Normotensive WKY Rats Research Articles

Modulation of left ventricular hypertrophy in spontaneously hypertensive rats by acetylcholinesterase and ACE inhibitors: physiological, biochemical, and proteomic studies

BackgroundThe consequences at the molecular level and the mechanisms involved in a possible cardioprotective effect of antihypertensive treatment are not yet fully understood. Here, the efficacy of pyridostigmine (PYR) and trandolapril (TRA) as antihypertensive and antihypertrophic agents was investigated and compared in hypertensive SHR and normotensive WKY rats. In parallel, we investigated the effects of these drugs on myocardial β-adrenergic and cholinergic signaling pathways and protein expression profiles.MethodsAge-matched male SHR and WKY rats were chronically (8 weeks) treated with PYR or TRA in drinking water. Blood pressure (BP) and heart rate (HR) were monitored telemetrically prior to tissue sampling for biochemical analysis. Baroreceptor reflex sensitivity (BRS) and methylatropine HR response as a measure of vagal tone were evaluated in separate groups of animals.ResultsPYR slightly lowered BP and HR in SHR rats during the dark phase of the day, while TRA effectively reduced BP during the light and dark phases without affecting HR. PYR enhanced BRS and improved vagal tone. There were no significant alterations in myocardial β-adrenergic and cholinergic signaling, with the exception of decreased forskolin-stimulated adenylyl cyclase (AC) activity in SHR rats, which was restored by TRA. Proteomic analysis revealed numerous differences induced by both treatments. Notable were changes in TGFβ-related signaling pathways as well as proteins involved in modifying hemodynamic parameters and cardiac hypertrophy.ConclusionsPYR is able to slightly decrease BP and HR in SHR rats but effectively increase BRS through vagal potentiation. The specific differences in protein expression profiles in rat myocardium induced by treatment with PYR and TRA reflect different mechanisms of action of these two agents at the molecular level.

Abstract P436: Increased West Nile Virus Infection And Pathology In The Spontaneously Hypertensive Rat

Introduction: West Nile virus (WNV), a mosquito-borne flavivirus, causes grave neuroinvasive disease and death, and has been reported in over 55,000 people in the US since 1999. Patients >55 years and those with hypertension are most at risk for WNV-associated mortality. Understanding how hypertension correlates with WNV morbidity and mortality may reduce the burden of this disease. Investigation of WNV in hypertensive people is limited to basic epidemiology. Without an animal model, understanding the causal relationship between hypertension and WNV-associated pathology remains elusive. Here, we tested a novel hypothesis that hypertension will promote WNV infection and pathology in a rodent model of age-dependent chronic hypertension, the spontaneously hypertensive rat (SHR). Methods: Male adult SHR and normotensive WKY rats were implanted with radiotelemetry transmitters for measurements of blood pressure and heart rate. Following baseline measurements, SHR and WKY rats (N=9/gp) were injected with WNV (1 X 10 4 PFU/ml, i.p.). Vehicle injection (PBS, i.p.) was performed in additional WKY and SHR (N=2-3/gp). Each rat was observed for clinical abnormalities, and body weight, blood pressure, and heart rate were measured daily. Rats were sacrificed on days 9 and 10 post-infection (PI). At endpoint, several tissues were processed for measurement of viral load by RT-qPCR and histopathology. Bone marrow and circulating immune cells (CD4 + , CD8 + , CD68 + ) were quantified at endpoint by flow cytometry. Results: One SHR rat developed neurological symptoms and was euthanized on day 4 PI. Viral load was significantly increased in the SHR kidney and spleen (P<0.001) in SHR compared to WKY rats. In addition, WNV-infection elevated heart rate in the SHR but not the WKY (P<0.0001). SHR also showed elevated circulating CD68 + monocytes (P<0.05), and a significant increase (P<0.05) in renal pathology was observed in the SHR following WNV infection. Conclusions: We demonstrate, for the first time, a positive interaction between high blood pressure and severity of WNV infection in a rodent model of chronic hypertension. Further studies are needed to elucidate the mechanisms of this interaction for new therapeutic targets of WNV disease.

Активация микроглии в головном мозге спонтанно гипертензивных крыс

Arterial hypertension is one of the most significant medical and social problems, being widespread and associated with the risk of renal failure, cardiovascular and cerebrovascular complications. The aim was to investigate the morphofunctional state of microglia in different regions of the rat brain in the setting of arterial hypertension. Brain samples from spontaneously hypertensive SHR rats aged 3–8 months (n = 4) were used as study material. Normotensive WKY rats of the same age (n = 3) were used as the control group. The work was performed using immunohistochemical analysis and confocal laser microscopy. During the quantitative analysis, we were seeking to determine the number of microgliocyte bodies and the area occupied by the bodies and processes of these cells per 1 mm2 of the nervous tissue. An immunohistochemical reaction for calcium-binding protein Iba1 revealed that in rats with arterial hypertension, microglia in the cerebral cortex, striatum, subcortical white matter and subfornical organ showed morphological signs of activation: increased body size and thickening of the processes of these cells. The strongest activation is demonstrated by microglia of the subfornical organ, which is in a preactivated state in normotensive rats. The performed statistical analysis revealed a trend towards an increase in the amount of microglia in the brain in SHR rats compared to animals in the control group. The Iba1/CD68 double immunofluorescence reaction showed no changes in the amount and/or distribution of lysosomal CD68 protein in spontaneously hypertensive rats compared to control group. The results obtained indicate chronic activation of microglia in the brain of spontaneously hypertensive rats. Activation of microglia in this case is not accompanied by an increase in the phagocytic activity of these cells.

Prophylactic Treatment with Hydrogen Sulphide Can Prevent Renal Ischemia-Reperfusion Injury in L-NAME Induced Hypertensive Rats with Cisplatin-Induced Acute Renal Failure.

(Background and Objectives): Renal ischemia perfusion injury is one of the major issues in kidney transplant. The aim of the study was to investigate the hypothesis that prophylactic treatment-with a hydrogen sulphide donor to an acute renal failure case of hypertensive rats-can minimize the ischemia reperfusion injury of the kidney which is beneficial for kidney transplant. To check this hypothesis, the present study was designed to investigate the effect of chronic administration of a hydrogen sulphide (H2S) donor and sodium hydrosulfide (NaHS) on nuclear factor kappa B (NF-kB) and inter cellular adhesion molecule-1 (ICAM-1) concentration in non-renal failure (NRF) and acute renal failure (ARF) rats in the ischemia-reperfusion injury (IRI) model of the kidney in both normotensive WKY and hypertensive rats (L-nitro arginine methyl ester (L-NAME-induced); (Materials and Methods): A total number of 48 Sprague-Dawley rats were recruited into eight groups each consisting of six animals. Each of these eight groups was used to measure systemic and renal parameters, H2S, antioxidant parameters in plasma, plasma concentration of NF-kB and ICAM-1 and renal cortical blood pressure. ARF was induced by single intraperitoneal (i.p.) cisplatin injection (5 mg/kg). Hypertension was induced by oral administration of L-NAME in drinking water for four weeks at 40 mg/kg/day. NaHS was administered (i.p) at 56 µmol/kg for five weeks while dL-propargylglycine (PAG), a H2S generation inhibitor, was administered as a single intra-peritoneal injection (50 mg/kg). An acute surgical experiment was performed for the induction of renal ischemia for 30 min by renal artery clamping followed by reperfusion for three hours; (Results): Chronic administration of NaHS attenuated the severity of ARF in both normotensive and hypertensive animals (L-NAME) along with lowering the blood pressure in hypertensive groups. NaHS improved the oxidative stress parameters such as total superoxide dismutase (T-SOD), glutathione (GSH) and reduced the malondialdehyde (MDA) concentration along with reduction of NF-kB and ICAM-1 following renal IRI; Conclusions: These findings demonstrate that H2S not only reduced the severity of cisplatin induced ARF but also reduced the severity of renal IRI by upregulating antioxidants along with decreased concentrations of NF-kB and ICAM-1 in normotensive and L-NAME induced hypertensive rats.

Abstract 14157: Effects of Follistatin on the Regulation of Vascular Function by Perivascular Adipose Tissue in a Model of Essential Hypertension

Introduction: We previously showed that follistatin (FST), a potent activin inhibitor, lowers BP in the spontaneously hypertensive rat (SHR), a model of essential HTN. This was associated with improved vessel structure (reduced collagen) and function (reduced hypercontractility and improved endothelium-dependent relaxation). FST also reduced vascular oxidative stress (ROS). Perivascular adipose tissue (PVAT) is known to regulate vascular tone. FST induces browning and thus increased thermogenic activity of peripheral adipose tissue. Transition from white to brown adipose tissue is associated with decreased risk of CVD. Here, we assess FST effects on PVAT and its regulation of SHR vascular function. Methods: SHR were treated with FST from 12-20 weeks of age. Normotensive WKY rats served as controls. Mesenteric resistance arteries were isolated with or without intact PVAT. Vessel function was assessed using wire myography. In separate studies, white mesenteric and brown aortic PVAT, or peripheral fat depots, were isolated from WKY rats. These were introduced to WKY mesenteric vessels to study PVAT-isolated function. Results: Intact PVAT augmented constriction to KCl in SHR, but not WKY arteries, an effect prevented by FST. Improvement in endothelium-dependent vasodilation in FST-treated SHR was PVAT-dependent. SHR PVAT had increased adipocyte size and ROS (by DHE), both reversed by FST. Isolated PVAT from WKY incubated with pyocyanin (30’) to induce ROS augmented the KCl response, suggesting that FST inhibition of PVAT oxidative stress in HTN restores beneficial vascular effects. Incubation of WKY vessels with brown PVAT inhibited constriction to KCl more effectively than did white PVAT. Interestingly, peripheral white adipose (inguinal) did not have any anticontractile effect. Brown adipose (interscapular) effects were intermediate. Conclusions: Improvement in anticontractile effects of PVAT by FST in resistance vessels is associated with reduced PVAT oxidative stress. Brown PVAT is more effective than white PVAT in inhibiting vessel contraction, and unlike white PVAT, peripheral white adipose does not have anticontractile effects. Future work will assess effects of FST on PVAT browning, and whether browning itself can improve BP.

Abstract T5: Molecular Mechanisms Involved In The Vascular Protective Effects Of Mas1 And Et B R Interaction

We demonstrated that Mas1 and ET B R physically interact in endothelial cells inducing Ang-(1-7) vascular protection. Using high throughput screening of >20,000 druggable compounds, we identified a number of molecules that enhance Mas1:ET B R interactions. Of these, 2 potently enhanced interaction between the receptors. These enhancers (Enh), termed Enh3 and Enh4 were used to assess Mas1:ET B R interaction and cellular functional responses in vascular smooth muscle cells (VSMCs) from normotensive WKY and hypertensive SHRSP rats. Cells were exposed to Enh 3 and Enh4 (10 -5 M) for short (5,15 and 30min) and long timepoints (5hours). Expression of signaling molecules was assessed by immunoblotting and Ca 2+ influx was evaluated using fluorescence microscopy. In WKY VSMCs, Enh 3 short term stimulation reduced basal ERK1/2 phosphorylation (26.8±5.9% vs veh, p<0.01), an effect absent in SHRSP VSMCs, while Enh 4 had no effect. Short term exposure to Enh 4, but not Enh3, reduced basal MLC20 phosphorylation (54.9±7.5% vs veh, p<0.001) in WKY but not in SHRSP VSMCs. In SHRSP VSMCs, Enh 3, but not Enh4, reduced basal AKT phosphorylation (63.5±8.9% vs veh, p<0.001). Long term stimulation with Enh 3 reduced expression of AKT (38.0±2.0% vs veh, p<0.001), PCNA (60.0±7.0% vs veh, p<0.001), and VCAM-1 (35.0±8.0% vs veh, p<0.001) in WKY VSMCs. Similarly, AKT (35.0±12.0% vs veh, p<0.05) expression was reduced by Enh 3, with no effect on other markers in SHRSP VSMCs. Enh 4 long-term stimulation reduced AKT expression (30.0±10.0% vs veh, p<0.05) in WKY VSMCs, without effect on SHRSP VSMCs. ET-1 induced Ca 2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. In conclusion, enhancing Mas1:ET B R interaction attenuates mitogenic and pro-inflammatory signaling pathways in WKY and SHRSP VSMCs. Enhancing interaction between these receptors does not increase Ca 2+ signaling, important in VSMC contraction. Our data suggest that Enh3 and Enh4 may have VSMC protective effects and that they do not amplify injurious signaling induced by ET-1/ETBR. These findings identify a potential new strategy in vasoprotection.

Apelin‐Induced Relaxation is Impaired in Coronary Arteries from Spontaneously Hypertensive Rats

Hypertension is a major modifiable risk factor for coronary artery disease. The pathophysiological mechanisms underlying hypertension as a risk factor for coronary artery disease are not fully understood. Apelin is a vasoactive peptide that binds to APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G-protein-dependent and -independent pathways, including activation of G-protein-coupled-receptor kinase 2 (GRK2), which can potentially lead to inhibition of eNOS and nitric oxide (NO) production. We have previously shown that apelin causes endothelium-dependent, NO-mediated relaxation of coronary arteries from normotensive animals (JPET 366:265, 2018), but its effects in hypertensive coronary arteries are unknown. Here, we tested the hypothesis that apelin-induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Western blot analysis demonstrated increased expression of GRK2 in cultured coronary endothelial cells from SHR in comparison with normotensive WKY (control) rats. Isolated coronary arterial rings were mounted in wire myographs for isometric tension recording and contracted with 5-HT (10-7 M). Apelin (10-9-10-6 M) caused endothelium-dependent relaxation of WKY coronary arteries (pD2 = 7.00 ± 0.11; Emax = 54 ± 4% relaxation; n=6), but had no effect in rings from SHR. Conversely, endothelium-dependent relaxations to an APJ receptor biased agonist, CMF-019 (10-9-10-5 M), which preferentially activates the G-protein-dependent pathway with minimal effect on GRK2, did not differ between SHR and WKY. In the presence of apelin (10-7 M), endothelium-dependent relaxation to acetylcholine (ACh) (10-9-10-5 M) was impaired in SHR coronary arteries (pD2 = 6.82 ± 0.10 vs 6.52 ± 0.18, and Emax = 81 ± 8 vs 60 ± 10% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05); however, apelin had no effect on ACh-induced relaxation of WKY coronary arteries. By contrast, ACh-induced relaxation of SHR and WKY coronary arterial rings was unaffected by CMF-019 (10-7 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 × 10-5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2 = 6.24 ± 0.30; Emax = 42 ± 13% relaxation; n=3). Relaxations in response to the NO-donor, DEA NONOate (10-9-10-4 M), were not altered by apelin (10-7 M) treatment in either WKY or SHR coronary arteries. Taken together, the data indicate that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. In SHR coronary arteries, apelin instead actively inhibited relaxation to another endothelium-dependent vasodilator; i.e. ACh. That the effects of the G-protein biased agonist, CMF-019, did not differ between WKY and SHR coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in hypertensive arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself, as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in coronary arteries from SHR.

Изменения клеток Колмера у крыс линии SHR после ишемии головного мозга

Kolmer cells (epiplexus macrophages) are the least studied population of phagocytic cells in the mammalian brain. In the present work, we studied the response of these cells to high blood pressure and ischemic brain injury caused by a short middle cerebral artery occlusion. We used spontaneously hypertensive SHR and normotensive WKY rats. Kolmer cells of normotensive WKY animals had a small volume of perinuclear cytoplasm with a cortical concentration of Iba-1 protein. These cells had characteristic thin, sometimes tortuous processes. Moderate activation due to high blood pressure in SHR rats was accompanied by an increase in the perinuclear cytoplasm volume and the appearance of numerous straight thin processes. Further activation caused by ischemia led to the disappearance of processes and the cells became round. The structural rearrangements were accompanied by the disappearance of the Iba-1 protein concentration gradient in the cytoplasm. Also, the intranuclear accumulation of the cytoplasmic Iba-1 protein in Kolmer cells was first discovered, independent of their activation. That indicates the polyfunctionality of this protein in the cell. The obtained results point to the involvement of Kolmer cells in the brain's response to injury.

Calcitriol inhibits COX-1 and COX-2 expressions of renal vasculature in hypertension: Reactive oxygen species involved?

ABSTRACT Vitamin D modulates about 3% human gene transcription besides the classical action on calcium/phosphorus homeostasis. The blood pressure-lowing and other protective action on cardiovascular disease have been reported. The present study aims to examine whether COX-1 and COX-2 were implicated in endothelial dysfunction in hypertension and calcitriol, an active form of vitamin D preserved endothelial function through regulating COX expression. Isometric study demonstrated the impaired endothelium-dependent relaxation (EDR) in renal arteries from spontaneously hypertensive rats were reversed by 12 h-calcitriol treatment and COX-1 and COX-2 inhibitors. Combined uses of COX-1 and COX-2 inhibitor induced more improved relaxations. Exaggerated expressions of COX-1 and COX-2 in renal artery from SHR were inhibited by 12 h-administration of calcitriol, NADPH oxidase inhibitor DPI, or reactive oxygen species (ROS) scavenger tempol. Furthermore, in normotensive WKY rats, calcitriol prevents against the blunted EDR in renal arteries by 12 h-Ang II exposure, with similar improvements by COX-1 and COX-2 inhibitors. Accordingly, increased COX-1 and COX-2 expressions by Ang II exposure were corrected by losartan, DPI, or tempol. Studies on human renal artery also revealed the beneficial action of calcitriol is mediated by suppressing COX-1 and COX-2 expressions, dependent on vitamin D receptor (VDR) activation. Taken together, our findings showed that COX-1 and COX-2 are positively involved in the renovascular dysfunction in hypertension and via VDR, calcitriol benefits renovasular function by suppressing COX-1 and COX-2 expressions. Furthermore, ROS is involved in the COX-1 and COX-2 up-regulations of renal arteries, maybe serving as a mediator in the inhibitory action of calcitriol on COX expression.

Group III Metabotropic Glutamate Receptors Regulate Excitability of Hypothalamic Presympathetic Neurons and Sympathetic Output in Hypertension

Enhanced glutamatergic input to the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. Group III metabotropic glutamate receptors (mGluRs) are expressed in the PVN. In the present study, we determined the role of group III mGluRs in the control of the excitability of spinally projecting PVN neurons and sympathetic outflow in hypertension. We found that stimulation of group III mGluRs with L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) consistently inhibited the firing activity of PVN neurons in normotensive WKY rats. However, L‐AP4 inhibited 37%, but excited 48%, of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Both the inhibitory and excitatory effects of L‐AP4 on the firing activity were abolished by the group III mGluR antagonist CPPG. Inclusion of GDP‐β‐S in the intracellular recording solution eliminated the excitatory effect of L‐AP4 on the firing activity of PVN neurons in SHRs, but it did not significantly change the inhibitory effect of L‐AP4 on PVN neurons in WKY rats or SHRs. L‐AP4 significantly reduced the frequency of spontaneous excitatory postsynaptic currents of PVN neurons in both WKY rats and SHRs. Interestingly, pretreatment of brain slices with the group I mGluR agonist DHPG converted the L‐AP4 effect on PVN neurons from inhibitory to excitatory in WKY rats. Also, L‐AP4 consistently inhibited PVN neurons in SHRs after treatment with the group I mGluR antagonist MPEP. In addition, microinjection of L‐AP4 into the PVN dose‐dependently decreased arterial Bp and lumbar sympathetic nerve activity (LSNA) in WKY rats and SHRs. Microinjection of L‐AP4 in the presence of MPEP produced a more significant reduction in arterial BP and LSNA than did with microinjection of L‐AP4 alone in SHRs. These findings suggest that group III mGluRs in the PVN regulate the synaptic plasticity and excitability of PVN presympathetic neurons and sympathetic vasomotor activity in hypertension. The signaling of postsynaptic group III mGluRs is influenced by increased group I mGluR activity in PVN presympathetic neurons in hypertension. Supported by NIH grants HL131161 and HL142133.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypertensive Neuropathy: A New Clinical Entity?

Hypertension is a main risk factor for stroke and vascular dementia and may cause important changes to the cerebrovascular tree, turning the brain more susceptible to infarcts, microaneurysms and ischemia. In spite of the well documented influence of hypertension on the brain, data on the sensitivity of peripheral nerves in hypertension is scarce. Our laboratory has been working for decades to help understanding how hypertension affects peripheral nerve morphology and function, using the spontaneously hypertensive rats (SHR) model. Spontaneously hypertensive rats (SHR), first inbred from Wistar Kyoto rats (WKY), are considered a good experimental model of human essential hypertension. The purpose of this study was to investigate the alterations on the morphology and morphometric data on sural, phrenic, vagus and recurrent laryngeal nerves of SHR, previous to the development of hypertension and with well‐established hypertension. Male and female SHR and normotensive WKY rats aged 5, 8 and 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural, vagus, phrenic and recurrent laryngeal nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Nerves from young animals did not show differences between sides and genders. Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR in all nerves. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified in nerves from adult hypertensive animals. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. These results indicate that hypertension altered important morphometric parameters related to nerve conduction in hypertensive animals. Moreover the comparison between males and females of WKY and SHR showed that the morphological and morphometric alterations due to hypertension are not gender related. The presence of a so called “hypertensive neuropathy” is documented with endoneural vascular lesions, axonal atrophy and loss of small myelinated fibers.Support or Funding InformationSupport: CNPq, FAPESP and FAEPAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Cerebellar adrenomedullin: A new target for blood pressure regulation

Adrenomedullin (AM) and their receptor components, CRLR, RAMP1, RAMP2 and RAMP3 are widely expressed in the central nervous system, including cerebellum. Recent evidence suggests a role for cerebellar adrenomedullinergic system in blood pressure (BP) regulation. We assessed AM, RAMP1, RAMP2, RAMP3 and CRLR expression; and AM receptor signaling pathway in the cerebellar vermis of WKY and SHR rats. Our findings demonstrated that CRLR, RAMP1 and RAMP3 expression was higher in cerebellum of SHR rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16 week old rats. In regard of AM signaling pathways, we show that AM activates ERK, increases cAMP production, increases cGMP production and NO accumulation. These effects are mediated through the activation of AM 1 receptor, since AM 22-52 blunted AM action, meanwhile AM increase of cAMP production is also mediated through stimulation of AM2 and CGRP receptors. Furthermore, AM decreased TBARS production and antioxidant enzymes basal activity: catalase (CAT), gluthation peroxidase (GPx) and superoxide dismutase (SOD). During hypertension, AM signaling pathways in the cerebellar vermis are altered. Effectively, in WKY rats AM decreased the activity of three antioxidant enzymes and TBARS production, while in SHR rats AM was unable to affect these signaling pathways; AM-induced ERK1/2 activation in cerebellum of the SHR was smaller in magnitude than in WKY; and AM increased cGMP/NO production in cerebellar vermis of the WKY rats, while in SHR rats AM was unable to stimulate this signaling pathway. We anticipated a possible functional role of cerebellar AM on BP regulation. Indeed, in vivo microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR but not in normotensive WKY rats. Co-injections of a putative AM receptor antagonist, AM 22–52 abolished the decreases in MAP evoked by AM, showing that AM acts through its AM1 receptor to reduce blood pressure. These findings demonstrate dysregulation of cerebellar AM system during hypertension, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise, they constitute a novel mechanism of BP control which has not been described so far.

In Hypertension, Global Endothelial Calcium Underlies a Protective Mechanism that Limits Arterial Vasoconstriction.

In small arteries, increased vascular reactivity contributes to the raised blood pressure that characterizes established hypertension. Endothelial function is altered in hypertension and may confer protection against the increased vascular reactivity but the precise mechanisms involved are unresolved. Here, in isolated small mesenteric arteries from hypertensive SHR (arterial BP, 190 ± 4 mmHg) and age‐matched normotensive WKY (arterial BP, 120 ± 4 mmHg) rats, the mechanisms of altered endothelial function were examined using simultaneous measurement of arterial contraction and endothelial Ca2+ signalling in large fields (~2000) of cells.At rest, average Ca2+ concentration was higher in endothelium from SHR rats, compared to that in normotensive WKY controls (n = 8 in each group). In both groups, phenylephrine (1 μM) evoked an arterial contraction that had no accompanying change in endothelial Ca2+ levels during the contraction. However, phenylephrine‐induced (1 μM) contraction was reduced in arteries from SHR, compared to WKY rats (p &lt; 0.05; n = 5 in each group). In both normotensive and hypertensive arteries, selective disruption of endothelial Ca2+ signalling, using BAPTA‐AM (30 μM), inhibited ACh‐mediated endothelial Ca2+ signalling and artery relaxation and increased the contractions evoked by phenylephrine (n = 5 per group). After BAPTA treatment, the response to phenylephrine was normalised among groups – i.e. 1 μM phenylephrine evoked a maximal contraction in both groups. The nitric oxide synthase inhibitor L‐NAME also normalised phenylephrine‐induced contractions in pressurised arteries. These results suggest that basal, Ca2+‐dependent, nitric oxide production may be upregulated in the endothelium of SHR rats to limit arterial contractions. To explore this possibility further, local spontaneous endothelial Ca2+ signals were examined using high spatial (100X, NA = 1.4) and temporal (20 Hz) resolution imaging. In the absence of stimulation with pharmacological agents, spontaneous local Ca2+ events occurred frequently in the endothelium of blood vessels from WKY rats. These events had a wide spectrum of amplitudes and durations. A spatial map of the location and distribution of the local Ca2+ sites showed the majority of local events occurred close to junctions with smooth muscle cells to a greater extent than would be expected if the Ca2+ events initiated randomly throughout the cytoplasm (p &lt; 0.05; n = 8 in each group). These findings suggest that the local endothelial Ca2+ rises may account for an endothelial‐dependent opposition to phenylephrine‐induced tone. However, the propensity of local Ca2+ signals was significantly decreased in arteries from SHR compared to those from WKY rats (p &lt; 0.05; n = 8 in each group). This result suggests that alterations in local endothelial Ca2+ signal dynamics are unlikely to explain the increased endothelium‐dependent negative feedback that opposes phenylephrine‐evoked contraction in hypertension.We suggest the elevated global Ca2+ concentration in endothelial cells at rest accounts for increased NO production and suppressed phenylephrine‐evoked contraction. The increased endothelial global Ca2+ concentration may be a compensatory mechanism to offset the increased contractile responsiveness of smooth muscle cells in the hypertensive rat arteries.Support or Funding InformationThis work was funded by the Wellcome Trust (202924/Z/16/Z) and the British Heart Foundation (PG/16/54/32230; PG16/82/32439), whose support is gratefully acknowledged. C.W. is supported by a Sir Henry Wellcome Postdoctoral Research Fellowship (204682/Z/16/Z).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Short hairpin RNA interference targeting interleukin 1 receptor type I in the paraventricular nucleus attenuates hypertension in rats.

Blood pressure is controlled by tonic sympathetic activities, excessive activation of which contributes to the pathogenesis and progression of hypertension. Interleukin (IL)-1β in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. Here, we investigated the therapeutic effects of IL-1 receptor type I (IL-1R1) gene silencing in the PVN on hypertension. Recombinant lentivirus vectors expressing a short hairpin RNA (shRNA) targeting IL-1R1 (Lv-shR-IL-1R1) or a control shRNA were microinjected into PVN of spontaneously hypertensive rats (SHRs) and normotensive WKY rats. The fluorescence of green fluorescent protein-labelled vectors appeared at 2weeks after injection and persisted for at least 8weeks. IL-1R1 protein expression in the PVN was reduced 4weeks after Lv-shR-IL-1R1 injection in SHRs. IL-1R1 interference also reduced basal sympathetic activity, cardiac sympathetic afferent reflex in SHRs. Depressor effects were observed from week 2 to 10 after Lv-shR-IL-1R1 treatment in SHRs, with the most prominent effects seen at the end of week 4. Furthermore, Lv-shR-IL-1R1 treatment decreased the ratio of left ventricular weight to body weight and cross-sectional areas of myocardial cells in SHRs. Additionally, Lv-shR-IL-1R1 treatment prevented an increase in superoxide anion and pro-inflammatory cytokines (PICs, TNF-α and IL-1β) in the PVN of SHR, and upregulated anti-inflammatory cytokine (AIC, IL-10) expression. These results indicate that shRNA interference targeting IL-1R1 in the PVN decreases arterial blood pressure, attenuates excessive sympathetic activity and cardiac sympathetic afferent reflex, and improves myocardial remodelling in SHRs by restoring the balance between PICs and AICs to attenuate oxidative stress.

Indonesian herbal medicine prevents hypertension-induced left ventricular hypertrophy by diminishing NADPH oxidase-dependent oxidative stress.

Indonesian herbal medicine Centella asiatica, Justicia gendarussa and Imperata cylindrica decoction (CJID) are known to be efficacious for hypertension. Oxidative stress plays an important role in hypertension-induced left ventricular hypertrophy (H-LVH). This study evaluated whether CJID inhibit cardiac remodeling in spontaneously hypertensive rats (SHRs) through mechanism of oxidative stress-related cardiac-NADPH oxidase (NOXs) pathway: NOX1, NOX2 and NOX4. Forty-weeks-old SHRs and normotensive-WKY rats, were both randomly divided into 2 groups: CJID and control. All rats were treated for 5 weeks. Systolic blood pressure (SBP) and heart rate (HR) were measured. LV morphology, function and performance were assessed by histological staining and echocardiography. Serum and cardiac superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were assessed. Cardiac superoxide and hydrogen peroxide (H2O2) productions, protein expressions of SOD2, SOD3, NOX1, NOX2 and NOX4 were also determined. We found that SBP and HR were significantly decreased in SHRs-treated group. Echocardiography showed that CJID significantly improved LV morphometry and function. CJID decreased MDA level, but increased SOD activity. Cardiac superoxide and H2O2 generation were decreased in SHRs-treated group. CJID caused cardiac SODs expressions to be increased but NOXs expressions to be suppressed. In conclusion, CJID prevents H-LVH by reducing reactive oxygen species production via the NOXs-dependent pathway.

Sex-difference in expression and function of beta-adrenoceptors in macrovessels: role of the endothelium.

Estrogen modulates adrenergic reactivity of macrovessels, resulting in weaker α-adrenergic vasoconstriction in females than males. However, the mechanisms governing this important sex-specific difference are not well understood. We hypothesized that vessels of females express more dilatory β-adrenoceptors, which counteract constrictive effects of α-adrenoceptors. This hypothesis was tested using aortas of normotensive (WKY) and hypertensive rats (SHR), along with human mammary artery. Selective blockade of β1 (CGP20712) or β3 (SR59230A), but not β2 (ICI118,551) adrenoceptors, greatly increased α-adrenergic constriction (norepinephrine) of aorta in female SHRs, but not in male SHRs at 12weeks of age. Consistently, the selective β1/β2 (isoproterenol) and β3-adrenergic (BRL37344) relaxation was stronger in female SHRs than in males. Removal of endothelium and use of L-NMMA abolished sex-difference in α-adrenergic constriction and β-adrenergic relaxation. Immunostainings revealed endothelial localization of β1- and β3-adrenoceptors. mRNA levels of aortic β1- and β3-, but not β2-adrenoceptors were markedly higher in female than in male SHRs. The sex-specific differences in α-adrenergic constriction and β-adrenoceptor mRNA levels were age-dependent, predominantly present up to 29weeks and disappeared at 36weeks of age. The sex-specific difference was not strain-dependent and was similarly present in normotensive WKY rats. Human mammary artery of women showed a weaker α-adrenergic constriction than arteries of men. This sex-specific difference was prominent at 45-65years and disappeared with aging. Our results convincingly demonstrate that female macrovessels express more dilatory β1- and β3-adrenoreceptors than male vessels with a predominant endothelial localization. This sex-specific difference is functionally relevant in young adults and is attenuated with aging.

The phosphoinositide-3 kinase signaling is involved in neuroinflammation in hypertensive rats.

It has been demonstrated that neuroinflammation is associated with cardiovascular dysfunction. The phosphoinositide-3 kinase (PI3K) signaling in the rostral ventrolateral medulla (RVLM), a key region for sympathetic outflow, is upregulated and contributes to increased blood pressure (BP) and sympathetic outflow in hypertension. This study was designed to determine the role of the PI3K signaling in neuroinflammation in the RVLM of hypertension. The normotensive WKY rats were performed by intracisternal infusion of lipopolysaccharide (LPS) or angiotensin II (Ang II) for inducing neuroinflammation. Elisa was used to determine the level of proinflammatory cytokines. Western blot was employed to detect the protein expression of PI3K signaling pathway. Gene silencing of PI3K p110δ subunit and overexpression of angiotensin-converting enzyme 2 (ACE2) were realized by injecting related lentivirus into the RVLM. In the spontaneously hypertensive rats (SHR), the PI3K signaling in the RVLM was upregulated compared with WKY, gene silencing of PI3K in the RVLM significantly reduced BP and renal sympathetic nerve activity (RSNA), but also decreased the levels of proinflammatory cytokines. In the WKY rats, central infusion of LPS and Ang II significantly elevated BP and RSNA, but also increased the levels of proinflammatory cytokines and PI3K signaling activation in the RVLM. These changes in the Ang II-induced hypertension were effectively prevented by gene silencing of PI3K in the RVLM. Furthermore, overexpression of ACE2 in the RVLM significantly attenuated high BP and neuroinflammation, as well as decreased the activation of PI3K signaling in hypertensive rats. This study suggests that the PI3K signaling in the RVLM is involved in neuroinflammation in hypertension and plays an important role in the renin-angiotensin system-mediated changes in neuroinflammation in the RVLM.

EFFECT OF ADDITION OF CALCIUM AND MAGNESIUM IN DRINKING WATER ON STATE OF CARDIOVASCULAR SYSTEM IN SPONTANEOUSHYPERTENSIVE RATS

The aim. To assess the effect of enrichment of calcium and magnesium in drinking water on the level of arterial pressure (BP), myocardial remodeling processes, and autorhythmic contractile activity of the portal vein (PV) of spontaneously hypertensive SHR rats and control WKY rats. Material and methods . One group of SHR rats received drinking water enriched with calcium (120 mg/L) and magnesium (45 mg/L) from a 6-week-old age within 2 months. Two other groups of SHR and WKY rats received St. Petersburg tap water with a low content of Са 2+ (8 mg/L) and Mg 2+ (3 mg/L). All groups received a standard diet. At the end of the observation period, rats were assessed with blood pressure, myocardial mass index, urea level (UR), total cholesterol (Chol), total calcium (totalCa) and albumin (Alb) in blood. A contractile activity of PV was investigated by the method of myography (in vitro). The total and maximal amplitude and frequency of contractions of PV, as well as the work done by PV was determined. Results. In SHR rats, the enrichment of drinking water of Са 2+ and Mg 2+ , prevented the progressive rise BP to the level characteristic of the SHR, resulting in its stabilization at 18% lower than in the SHR of the mineral deficient group. Theincreaseinthecontent of Са 2+ and Mg 2+ in drinking water did not affect the degree of myocardial hypertrophy, as well as on the level of UR, totalCa and Alb of blood, but led to a decrease in the level of total cholesterol compared to animals that received low-mineralized water. The options of the autorhythmic contractile activity depended on the mineralization of drinking water. The addition of Са 2+ and Mg 2+ to the drinking water of SHR rats resulted in a decrease in both the amplitude of contractions of PV and the work performed by PV as compared with rats of mineral deficient groups. Conclusion. The researches have shown the important role of mineral composition of drinking water in processes of regulation of a level of arterial pressure. In spontaneously hypertensive SHR rats, the antihypertensive effect of drinking water enrichment with Са 2+ and Mg 2+ has been revealed. The increase in the intake of exogenous calcium and magnesium with water modifies the autorhythmic contractile activity of the portal vein of spontaneously hypertensive SHR rats, normalizing it to a level characteristic of normotensive WKY rats.

Alterations in the gut microbiota can elicit hypertension in rats.

Gut dysbiosis has been linked to cardiovascular diseases including hypertension. We tested the hypothesis that hypertension could be induced in a normotensive strain of rats or attenuated in a hypertensive strain of rats by exchanging the gut microbiota between the two strains. Cecal contents from spontaneously hypertensive stroke prone rats (SHRSP) were pooled. Similarly, cecal contents from normotensive WKY rats were pooled. Four-week-old recipient WKY and SHR rats, previously treated with antibiotics to reduce the native microbiota, were gavaged with WKY or SHRSP microbiota, resulting in four groups; WKY with WKY microbiota (WKY g-WKY), WKY with SHRSP microbiota (WKY g-SHRSP), SHR with SHRSP microbiota (SHR g-SHRSP), and SHR with WKY microbiota (SHR g-WKY). Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. At 11.5 wk of age systolic blood pressure increased 26 mmHg in WKY g-SHRSP compared with that in WKY g-WKY (182 ± 8 vs. 156 ± 8 mmHg, P = 0.02). Although the SBP in SHR g-WKY tended to decrease compared with SHR g-SHRSP, the differences were not statistically significant. Fecal pellets were collected at 11.5 wk of age for identification of the microbiota by sequencing the 16S ribosomal RNA gene. We observed a significant increase in the Firmicutes:Bacteroidetes ratio in the hypertensive WKY g-SHRSP, as compared with the normotensive WKY g-WKY (P = 0.042). Relative abundance of multiple taxa correlated with SBP. We conclude that gut dysbiosis can directly affect SBP. Manipulation of the gut microbiota may represent an innovative treatment for hypertension.