Mesenteric Arteries Of Spontaneously Hypertensive Rats Research Articles

Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Follistatin-like 1 protects endothelial function in the spontaneously hypertensive rat by inhibition of endoplasmic reticulum stress through AMPK-dependent mechanism

ABSTRACT Objective Endothelial dysfunction is a critical initiating factor in the development of hypertension and related complications. Follistatin-like 1 (FSTL1) can promote endothelial cell function and stimulates revascularization in response to ischemic insult. However, it is unclear whether FSTL1 has an effect on ameliorating endothelial dysfunction in spontaneously hypertensive rats (SHRs). Methods Wistar Kyoto (WKY) and SHRs were treated with a tail vein injection of vehicle (1 mL/day) or recombinant FSTL1 (100 μg/kg body weight/day) for 4 weeks. Blood pressure was measured by tail-cuff plethysmograph, and vascular reactivity in mesenteric arteries was measured using wire myography. Results We found that treatment with FSTL1 reversed impaired endothelium-dependent relaxation (EDR) in mesenteric arteries and lowered blood pressure of SHRs. Decreased AMP-activated protein kinase (AMPK) phosphorylation, elevated endoplasmic reticulum (ER) stress markers, increased reactive oxygen species (ROS), and reduction of nitric oxide (NO) production in mesenteric arteries of SHRs were also reversed by FSTL1 treatment. Ex vivo treatment with FSTL1 improved the impaired EDR in mesenteric arteries from SHRs and reversed tunicamycin (ER stress inducer)-induced ER stress and the impairment of EDR in mesenteric arteries from WKY rats. The effects of FSTL1 were abolished by cotreatment of compound C (AMPK inhibitor). Conclusions These results suggest that FSTL1 prevents endothelial dysfunction in mesenteric arteries of SHRs through inhibiting ER stress and ROS and increasing NO production via activation of AMPK signaling.

Treatment with Ligilactobacillus murinus lowers blood pressure and intestinal permeability in spontaneously hypertensive rats

One feature of hypertension is a microbial imbalance with increased intestinal permeability. In this study, we examined whether an alteration in the microbiota affects blood pressure and intestinal permeability in spontaneously hypertensive rats (SHRs). We performed a 16S metagenome analysis of feces from 10- to 15-week-old SHRs using a synthetic long-read sequencing approach, and found a candidate for the microbiome treatment, Ligilactobacillus murinus (L. murinus), that was robustly decreased. Oral administration of L. murinus to SHRs for 2 weeks significantly inhibited blood pressure elevation and improved endothelium-dependent vasodilation but did not attenuate enhanced vascular contraction in SHR mesenteric arteries. The proximal colon of SHRs exhibited increased intestinal permeability with decreased levels of the tight junction protein claudin 4, morphological changes such as decreased intestinal crypts and elevated TNF-α levels, which was reversed by treatment with L. murinus. Consistent with these intestinal phenotypes, plasma lipopolysaccharides levels were elevated in SHR but decreased following L. murinus administration. We concluded that oral administration of L. murinus to SHRs exerts protective effects on intestinal permeability via restoration of claudin 4 expression and reversal of morphologic disorder, which may improve low-grade endotoxemia and thus reduce development of hypertension via recovery of endothelial vasodilating functions.

Impaired vascular β-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries

It was suggested that impaired β-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased β-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of β-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (β2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (β1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated β-adrenergic vasodilatation of conduit arteries of SHR but similar β-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to β-adrenergic agonists do not support major role of altered β-adrenergic vasodilatation for high BP in genetic hypertension.

Abstract 003: Treatment With Ligilactobacillus Improves Gut Permeability And Hypertension In Spontaneously Hypertensive Rats

Emerging evidence provide that a microbial imbalance (dysbiosis) is linked to several diseases including cardiovascular diseases. One of the features in hypertension is increased gut permeability. Supporting this, plasma endotoxin level is increased in hypertension and our previous study indicated that the plasma level of exotoxin from Streptococcus was increased in spontaneously hypertensive rats (SHR). Thus, we hypothesized that improvement of gut permeability in hypertension might be expected to prevent development of hypertension. In this study, we performed a 16S metagenome analysis using a synthetic long read sequencing approach by LoopSeq in feces from 10- to 15-week old SHR and Wistar-Kyoto rat (WKY). The analysis revealed that two difference species of anti-inflammatory microbiome were strongly decreased in SHR compared with WKY (n=4). We focus on the decreased Ligilactobacillus sp. (LAB) in SHR and oral administration of LAB to SHR for 2 weeks. SHR (10-15-week-old) exhibited increased gut permeability in proximal colon (P<0.05, n=4), which was improved by treatment with LAB for 2 weeks (P<0.05, n=4). The expression level of tight junction protein Claudin4 was decreased in SHR proximal colon (SHR vs WKY, 1.0±0.1 vs 1.7±0.2-fold increase to SHR, P<0.05, n=8), which was significantly increased by LAB treatment (SHR vs SHR+LAB, 1.0±0.1 vs 2.8±0.5-fold increase to SHR, P<0.05, n=10). Proximal colon in SHR increased the expression level of TNF-α mRNA (1.0±0.2 vs 0.2±0.1, P<0.05, n=8), which was reversed by LAB (1.0±0.2 vs 0.3±0.1, P<0.05, n=10). The level of mucin 2 was lower in SHR proximal colon (1.0±0.2 vs 4.3±0.8, P<0.05, n=8), which was not reversed by LAB (1.0±0.1 vs 0.8±0.3, n=10). We observed that high blood pressure in SHR was attenuated by treatment with LAB for 2 weeks (194.0±5.9 vs 149.7±4.8 mmHg, P<0.05, n=10). LAB also improved decrease in acetylcholine-induced relaxation in SHR mesenteric artery (P<0.05, n=5-8). We conclude that treatment of SHR with the decreased anti-inflammatory microbiota, LAB improves gut permeability in proximal colon possibly through inhibition of inflammation, resulting in lowering blood pressure. These results suggest that anti-inflammatory microbiome might be a new therapeutic target for hypertension.

Impact of Selective Renal Afferent Denervation on Oxidative Stress and Vascular Remodeling in Spontaneously Hypertensive Rats.

Oxidative stress and sustained sympathetic over-activity contribute to the pathogenesis of hypertension. Catheter-based renal denervation has been used as a strategy for treatment of resistant hypertension, which interrupts both afferent and efferent renal fibers. However, it is unknown whether selective renal afferent denervation (RAD) may play beneficial roles in attenuating oxidative stress and sympathetic activity in hypertension. This study investigated the impact of selective RAD on hypertension and vascular remodeling. Nine-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were subjected to selective renal afferent denervation (RAD) with 33 mM of capsaicin for 15 min. Treatment with the vehicle of capsaicin was used as a control. The selective denervation was confirmed by the reduced calcitonin gene-related peptide expression and the undamaged renal sympathetic nerve activity response to the stimulation of adipose white tissue. Selective RAD reduced plasma norepinephrine levels, improved heart rate variability (HRV) and attenuated hypertension in SHR.It reduced NADPH oxidase (NOX) expression and activity, and superoxide production in the hypothalamic paraventricular nucleus (PVN), aorta and mesenteric artery of SHR. Moreover, the selective RAD attenuated the vascular remodeling of the aorta and mesenteric artery of SHR. These results indicate that selective removal of renal afferents attenuates sympathetic activity, oxidative stress, vascular remodeling and hypertension in SHR. The attenuated superoxide signaling in the PVN is involved in the attenuation of sympathetic activity in SHR, and the reduced sympathetic activity at least partially contributes to the attenuation of vascular oxidative stress and remodeling in the arteries of hypertensive rats.

Reduced Relaxant Response to Adenine in the Superior Mesenteric Artery of Spontaneously Hypertensive Rats.

We investigated the vascular response to nucleobase adenine using freshly isolated superior mesenteric arteries of spontaneously hypertensive rats (SHR) and its control, Wistar Kyoto (WKY) rats. Endothelium-dependent and endothelium-independent relaxations were assessed in isolated segments in an organ bath. The releases of the metabolites of thromboxane A2 and prostaglandin I2 were also detected. Adenine induced vasorelaxation in both the endothelium-intact and endothelium-denuded arteries in a concentration-dependent manner. In the SHR group, the adenine-induced relaxation was slightly but significantly reduced in the endothelium-intact rings when compared with that in the WKY group. However, the relaxation in the endothelium-denuded rings were similar between the two groups. The difference in the adenine-mediated relaxation in the superior mesenteric arteries between the SHR and WKY groups was eliminated by endothelial denudation and a nitric oxide (NO) synthase inhibitor. In the absence and presence of adenine, SHR tended to have higher levels of metabolites of thromboxane A2 and prostaglandin I2 compared with WKY. However, adenine did not induce the release of these substances in the arteries in both the SHR and WKY groups. These results suggest that the reduced adenine-mediated relaxation in the superior mesenteric arteries in SHR is due to a lack of contribution from the endothelium-derived NO and not from the release of prostanoids.

Abstract 37: Loss Of BK Ca - IP3R Functional Coupling Underlies Vascular Hypercontractility In Spontaneously Hypertensive Rats

The cellular mechanisms underlying enhanced vascular contractility in hypertension are still not fully clear. Recent studies have demonstrated that functional coupling between the large-conductance Ca 2+ -activated K + (BK Ca ) channel in the cell membrane and inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum is involved in the regulation of intracellular Ca 2+ in vascular smooth muscle cells (VSMC) and vascular contractility. Here, we examined the role of BK Ca -IP3R coupling in vascular hypercontractility in Spontaneously Hypertensive Rats (SHR), as compared with Sprague Dawley (SD) rats. In pressurized mesenteric arteries, paxilline (1μM), a selective BK Ca channel blocker, significantly increased norepinephrine (1μM)-induced vasoconstriction in both SHR (% initial diameter: 71.6±6.8% [95.9±13 of 132.8±6.2 μm] and 65.0±4.8% [80.6±7.7 of 123.6±3.9 μm] in arteries treated with vehicle and paxilline respectively, n=4, P<0.05) and in SD rats (% initial diameter: 84.2±7.9% [108.4±12.3 of 128.5±2.5 μm] and 60.5±0.2% [77.4±0.7 of 127.9±0.6 μm] in arteries treated with vehicle and paxilline respectively, n=3, P<0.05). Paxilline-induced increases in vasoconstriction were significantly greater in arteries of SD as compared with SHR rats, suggesting that BK Ca channels are involved in hypercontractility in SHR. In inside-out patch-clamp, BK Ca channels in VSMCs isolated from mesenteric arteries of SHR and SD rats displayed identical calcium and voltage sensitivity. We next examined the effect of the selective IP3R-agonist, Adenophostin A, on BK Ca current density in VSMCs, using patch-clamp whole-cell mode. The results demonstrated that Adenophostin A (5μM) significantly increased BK Ca current density in VSMCs from both SHR (from 41.9± 5.7 to 61.5±9.1 pA/pF; n=6 cells, p<0.05) and SD rats (from 29.9 ± 8.0 to 92.6 ± 9.1 pA/pF, n=5 cells, P<0.01). There was no significant difference in basal BKca current density between SHR and SD rats; however, the Adenophostin-induced elevation in BK Ca current density was significantly diminished in VSMCs of SHR as compared with SD rats. In conclusion, the results indicate that loss of functional coupling between BKca and IP3R is involved in vascular hypercontractility in SHR arteries.

Improvement of Vascular Function by Knockdown of Salusin-β in Hypertensive Rats via Nitric Oxide and Reactive Oxygen Species Signaling Pathway.

PurposeSalusin-β, a multifunctional vasoactive peptide, has a potentially important function in the pathological development of hypertension. However, the exact functional role of salusin-β and the underlying mechanism in this process are still not fully understood. The current study aimed to investigate the effects of silencing salusin-β on vascular function and vascular remodeling, as well as its signaling pathways in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).MethodsSilencing salusin-β was performed by caudal vein injection of adenovirus expressing salusin-β short hairpin RNA (shRNA). Acetylcholine (ACh)-induced endothelium-dependent relaxation was used to evaluate vasodilator function, and high K+ solution-induced constriction was used to evaluate vasoconstriction function.ResultsSalusin-β levels in plasma and its protein expression in mesenteric artery (MA), coronary artery (CA), and pulmonary artery (PA) of SHR were higher than those in WKY. The salusin-β level and expression were decreased effectively by salusin-β shRNA. Knockdown of salusin-β decreased arterial blood pressure (ABP) and high K+ solution-induced vascular constrictions, and improved the endothelium-dependent relaxation and vascular remodeling in SHR. The improved effect of silencing salusin-β on ACh-induced relaxation in SHR was almost blocked by the nitric oxide synthase (NOS) inhibitor L-NAME. Compared to WKY, the endothelial NOS (eNOS) activity and level, and nitric oxide (NO) level were decreased, while NAD(P)H oxidase activity and reactive oxygen species (ROS) levels in MA, CA, and PA of SHR were increased, which were all redressed by salusin-β knockdown.ConclusionThese results indicate that knockdown of salusin-β improves endothelium-dependent vascular relaxation and vascular remodeling and decreases ABP and vasoconstriction in SHR, which might be accomplished by increasing eNOS activation and NO release while inhibiting NAD(P)H oxidase derived-ROS generation. Scavenging salusin-β improves vascular function and then prevents the development and progression of vasculopathy of hypertension.

Chronic Intermittent Hypobaric Hypoxia Decreases High Blood Pressure by Stabilizing the Vascular Renin-Angiotensin System in Spontaneously Hypertensive Rats.

Background and AimsPrevious studies have demonstrated the anti-hypertensive effect of chronic intermittent hypobaric hypoxia (CIHH) in hypertensive rats. The present study investigated the anti-hypertensive effect of CIHH in spontaneously hypertensive rats (SHR) and the role of the renin-angiotensin system (RAS) in anti-hypertensive effect of CIHH.MethodsFifteen-week-old male SHR and WKY rats were divided into four groups: the SHR without CIHH treatment (SHR-CON), the SHR with CIHH treatment (SHR-CIHH), the WKY without CIHH treatment (WKY-CON), and the WKY with CIHH treatment (WKY-CIHH) groups. The SHR-CIHH and WKY-CIHH rats underwent 35-days of hypobaric hypoxia simulating an altitude of 4,000 m, 5 h per day. Arterial blood pressure and heart rate were recorded by biotelemetry, and angiotensin (Ang) II, Ang1–7, interleukin (IL)-6, tumor necrosis factor-alpha (TNF)-α, and IL-10 in serum and the mesenteric arteries were measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The microvessel tension recording technique was used to determine the contraction and relaxation of the mesenteric arteries. Hematoxylin and eosin and Masson’s staining were used to observe vascular morphology and fibrosis. Western blot was employed to detect the expression of the angiotensin-converting enzyme (ACE), ACE2, AT1, and Mas proteins in the mesenteric artery.ResultsThe biotelemetry result showed that CIHH decreased arterial blood pressure in SHR for 3–4 weeks (P < 0.01). The ELISA and immunohistochemistry results showed that CIHH decreased Ang II, but increased Ang1–7 in serum and the mesenteric arteries of SHR. In the CIHH-treated SHR, IL-6 and TNF-α decreased in serum and the mesenteric arteries, and IL-10 increased in serum (P < 0.05–0.01). The microvessel tension results revealed that CIHH inhibited vascular contraction with decreased Ang1–7 in the mesenteric arteries of SHR (P < 0.05–0.01). The staining results revealed that CIHH significantly improved vascular remodeling and fibrosis in SHR. The western blot results demonstrated that CIHH upregulated expression of the ACE2 and Mas proteins, and downregulated expression of the ACE and AT1 proteins (P < 0.05–0.01).ConclusionCIHH decreased high blood pressure in SHR, possibly by inhibiting RAS activity, downregulating the ACE-Ang II-AT1 axis and upregulating the ACE2-(Ang1-7)-Mas axis, which resulted in antagonized vascular remodeling and fibrosis, reduced inflammation, and enhanced vascular relaxation.

Enhanced Myogenic Constriction in the SHR Preglomerular Vessels Is Mediated by Thromboxane A2 Synthesis.

BackgroundSpontaneously Hypertensive Rats (SHR) have chronically elevated blood pressures at 30 weeks of age (systolic: 191.0 ± 1.0, diastolic: 128.8 ± 0.9). However, despite this chronic malignant hypertension, SHR kidneys remain relatively free of pathology due to having an augmented myogenic constriction (MC). We hypothesized that the enhanced MC in the SHR preglomerular vessels was due to increased prostaglandin and decreased nitric oxide (NO) synthesis, providing renal protection.MethodsSHR and Wistar Kyoto (WKY) arcuate and mesenteric arteries were treated with indomethacin (prostaglandin synthesis inhibitor), N omega-nitro-L-arginine (L-NNA, NO synthase inhibitor), and nifedipine (L-type calcium channel blocker); and MC was measured in these vessels. The role of endothelium in MC was examined by removing endothelium from WKY and SHR preglomerular and mesenteric arteries using human hair, and measuring MC. We also studied the source of prostaglandin in the SHR by treating endothelium-removed arcuate arteries with indomethacin and furegrelate (thromboxane synthase inhibitor).ResultsMC was enhanced in the SHR preglomerular vessels but not the mesenteric arteries. Indomethacin and LNNA removed the enhanced MC in the SHR. Nifedipine also inhibited MC in both WKY and SHR arcuate and mesenteric arteries. Removing endothelium did not change MC in either arcuate or mesenteric arteries of WKY and SHR rats; and did not remove the augmented MC in the SHR arcuate arteries. Indomethacin and furegrelate decreased MC in endothelium-removed SHR arcuate arteries and obliterated the enhanced MC in the SHR.ConclusionThe enhanced MC in the SHR arcuate arteries was due to thromboxane A2 synthesis from the tunica media and/or adventitia layers. MC was not dependent on endothelium, but was dependent on L-type calcium channels. Nevertheless, SHR arcuate arteries displayed differential intracellular calcium signaling compared to the WKYs.

Ketogenic diet aggravates hypertension via NF-κB-mediated endothelial dysfunction in spontaneously hypertensive rats

AimsKetogenic diet (KD) has been proposed to be an effective lifestyle intervention for metabolic syndrome. However, the effects of KD on hypertension have not been well investigated. The present study aimed to investigate the effects and underling mechanisms of KD on hypertension in spontaneously hypertensive rats (SHRs). Materials and methodsSHRs were subjected to normal diet or KD for 4 weeks, starting at the age of 10 weeks. Then, the blood pressure and vascular function were assessed. Next, the eNOS expression, inflammatory factors and relative signaling pathway were examined. Human umbilical vein endothelial cells were used to investigate the underlying mechanism account for the effect of ketone on inflammation and eNOS expression. Key findingsCompared with the normal diet, KD was indicated to aggravate hypertension and impaire endothelium-dependent relaxation in mesenteric arteries of SHRs. eNOS and CD31 expression in mesenteric arteries were also significantly suppressed by KD. In addition, KD markedly increased the activation of NF-κB pathway and the expression of IL1-β and TNF-α. In vitro, results showed that inhibition of NF-κB could rescue the adverse effects of ketone body and TGF-β on eNOS expression and inflammation response. SignificanceOur study indicated that KD impaired endothelium-dependent relaxation in mesenteric arteries and aggravated the development of hypertension in SHRs, suggesting that it should be more cautious to apply KD into clinical application in hypertensive individuals.

Effects of Angiotensin-(1-7) and Angiotensin II on Acetylcholine-Induced Vascular Relaxation in Spontaneously Hypertensive Rats.

Endothelial dysfunction of small arteries occurs in patients with hypertension and in various hypertensive models. Endothelial function is usually evaluated by the degree of acetylcholine- (ACh-) induced vascular relaxation. Our previous study has found that compared to Wistar-Kyoto rats (WKY), ACh-induced vasodilatation was attenuated significantly in the mesenteric artery (MA), coronary artery (CA), and pulmonary artery (PA) of spontaneously hypertensive rats (SHR). This study investigated the influence of angiotensin- (Ang-) (1-7) and Ang II on blood pressure and ACh-induced vascular relaxation, as well as their interactive roles and downstream signal pathways in SHR and WKY. Intravenous injection of Ang II significantly increased, while Ang-(1-7) decreased the mean arterial pressure (MAP) in SHR. Ang-(1-7) improved ACh-induced relaxation in the MA, CA, and PA of SHR, while Ang II further attenuated it, which were inhibited by pretreatment with Mas receptor antagonist A-779 or AT1 receptor antagonist losartan, respectively. Ang-(1-7) decreased the basal arterial tension, and Ang II induced great vasoconstriction in SHR. Pretreatment with Ang-(1-7) inhibited the Ang II-induced pressor response, vasoconstriction, and the effects on ACh-induced relaxation in SHR. AT1 receptor expression was higher, while nitric oxide (NO), cGMP, and protein kinase G (PKG) levels of arteries were lower in SHR than in WKY. Ang II decreased, while Ang-(1-7) increased, the levels of NO, cGMP, and PKG of arteries. In addition, pretreatment with Ang-(1-7) inhibited the Ang II-induced reduction of NO, cGMP, and PKG in SHR. These results indicate that the activation of the Mas receptor by Ang-(1-7) can improve endothelial function and decrease MAP in SHR and inhibit the deteriorative effect of Ang II on endothelial function through the NO-cGMP-PKG pathway.

CaSR participates in the regulation of vascular tension in the mesentery of hypertensive rats via thePLC‑IP3/AC‑V/cAMP/RAS pathway.

Hypertension is a cardiovascular disease that severely impairs human health; however, its specific etiology and pathogenesis are complex. The present study investigated the effects of the calcium sensing receptor (CaSR) on vascular tone in spontaneously hypertensive rats (SHRs), and clarified the role and mechanism of CaSR in regulating this property with respect to the phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3)/adenylate cyclase-V(AC-V)/cyclic adenosine monophosphate (cAMP)/renin-angiotensin system (RAS) pathway in these animals. CaSR protein expression in the mesenteric artery (MA) of rats and CaSR protein expression in SHRs were significantly reduced. Based on wire myography studies, vasoconstriction was significantly augmented and vasodilatation was attenuated in SHRs, and this effect was endothelium-independent. The CaSR calcimimetic NPSR568 and inhibitor NPS2143 reduced vasoconstriction and enhanced vasodilation in SHRs. Furthermore, pretreatment with PLC-IP3/AC-V/cAMP/RAS pathway blockers significantly reduced the vasoconstriction response and enhanced the vasodilator response in SHRs and Wistar-Kyoto rats (WKY), and these effects were partially dependent on the endothelium. Additionally, pretreatment with CaSR inhibitors were determined to cooperate with the PLC-IP3/AC-V/cAMP/RAS pathway inhibitors to significantly reduce vasoconstriction and enhance vasodilation in SHRs and WKY. Our results demonstrated that CaSR is functionally expressed in the MA of SHRs, and that CaSR expression is decreased in SHRs. Additionally, vasoconstriction was enhanced while vasodilatation was attenuated in SHRs; these processes were determined to be endothelium-independent. CaSR is involved in the regulation of blood pressure and vascular tension in SHRs and WKYs. In association with mechanistic differences, this effect was proposed to be partially endothelium-dependent and mediated by the PLC-IP3/AC-V/cAMP/RAS pathway.

A4646 A novel endothelial NO-dependent vasorelaxing and anti-hypertensive peptide derived from rice bran

Objectives: We recently identified a novel anti-hypertensive peptide Leu-Arg-Ala (LRA) from the enzymatic digestion of rice bran protein (Shobako et al., Mol. Nutr. Food res., 2018). Orally administered LRA at a dose of 0.25 mg/kg potently reduced the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs), but had low ACE inhibitory activity (IC50 = 62 μM), explaining its potent hypotensive activity. In this study, we investigated vasorelaxing activity of LRA and the involvement of nitric oxide (NO) signaling. Methods: The vasorelaxing activity was determined by the mesenteric artery isolated from SHRs. Changes in SBP of SHRs were measured by tail-cuff method. Results: A novel rice bran-derived hypotensive peptide LRA exhibited vasorelaxing activity in the mesenteric artery of SHRs. Pretreatment with NG-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS), diminished the LRA-induced vasorelaxing activity. L-NAME also inhibited the anti-hypertensive effects of LRA. LRA also activated phosphorylation of endothelial NOS in human umbilical vein endothelial cells (HUVECs). Thus, these data suggest that LRA-induced vasorelaxing and anti-hypertensive activites were mediated by the activation of endothelial NO signaling. Next, we investigated mechanisms upstream of NO signaling. The LRA-induced vasorelaxant activity was not blocked by HOE 140 (bradykinin B2 antagonist), A779 (MAS antagonist) and wortomannin (PI3 kinase inhibitor), implying that NO-mediated vasorelaxing activity by LRA is independent of the bradykinin B2/MAS receptor-PI3 kinase activation. Conclusion: We found that LRA possibly exhibited vasorelaxing and hypotensive effects in a novel endothelial NO signaling-dependent manner. This is the first example of a NO-dependent vasorelaxing peptide identified from rice bran.

Silencing salusin-\u03b2 attenuates cardiovascular remodeling and hypertension in spontaneously hypertensive rats

Salusin-β is a bioactive peptide involved in vascular smooth muscle cell proliferation, vascular fibrosis and hypertension. The present study was designed to determine the effects of silencing salusin-β on hypertension and cardiovascular remodeling in spontaneously hypertensive rats (SHR). Thirteen-week-old male SHR and normotensive Wistar-Kyoto rats (WKY) were subjected to intravenous injection of PBS, adenoviral vectors encoding salusin-β shRNA (Ad-Sal-shRNA) or a scramble shRNA. Salusin-β levels in plasma, myocardium and mesenteric artery were increased in SHR. Silencing salusin-β had no significant effect on blood pressure in WKY, but reduced blood pressure in SHR. It reduced the ratio of left ventricle weight to body weight, cross-sectional areas of cardiocytes and perivascular fibrosis, and decreased the media thickness and the media/lumen ratio of arteries in SHR. Silencing salusin-β almost normalized plasma norepinephrine and angiotensin II levels in SHR. It prevented the upregulation of angiotensin II and AT1 receptors, and reduced the NAD(P)H oxidase activity and superoxide anion levels in myocardium and mesenteric artery of SHR. Knockdown of salusin-β attenuated cell proliferation and fibrosis in vascular smooth muscle cells from SHR. These results indicate that silencing salusin-β attenuates hypertension and cardiovascular remodeling in SHR.

Choline ameliorates cardiovascular damage by improving vagal activity and inhibiting the inflammatory response in spontaneously hypertensive rats

Autonomic dysfunction and abnormal immunity lead to systemic inflammatory responses, which result in cardiovascular damage in hypertension. The aim of this report was to investigate the effects of choline on cardiovascular damage in hypertension. Eight-week-old male spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats were intraperitoneally injected with choline or vehicle (8 mg/kg/day). After 8 weeks, choline restored the cardiac function of the SHRs, as evidenced by decreased heart rate, systolic blood pressure, left ventricle systolic pressure, and ±dp/dtmax and increased ejection fraction and fractional shortening. Choline also ameliorated the cardiac hypertrophy of the SHRs, as indicated by reduced left ventricle internal dimensions and decreased cardiomyocyte cross-sectional area. Moreover, choline improved mesenteric arterial function and preserved endothelial ultrastructure in the SHRs. Notably, the protective effect of choline may be due to its anti-inflammatory effect. Choline downregulated expression of interleukin (IL)-6 and tumour necrosis factor-α and upregulated IL-10 in the mesenteric arteries of SHRs, possibly because of the inhibition of Toll-like receptor 4. Furthermore, choline restored baroreflex sensitivity and serum acetylcholine level in SHRs, thus indicating that choline improved vagal activity. This study suggests that choline elicits cardiovascular protective effects and may be useful as a potential adjunct therapeutic approach for hypertension.

Gene Expression and MicroRNA Expression Analysis in Small Arteries of Spontaneously Hypertensive Rats. Evidence for ER Stress.

Small arteries are known to develop functional and structural alterations in hypertension. However, the mechanisms of this remodeling are not fully understood. We hypothesized that altered gene expression is associated with the development of hypertension in mesenteric arteries of spontaneously hypertensive rats (SHR). Three sublines of SHR and normotensive Wistar Kyoto rats (WKY) were studied at 6 weeks and 5 months of age. MiRNA and mRNA microarray experiments were performed and analyzed with bioinformatical tools, including Ingenuity Pathway Analysis (IPA). Principal component analysis showed a clear separation in both miRNA and mRNA expression levels between both ages studied, demonstrating strong age-related changes in expression. At the miRNA level, IPA identified differences between SHR and WKY related to metabolic diseases, cellular growth, and proliferation. The mRNAs differentially expressed between SHR and WKY were related to metabolism, cellular movement and proliferation. The most strongly upregulated gene (9.2-fold) was thrombospondin 4 (Thbs4), a protein involved in the endoplasmic reticulum (ER) stress response that activates transcription factor 6α (ATF6α). ATF6α downstream targets were also differentially expressed in SHR vs. WKY. Differential expression of THBS4, the cleaved form of ATF6α, and two of its targets were further confirmed at the protein level by western blot. In summary, these data revealed a number of genes (n = 202) and miRNAs (n = 3) in mesenteric arteries of SHR that had not been related to hypertension previously. The most prominent of these, Thbs4, is related to vascular ER stress that is associated with hypertension.

Abstract P125: Visceral Adipose Tissue-derived Serine Protease Inhibitor Prevents the Development of Hypertension Through the Inhibition of Vascular Remodeling via Anti-Oxidative Mechanisms in Spontaneously Hypertensive Rats

Visceral adipose tissue-derived serine protease inhibitor (vaspin) is an adipokine originally identified in visceral adipose tissue of obese type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats. We have been focusing on the direct vascular effects of vaspin and found that vaspin inhibits: 1) tumor necrosis factor-α-induced inflammatory responses in vascular smooth muscle cells (SMCs) via the anti-oxidative mechanisms, 2) platelet derived growth factor-BB-induced migration of SMCs, 3) apoptosis of vascular endothelial cells (ECs) mediated by methylglyoxal, a metabolite of glucose. Since vascular remodeling via inflammation and migration of vascular SMCs as well as apoptosis of ECs is an important process for the development of hypertension, it is suggested that vaspin has preventive roles on the pathogenesis of hypertension. However, it is not revealed whether vaspin affects the development of hypertension in in vivo hypertensive model. The aim of the present study was to explore it. Five-week-old male spontaneously hypertensive rats (SHR) were received an intraperitoneal injection of vaspin (1 μg/kg) or saline once daily for four weeks. Age-matched male Wistar-Kyoto (WKY) rats were used as a control. Blood pressure (BP) was measured using a tail-cuff method weekly. Isolated superior mesenteric arteries were used for the examination of vascular structural changes by a hematoxylin and eosin staining. Reactive oxygen species (ROS) generation was examined by an immunohistochemical staining to 4-hydroxy-2-nonenal (4-HNE), an end-product of lipid peroxidation by ROS. Vaspin significantly inhibited age-dependent elevation of BP in SHR (from 165.5 ± 3.9 mmHg to 153.1 ± 2.5 mmHg, n = 3, P &lt; 0.05). Vaspin significantly inhibited vascular wall hypertrophy in SHR mesenteric artery (from 1.35±0.05 to 0.98±0.08-fold relative to WKY, n = 3, P &lt; 0.05). Moreover, vaspin inhibited an increase of 4-HNE-positive area to vessel area ratio in SHR mesenteric artery (from 13.1±4.4 % to 4.6±0.4 %, n = 3). The present results demonstrate that vaspin inhibits the increase of BP through inhibiting vascular remodeling via anti-oxidative mechanisms in SHR.

Sex-specific alterations in NOS regulation of vascular function in aorta and mesenteric arteries from spontaneously hypertensive rats compared to Wistar Kyoto rats.

The present study tested the hypothesis that spontaneously hypertensive rats (SHR) have impaired nitric oxide synthase (NOS)‐mediated regulation of vascular function versus Wistar‐Kyoto rats (WKY). Aorta and small mesenteric arteries were studied from male and female SHR (M SHR and F SHR) and WKY (M WKY and F WKY). Phenylephrine (PE)‐induced vasoconstriction was greater in aorta of M SHR versus all others (P < 0.05); there were neither sex nor strain differences in PE contraction in mesenteric arteries. The NOS inhibitor l‐Nitro‐Arginine Methyl Ester (l‐NAME) increased PE‐induced vasoconstriction in all rats, although the increase was the least in male SHR (P < 0.05), revealing a blunted vasoconstrictor buffering capacity of NOS. l‐NAME increased sensitivity to PE‐induced constriction only in mesenteric arteries of SHR, although, the maximal percent increase in contraction was comparable among groups. ACh‐induced relaxation was also less in aorta from M SHR versus all others (P < 0.05). ACh relaxation was comparable among groups in mesenteric arteries, although SHR exhibited a greater NOS component to ACh‐induced relaxation than WKY. To gain mechanistic insight into sex and strain differences in vascular function, NOS activity and NOS3 protein expression were measured. Aortic NOS activity was comparable between groups and M SHR had greater NOS3 expression than M WKY. In contrast, although vascular function was largely maintained in mesenteric arteries of SHR, NOS activity was less in SHR versus WKY. In conclusion, M SHR exhibit a decrease in NOS regulation of vascular function compared to F SHR and WKY, although this is not mediated by decreases in NOS activity and/or expression.