth cells promote angiogenesis and regulate portal hypertension in response to microbial signals

Paneth cells (PCs) synthesize and secrete antimicrobial peptides that are key mediators of host-microbe interactions, establishing a balance between intestinal microflora and enteric pathogens. We observed that their number increases in experimental portal hypertension and aimed to investigate the mechanisms by which these cells can contribute to the regulation of portal pressure. We first treated Math1Lox/LoxVilcreERT2 mice with tamoxifen to induce the complete depletion of intestinal PCs. Subsequently, we performed partial portal vein or bile duct ligation. We then studied the effects of these interventions on hemodynamic parameters, proliferation of blood vessels and the expression of genes regulating angiogenesis. Intestinal organoids were cultured and exposed to different microbial products to study the composition of their secreted products (by proteomics) and their effects on the proliferation and tube formation of endothelial cells (ECs). Invivo confocal laser endomicroscopy was used to confirm the findings on blood vessel proliferation. Portal hypertension was significantly attenuated in PC-depleted mice compared to control mice and was associated with a decrease in portosystemic shunts. Depletion of PCs also resulted in a significantly decreased density of blood vessels in the intestinal wall and mesentery. Furthermore, we observed reduced expression of intestinal genes regulating angiogenesis in Paneth cell depleted mice using arrays and next generation sequencing. Tube formation and wound healing responses were significantly decreased in ECs treated with conditioned media from PC-depleted intestinal organoids exposed to intestinal microbiota-derived products. Proteomic analysis of conditioned media in the presence of PCs revealed an increase in factors regulating angiogenesis and additional metabolic processes. Invivo endomicroscopy showed decreased vascular proliferation in the absence of PCs. These results suggest that in response to intestinal flora and microbiota-derived factors, PCs secrete not only antimicrobial peptides, but also pro-angiogenic signaling molecules, thereby promoting intestinal and mesenteric angiogenesis and regulating portal hypertension. Paneth cells are present in the lining of the small intestine. They prevent the passage of bacteria from the intestine into the blood circulation by secreting substances to fight bacteria. In this paper, we discovered that these substances not only act against bacteria, but also increase the quantity of blood vessels in the intestine and blood pressure in the portal vein. This is important, because high blood pressure in the portal vein may result in several complications which could be targeted with novel approaches.

Critical closing pressure during experimental intracranial hypertension: comparison of three calculation methods

ABSTRACT Objectives: The critical closing pressure (CrCP) defines arterial blood pressure below which cerebral arteries collapse. It represents a clinically relevant parameter for the estimation of cerebrovascular tone. Although there are few methods to assess CrCP, there is no consensus which of them estimates this parameter most accurately. The aim of present retrospective, experimental study was to compare three methods of CrCP estimation: conventional Aaslid’s formula and methods based on the cerebrovascular impedance: the established continuous flow forward (CFF) and a new pulsatile flow forward (PFF) model. Methods: The effects of the following physiological manoeuvres on the CrCP were studied in New Zealand white rabbits: lumbar infusion of Hartmann’s solution to induce mild intracranial hypertension, sympathetic blockade to induce arterial hypotension, and modulation of respiratory tidal volume to induce hypocapnia or hypercapnia. Results: During intracranial hypertension, all CrCP estimates were significantly higher than at baseline, decreased with decreasing ABP and increased with gradual hypocapnia. During hypercapnia, all CrCP estimates were significantly decreased but only in the case of CrCPA the negative, non-physiological values were observed (16% of the cases). The Bland–Altman analysis revealed that a good agreement between each impedance method and Aaslid’s method deteriorated significantly in the low range of the average numerical value of the estimates. Discussion: Our results confirm the limited usage of Aaslid’s formula for the calculation of CrCP. Although both impedance methods seem to be equivalent, the fact that PFF model better describes cerebrovascular hemodynamic allows the recommendation of this model for the calculation of CrCP.

SUN-172 SELECTIVE INHIBITION OF PKR AMELIORATES HYPERTENSIVE NEPHROPATHY AND AORTIC REMODELLING IN L-NAME TREATED WISTAR RATS

Arterial hypertension shares a global prevalence and is associated with hypertensive nephropathy and severe damage to the vasculature in the general population. Hypertensive Nephrosclerosis is often associated with fibrosis, inflammation and tubular cell death due to apoptosis accompanied by structural alterations in the vasculature. Double-stranded RNA dependent protein kinase (PKR) is a known inducer of inflammation and apoptosis. However, no research has been done so far to elucidate the role of PKR in hypertensive nephropathy and vascular complications. Thus, the aim of the present study was to investigate the role of PKR in nephropathy/vascular complications and the underlying molecular mechanism. Experimental hypertension was induced by administration of L-NAME (40 mg/kg b.w, p.o) along with selective PKR inhibitor imoxin (0.5mg/kg, i.p.) to Wistar rats for four weeks. Changes in physiological parameters were assessed by recording non-invasive blood pressure (BP), heart rate (HR) and body weight (BW) on days 0, 7, 14, 21, and 28 for all the rats. Serum creatinine, BUN, intracellular calcium, HDL, LDL, and TGs were measured for biochemical estimations. Expression of PKR and its downstream markers for inflammation, fibrosis, apoptosis and vascular damage (JNK, p-JNK, NLRP3, Ang-II, TGF-β, eNOS, p-CREB, and caspase-3,) in rat kidney and aorta were determined by western blot and immunohistochemistry (IHC). Dilation in renal corpuscles, tubules, glomerulosclerosis and collapsed glomeruli in kidney sections along with changes in the luminal diameter of aortic rings were studied by histological examination. Renal fibrosis was determined by Sirius red and Masson’s Trichrome staining. TUNEL assay was done for tubular cell death and apoptosis. L-NAME treated rats showed a significant increase in BP, biochemical parameters together with a decrease in HR and BW compared to control, attenuation was observed with imoxin administration, however, lipid profile remained unaltered. Results of western blot and IHC indicate a significant increase in expression of PKR and its downstream markers in kidney and aorta when compared to control, attenuation was observed in imoxin co-treated group. PKR is a major contributor involved in hypertensive nephropathy and aortic remodeling in L-NAME Treated Wistar Rats. The observed alterations in the kidney and vasculature could be mediated in part by activation of the JNK/TGF-β/eNOS pathway in a PKR dependent manner. Additionally, we also propose that these PKR mediated perturbations are blocked by selective inhibition of PKR by imoxin.

Platelet activation in experimental murine neonatal pulmonary hypertension.

Serotonin (5‐HT) contributes to the pathogenesis of experimental neonatal pulmonary hypertension (PH) associated with bronchopulmonary dysplasia (BPD). Platelets are the primary source of circulating 5‐HT and is released upon platelet activation. Platelet transfusions are associated with neonatal mortality and increased rates of BPD. As BPD is often complicated by PH, we tested the hypothesis that circulating platelets are activated and also increased in the lungs of neonatal mice with bleomycin‐induced PH associated with BPD. Newborn wild‐type mice received intraperitoneal bleomycin (3 units/kg) three times weekly for 3 weeks. Platelets from mice with experimental PH exhibited increased adhesion to collagen under flow (at 300 s−1 and 1,500 s−1) and increased expression of the αIIbβ3 integrin and phosphatidylserine, markers of platelet activation. Platelet‐derived factors 5‐HT and platelet factor 4 were increased in plasma from mice with experimental PH. Pharmacologic blockade of the 5‐HT 2A receptor (5‐HT 2A R) prevents bleomycin‐induced PH and pulmonary vascular remodeling. Here, platelets from mice with bleomycin‐induced PH demonstrate increased 5‐HT 2A R expression providing further evidence of both platelet activation and increased 5‐HT signaling in this model. In addition, bleomycin treatment increased lung platelet accumulation. In summary, platelets are activated, granule factors are released, and are increased in numbers in the lungs of mice with experimental neonatal PH. These results suggest platelet activation and release of platelet‐derived factors may increase vascular tone, promote aberrant angiogenesis, and contribute to the development of neonatal PH.

Adrenomedullin Is Necessary to Resolve Hyperoxia-Induced Experimental Bronchopulmonary Dysplasia and Pulmonary Hypertension in Mice

Bronchopulmonary dysplasia (BPD)-associated pulmonary hypertension (PH) is an infantile lung disease characterized by aberrant angiogenesis and impaired resolution of lung injury. Adrenomedullin (AM) signals through calcitonin receptor-like receptor and receptor activity-modifying protein 2 and modulates lung injury initiation. However, its role in lung injury resolution and the mechanisms by which it regulates angiogenesis remain unclear. Consequently, we hypothesized that AM resolves hyperoxia-induced BPD and PH via endothelial nitric oxide synthase (NOS3). AM-sufficient (ADM+/+) or-deficient (ADM+/-) mice were exposed to normoxia or hyperoxia through postnatal days (PNDs) 1 to 14, and the hyperoxia-exposed mice were allowed to recover in normoxia for an additional 56 days. Lung injury and development and PH were quantified at different time points. Human pulmonary microvascular endothelial cells were also used to examine the effects of AM signaling on the NOS3 pathway and angiogenesis. Lung blood vessels and NOS3 expression decreased and the extent of hyperoxia-induced BPD and PH increased in ADM+/- mice compared with ADM+/+ mice. Hyperoxia-induced apoptosis and PH resolved by PND14 and PND70, respectively, in ADM+/+ mice but not in ADM+/- mice. Knockdown of ADM, calcitonin receptor-like receptor, and receptor activity-modifying protein 2 invitro decreased NOS3 expression, nitric oxide generation, and angiogenesis. Furthermore, NOS3 knockdown abrogated the angiogenic effects of AM. Collectively, these results indicate that AM resolves hyperoxic lung injury via NOS3.

\u03b1-Linolenic acid but not linolenic acid protects against hypertension: critical role of SIRT3 and autophagic flux

Although dietary α-linolenic acid (ALA) or linolenic acid (LA) intake was reported to be epidemiologically associated with a lower prevalence of hypertension, recent clinical trials have yielded conflicting results. Comparable experimental evidence for the roles of these two different fatty acids is still lacking and the underlying mechanisms need to be further elucidated. Our data showed that ALA but not LA supplementation alleviated systolic blood pressure elevation and improved ACh-induced, endothelium-dependent vasodilation in both spontaneously hypertensive rats (SHRs) and AngII-induced hypertensive mice. In addition, SHRs displayed reduced vascular Sirtuin 3 (SIRT3) expression, subsequent superoxide dismutase 2 (SOD2) hyperacetylation and mitochondrial ROS overproduction, all of which were ameliorated by ALA but not LA supplementation. In primary cultured endothelial cells, ALA treatment directly inhibited SIRT3 reduction, SOD2 hyperacetylation, mitochondrial ROS overproduction and alleviated autophagic flux impairment induced by AngII plus TNFα treatment. However, these beneficial effects of ALA were completely blocked by silencing SIRT3. Restoration of autophagic flux by rapamycin also inhibited mitochondrial ROS overproduction in endothelial cells exposed to AngII plus TNFα. More interestingly, SIRT3 KO mice developed severe hypertension in response to a low dose of AngII infusion, while ALA supplementation lost its anti-hypertensive and endothelium-protective effects on these mice. Our findings suggest that ALA but not LA supplementation improves endothelial dysfunction and diminishes experimental hypertension by rescuing SIRT3 impairment to restore autophagic flux and mitochondrial redox balance in endothelial cells.

The SGLT2 inhibitor empagliflozin reduces mortality and prevents progression in experimental pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a rare, but progressive and devastating vascular disease with few treatment options to prevent the advancement to right ventricular dysfunction hypertrophy and failure. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, enhances urinary glucose excretion as well as reduces cardiovascular events and mortality in individuals with type 2 diabetes. While empagliflozin has been reported to lower systemic hypertension due to increased diuresis, the effect of empagliflozin on PAH is unknown. We used monocrotaline (MCT)-treated Sprague-Dawley rats to determine if empagliflozin alters PAH-associated outcomes. Compared to vehicle control, daily empagliflozin administration significantly improved survival in rats with severe MCT-induced PAH. Hemodynamic assessments showed that empagliflozin treatment significantly reduced mean pulmonary artery pressure, right ventricular systolic pressure, and increased pulmonary acceleration time. Empagliflozin treatment resulted in reduced right ventricular hypertrophy and fibrosis. Histological and molecular assessments of lung vasculature revealed significantly reduced medial wall thickening and decreased muscularization of pulmonary arterioles after empagliflozin treatment compared to vehicle-treated rats. In summary, SGLT2 inhibition with empagliflozin lowered mortality, reduced right ventricle systolic pressure, and attenuated maladaptive pulmonary remodeling in MCT-induced PAH. Clinical studies evaluating the efficacy of SGLT-2 inhibition should be considered for patients with PAH.

Lack of LRP8 triggers the formation of aortic dissection in angiotensin II infused mice

Factor XI-thrombin amplification loop depends on platelets and monocytes and augments angiotensin II (AngII) driven vascular injury in experimental hypertension in mice. Low-density lipoprotein receptor-related protein 8 (LRP8) plays an important role in apolipoprotein E homeostasis and was also found to be important in endothelial barrier stabilization. Moreover, LRP8 is able to form a complex with the platelet glycoprotein Ib alpha (GPIbalpha) which is the main receptor for von Willebrand factor. To explore the role of LRP8 and GPIbalpha in the development of hypertension related to platelet-dependent thrombin generation. We infused LRP8 KO mice with AngII for either 1 or 4 weeks using osmotic minipumps. Blood pressure was recorded using tail cuff measurement. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging (IVM). Histology of aortas was performed with sirius red staining. Infusion of AngII for 1 week in LRP8 KO mice altered endothelial dependent and independent vascular relaxation to the same extend as in control mice. Vascular rolling and adherence to the carotid endothelium, visualized by IVM was similar between LRP8 KO and control mice infused with AngII. There were no significant differences in AngII-induced blood pressure increase between the LRP8 KO and control mice. Unexpectedly, LRP8 KO mice demonstrated a drastically increased mortality rate in response to AngII infusion. In the 4 weeks AngII infusion experiments, 80% of the LRP8 KO mice died within the first 10 days of AngII infusion due to aortic dissection complicated by hemorrhage. LRP8 receptor dysfunction lead to the formation of aortic dissection in AngII infused mice. Further studies are needed to better characterize the role of immune cells and platelets in the development of these dissections.

БИОЭЛЕКТРИЧЕСКИЙ ИМПЕДАНС ТЕЛА КРЫС ЛИНИИ ВИСТАР РАЗНОГО ПОЛА ПРИ МОНОКРОТАЛИНОВОЙ МОДЕЛИ ЛЕГОЧНОЙ АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИИ

The aim of the work was to study the bioelectric impedance of the body in rats of both genders with monocrotaline-induced pulmonary hypertension. Materials and methods. Multi-frequency bioimpedance studies of the body of rats of both genders were carried out one month after the administration of monocrotaline (dose of 60 mg/kg, subcutaneously). Results. Hypertrophy of the right ventricle of the heart was revealed in animals with experimentally induced pulmonary hypertension, with a significantly greater thickness of the free wall of the right ventricle in males compared to females. Significant influence of the interaction of the gender factor and the effect of monocrotaline on the indicators of the bioelectric impedance of the body (amplitude, ratio of resistances at low and high current frequencies) was shown using the method of analysis of variance. The body bioelectric impedance amplitudes, normalized to body length, were significantly lower in the male rats and higher in the female rats with experimental pulmonary hypertension compared to the control animals of the same gender. In the female rats with the monocrotaline model of pulmonary hypertension, the ratio of the amplitudes of the bioelectric impedance of the body at low and high current frequencies was significantly lower than in the control animals. Conclusion. Changes in the body resistance of the rats with pulmonary hypertension indicate an inflammatory process. Gender differences in the changes in the bioelectrical impedance of the body indicate a more severe manifestation of pulmonary hypertension in the males.