The orphan receptor GPR35 contributes to angiotensin II–induced hypertension and cardiac dysfunction in mice

BACKGROUND: The orphan receptor G protein–coupled receptor 35 (GPR35) has been associated with a range of diseases, including cancer, inflammatory bowel disease, diabetes, hypertension, and heart failure. To assess the potential for GPR35 as a therapeutic target in cardiovascular disease, this study investigated the cardiovascular phenotype of a GPR35 knockout mouse under both basal conditions and following pathophysiological stimulation. METHODS: Blood pressure was monitored in male wild-type and GPR35 knockout mice over 7–14 days using implantable telemetry. Cardiac function and dimensions were assessed using echocardiography, and cardiomyocyte morphology evaluated histologically. Two weeks of angiotensin II (Ang II) infusion was used to investigate the effects of GPR35 deficiency under pathophysiological conditions. Gpr35 messenger RNA expression in cardiovascular tissues was assessed using quantitative polymerase chain reaction. RESULTS: There were no significant differences in blood pressure, cardiac function, or cardiomyocyte morphology in GPR35 knockout mice compared with wild-type mice. Following Ang II infusion, GPR35 knockout mice were protected from significant increases in systolic, diastolic, and mean arterial blood pressure or impaired left ventricular systolic function, in contrast to wild-type mice. There were no significant differences in Gpr35 messenger RNA expression in heart, kidney, and aorta following Ang II infusion in wild-type mice. CONCLUSIONS: Although GPR35 does not appear to influence basal cardiovascular regulation, these findings demonstrate that it plays an important pathological role in the development of Ang II–induced hypertension and impaired cardiac function. This suggests that GPR35 is a potential novel drug target for therapeutic intervention in hypertension

Pathophysiological Relationship between Lipoproteins and Cation Transport Properties of Platelets

Intracellular free calcium ions, ([Ca2+]i), and sodium/ hydrogen ion exchange, (Na+/H+ exchange), across the cell membrane play important second messenger roles in platelet activation and vascular smooth muscle cell contraction and growth. Changes in the normal activity of these second messengers may be responsible for enhanced platelet aggregability and increased vascular smooth muscle tone and hypertrophy which are associated with two major CHD risk factors, hypertension and dyslipidaemia. Several studies were undertaken using human platelets to identify possible pathophysiological control processes involved in cation transport. 1. Two methods for studying the Na+/H+ exchanger in human platelets were investigated, i) Amiloride-sensitive 22Na uptake was measured in platelets which had been acid loaded, by suspension in isotonic potassium propionate buffer (pH 6.7), to stimulate Na+/H+ exchange. Intraplatelet radioactivity was used to calculate the affinity (Km) and the capacity (Vmax) of Na+ uptake, ii) Binding studies using the radioactive amiloride analogue 3H-5'-(N-methyl-N-isobutyl)amiloride (MIA), to identify Na+/H+ exchanger numbers in platelet plasma membranes, were unsuccessful. 2. Platelet calcium metabolism was measured using three different techniques, i) [Ca2+]i was measured basally and after addition of 1 mumoI/I AVP in the presence and absence of 5mmol/l EGTA, was determined using the calcium- sensitive fluorescent probe Quin-2, ii) Calcium uptake was measured 1-30 min after adding 0.1ml aliquots of washed platelet suspensions (20x10e6cells) to buffer containing 0.15MBq 45Ca, with and without the presence of 1mumo1/1 arginine vasopressin (AVP). iii) Ca2+ efflux was measured in platelets loaded with 45Ca (30 min incubation with 0.48MBq/m1). After removal of excess 45Ca by washing and resuspension in fresh platelet buffer, aliquots were harvested at 5 min intervals. 3. Part of the protective effect of high density lipoproteins (HDL) in cardiovascular diseases may be due to their anti-aggregatory properties. These properties were examined in platelets by comparing the effects of HDL2 and HDL3 on (i) basal and AVP-stimulated changes in [Ca2+]i, (ii) 45Ca uptake and (iii) 45Ca efflux. In addition the effects of HDL2 and HDL3 on platelet Na+/H+ exchange kinetics were examined. Platelets from normal volunteers were preincubated with vehicle, HDL2 (50-500?g protein/ml) or HDL3 (500-3000mug protein/ml) for 30 min during Quin-2 or 45Ca loading periods, or for 30 min prior to Ca+ uptake or Na+/H+ exchange measurements. Amiloride-sensitive 22Na uptake was measured as previously described. HDL2 reduced and HDL3 (500?g protein/ml) enhanced AVP-stimulated increases in [Ca2+]i by 21% and 30% respectively (p<0.05). Since neither HDL fraction altered the [Ca2+]i response to AVP in platelets treated with EGTA, the differential effects of HDL appear to be due to changes in Ca2+ influx or efflux rather than release of stored Ca2+. Both HDL2 and HDL3 reduced the initial rate of basal 45Ca uptake in a dose-dependent manner (p<0.01); uptake by AVP-stimulated platelets was similarly reduced but the effect was not statistically significant. The Ca2+ content of platelets equilibrated with 45Ca for 30 min was less in the presence of either HDL2 or HDL3. Neither HDL2 nor HDL3 appeared to significantly affect platelet 45Ca efflux rate. Km and Vmax values for platelet Na+/H+ exchange in the presence of HDL2 were not different from that in untreated platelets (38.3+/-4.5 mmo1/1 and 377.4+/-29.6 pmo1/106cells/min versus 46.5+/-6.3 mmo1/1 and 428.8+/-25.5 pmo1/106cells/min respectively). Vmax was increased in the presence of HDL3 (545.9+/-43.5 pmo1/106cells/min) compared to that in untreated platelets (p<0.05). Km values remained unchanged (48.1+/-2.7 mmo1/1). The present results showing increases and decreases respectively in AVP-stimulated increases in [Ca2+]i in response to HDL2 and HDL3 explain previous observations of pro- and anti-aggregatory effects of these HDL subfractions. Effects on transport of calcium in and out of the cell might account for differences in responses to HDL subfractions. Na+/H+ exchanger activity, which may enhance Ca2+ mobilization through increases in pHi was enhanced by HDL3 but not by HDL2.4. In view of the in vitro findings of lipoproteins (above) we investigated whether relationships between circulating lipids and cation transport might explain aspects of cardiovascular disease and diabetes. Na+/H+ exchange and [Ca2+]i metabolism in platelets from normotensive subjects were compared with plasma lipoprotein and apolipoprotein profiles from normotensive subjects. In the same subjects, the impact of Apo E phenotype on plasma lipids and platelet cation transport were also considered. (Abstract shortened by ProQuest.)

From animal models to patients : the role of placental microRNAs, miR-210, miR-126, and miR-148a/152 in preeclampsia

Placental microRNAs (miRNAs) regulate the placental transcriptome and play a pathological role in preeclampsia (PE), a hypertensive disorder of pregnancy. Three PE rodent model studies explored the role of placental miRNAs, miR-210, miR-126, and miR-148/152 respectively, by examining expression of the miRNAs, their inducers, and potential gene targets. This review evaluates the role of miR-210, miR-126, and miR-148/152 in PE by comparing findings from the three rodent model studies with in vitro studies, other animal models, and preeclamptic patients to provide comprehensive insight into genetic components and pathological processes in the placenta contributing to PE. The majority of studies demonstrate miR-210 is upregulated in PE in part driven by HIF-1a and NF-?Bp50, stimulated by hypoxia and/or immune-mediated processes. Elevated miR-210 may contribute to PE via inhibiting anti-inflammatory Th2-cytokines. Studies report an up- and downregulation of miR-126, arguably reflecting differences in expression between cell types and its multifunctional capacity.MiR-126 may play a pro-angiogenic role bymediating the PI3K-Akt pathway. Most studies report miR-148/152 family members are upregulated in PE. Evidence suggests they may inhibit DNA methylation of genes involved in metabolic and inflammatory pathways. Given the genetic heterogeneity of PE, it is unlikely that a single placental miRNA is a suitable therapeutic target for all patients. Investigating miRNAs in PE subtypes in patients and animal models may represent a more appropriate approach going forward. Developing methods for targeting placental miRNAs and specific placental cell types remains crucial for research seeking to target placental miRNAs as a novel treatment for PE

Sex differences in preclinical models of hypertension

Hypertension remains the primary contributor in the development of cardiovascular disease which is rapidly increasing worldwide. High blood pressure affects men and women differently and understanding these sex differences is the ultimate unmet need for researchers in this field. Due to the inherent differences in hypertension prevalence, control and outcomes between men and women, novel research needs to be carried out to tackle these disparities and improve targeted treatment. Animal models of hypertension have provided valuable insights into the sexual dimorphism of blood pressure mechanisms. The availability of genetic and non-genetic hypertensive strains allows the opportunity to study diverse environmental and genetic factors that affect blood pressure, therefore presenting a valuable tool for researchers. Sex differences are present before birth and throughout life, which presents a challenge for the study of disease development in humans, but these complexities can be resolved with the use of in vivo models that display similarities to human disease. The aim of the present review is to provide an overview of the different available animal models of hypertension that present sexual dimorphisms and to discuss their relevance to humans

The role of tumor necrosis factor-α and natural killer cells in uterine artery function and pregnancy outcome in the stroke prone spontaneously hypertensive rat

Women with chronic hypertension are at increased risk of maternal and fetal morbidity and mortality. We have previously characterized the stroke-prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery function and adverse pregnancy outcome compared with the control Wistar–Kyoto. The activation of the immune system plays a role in hypertension and adverse pregnancy outcome. Therefore, we investigated the role of tumor necrosis factor-[alpha] in the SHRSP phenotype in an intervention study using etanercept (0.8 mg/kg SC) at gestational days 0, 6, 12, and 18 in pregnant SHRSP compared with vehicle-treated controls (n=6). Etanercept treatment significantly lowered systolic blood pressure after gestational day 12 and increased litter size in SHRSP. At gestational day 18, etanercept improved the function of uterine arteries from pregnant SHRSP normalizing the contractile response and increasing endothelium-dependent relaxation, resulting in increased pregnancy-dependent diastolic blood flow in the uterine arteries. We identified that the source of excess tumor necrosis factor-[alpha] in the SHRSP was a pregnancy-dependent increase in peripheral and placental CD3– CD161+ natural killer cells. Etanercept treatment also had effects on placental CD161+ cells by reducing the expression of CD161 receptor, which was associated with a decrease in cytotoxic granzyme B expression. Etanercept treatment improves maternal blood pressure, pregnancy outcome, and uterine artery function in SHRSP by antagonizing signaling from excess tumor necrosis factor-[alpha] production and the reduction of granzyme B expression in CD161+ natural killer cells in SHRSP

Utilising proteomics to understand and define hypertension: where are we and where do we go?

Introduction: Hypertension is a complex and multifactorial cardiovascular disorder. With different mechanisms contributing to a different extent to an individual’s blood pressure the discovery of novel pathogenetic principles of hypertension is challenging. However, there is an urgent and unmet clinical need to improve prevention, detection and therapy of hypertension in order to reduce the global burden associated with hypertension-related cardiovascular diseases. Areas covered: Proteomic techniques have been applied in reductionist experimental models including angiotensin II infusion models in rodents and the spontaneously hypertensive rat in order to unravel mechanisms involved in blood pressure control and end organ damage. In humans proteomic studies mainly focus on prediction and detection of organ damage, particularly of heart failure and renal disease. Whilst there are only few proteomic studies specifically addressing human primary hypertension there are more data available in hypertensive disorders in pregnancy such as preeclampsia. We will review these studies and discuss implications of proteomics on precision medicine approaches. Expert commentary: Despite the potential of proteomic studies in hypertension there has been moderate progress in this area of research. Standardised large-scale studies are required in order to make best use of the potential that proteomics offers in hypertension and other cardiovascular diseases

Modeling superimposed preeclampsia using Ang II (Angiotensin II) infusion in pregnant stroke-prone spontaneously hypertensive rats

Hypertensive disorders of pregnancy are the second leading cause of maternal deaths worldwide. Superimposed preeclampsia is an increasingly common problem and often associated with impaired placental perfusion. Understanding the underlying mechanisms and developing treatment options are crucial. The pregnant stroke-prone spontaneously hypertensive rat has impaired uteroplacental blood flow and abnormal uterine artery remodeling. We used Ang II (angiotensin II) infusion in pregnant stroke-prone spontaneously hypertensive rats to mimic the increased cardiovascular stress associated with superimposed preeclampsia and examine the impact on the maternal cardiovascular system and fetal development. Continuous infusion of Ang II at 500 or 1000 ng/kg per minute was administered from gestational day 10.5 until term. Radiotelemetry and echocardiography were used to monitor hemodynamic and cardiovascular changes, and urine was collected prepregnancy and throughout gestation. Uterine artery myography assessed uteroplacental vascular function and structure. Fetal measurements were made at gestational day 18.5, and placentas were collected for histological and gene expression analyses. The 1000 ng/kg per minute Ang II treatment significantly increased blood pressure (P&lt;0.01), reduced cardiac output (P&lt;0.05), and reduced diameter and increased stiffness of the uterine arteries (P&lt;0.01) during pregnancy. The albumin:creatinine ratio was increased in both Ang II treatment groups (P&lt;0.05; P&lt;0.0001). The 1000 ng/kg per minute–treated fetuses were significantly smaller than vehicle treatment (P&lt;0.001). Placental expression of Ang II receptors was increased in the junctional zone in 1000 ng/kg per minute Ang II–treated groups (P&lt;0.05), with this zone showing depletion of glycogen content and structural abnormalities. Ang II infusion in pregnant stroke-prone spontaneously hypertensive rats mirrors hemodynamic, cardiac, and urinary profiles observed in preeclamptic women, with evidence of impaired fetal growth

Vascular dysfunction and fibrosis in stroke-prone spontaneously hypertensive rats: the aldosterone-mineralocorticoid receptor-Nox1 Axis

Aims: We questioned whether aldosterone and oxidative stress play a role in vascular damage in severe hypertension and investigated the role of Nox1 in this process. Materials and methods: We studied mesenteric arteries, aortas and vascular smooth muscle cells (VSMC) from WKY and SHRSP rats. Vascular effects of eplerenone or canrenoic acid (CA) (mineralocorticoid receptor (MR) blockers), ML171 (Nox1 inhibitor) and EHT1864 (Rac1/2 inhibitor) were assessed. Nox1-knockout mice were also studied. Vessels and VSMCs were probed for Noxs, reactive oxygen species (ROS) and pro-fibrotic/inflammatory signaling. Key findings: Blood pressure and plasma levels of aldosterone and galectin-3 were increased in SHRSP versus WKY. Acetylcholine-induced vasorelaxation was decreased (61% vs 115%) and phenylephrine-induced contraction increased in SHRSP versus WKY (Emax 132.8% vs 96.9%, p &lt; 0.05). Eplerenone, ML171 and EHT1864 attenuated hypercontractility in SHRSP. Vascular expression of collagen, fibronectin, TGFβ, MCP-1, RANTES, MMP2, MMP9 and p66Shc was increased in SHRSP versus WKY. These changes were associated with increased ROS generation, 3-nitrotyrosine expression and Nox1 upregulation. Activation of vascular p66Shc and increased expression of Nox1 and collagen I were prevented by CA in SHRSP. Nox1 expression was increased in aldosterone-stimulated WKY VSMCs, an effect that was amplified in SHRSP VSMCs (5.2vs9.9 fold-increase). ML171 prevented aldosterone-induced VSMC Nox1-ROS production. Aldosterone increased vascular expression of fibronectin and PAI-1 in wild-type mice but not in Nox1-knockout mice. Significance: Our findings suggest that aldosterone, which is increased in SHRSP, induces vascular damage through MR-Nox1-p66Shc-mediated processes that modulate pro-fibrotic and pro-inflammatory signaling pathways

Ultrasound and microbubble gene delivery for targeting altered placental microRNAs in preeclampsia

Ultrasound (US) and microbubble (MB) gene delivery has attracted growing interest as a clinically applicable gene therapy (GT). Though preclinical studies have investigated the system in various tissues, there is limited research in targeting the placenta. This is a potential therapeutic strategy for preeclampsia (PE), which has an underlying genetic basis and ineffective management strategies. Differentially expressed placental microRNAs (miRNAs) in PE may represent suitable targets for GT. Microbubbles (SonoVue) and plasmid (pGL3 or pGL4.13) were administered systemically to CD1 mice, followed by exposure of the heart to US (H14, 1.8 M.I., 1cm focal depth, 2 minutes), using Siemens Acuson Sequoia-512 system and 15L8 probe. Luciferase assays were performed to evaluate gene transfection. Significantly differentially expressed placental miRNAs in PE patients were identified as candidates based on detection by three or more screening studies. Expression of candidate miRNAs was measured by qRT-PCR in PE rat model placentas. In trial 1, low levels of luciferase activity were detected in the heart of treatment mouse 1, 2 and 3. In trial 2, luciferase activity was evident in the atria of treatment mouse 2. In trial 3, higher luciferase activity was detected in the ventricles of the treatment mouse and activity was also detected in the atria. The literature review identified eight candidate miRNAs. MiR-223 (1.46-fold increase) and miR-181a (0.81-fold decrease) were significantly differentially expressed in PE rat model placentas. MiR-223 and -181a may represent targets for US and MB gene delivery. Future studies will apply the US and MB gene delivery protocol for translation to targeting the placenta in our PE rodent model

Dissecting the genetic components of a quantitative trait locus for blood pressure and renal pathology on rat chromosome 3

Background: We have previously confirmed the importance of rat chromosome 3 (RNO3) genetic loci on blood pressure elevation, pulse pressure (PP) variability and renal pathology during salt challenge in the stroke-prone spontaneously hypertensive (SHRSP) rat. The aims of this study were to generate a panel of RNO3 congenic sub-strains to genetically dissect the implicated loci and identify positional candidate genes by microarray expression profiling and analysis of next-generation sequencing data. Method and results: A panel of congenic sub-strains were generated containing Wistar-Kyoto (WKY)-introgressed segments of varying size on the SHRSP genetic background, focused within the first 50 Mbp of RNO3. Haemodynamic profiling during salt challenge demonstrated significantly reduced systolic blood pressure, diastolic blood pressure and PP variability in SP.WKYGla3a, SP.WKYGla3c, SP.WKYGla3d and SP.WKYGla3e sub-strains. Only SBP and DBP were significantly reduced during salt challenge in SP.WKYGla3b and SP.WKYGla3f sub-strains, whereas SP.WKYGla3g rats did not differ in haemodynamic response to SHRSP. Those sub-strains demonstrating significantly reduced PP variability during salt challenge also demonstrated significantly reduced renal pathology and proteinuria. Microarray expression profiling prioritized two candidate genes for blood pressure regulation (Dnm1, Tor1b), localized within the common congenic interval shared by SP.WKYGla3d and SP.WKYGla3f strains, and one candidate gene for salt-induced PP variability and renal pathology (Rabgap1), located within the region unique to the SP.WKYGla3d strain. Comparison of next-generation sequencing data identified variants within additional positional genes that are likely to affect protein function. Conclusion: This study has identified distinct intervals on RNO3-containing genes that may be important for blood pressure regulation and renal pathology during salt challenge