Relaxation Of Arteries Research Articles

Modulation of Vasomotor Function by Perivascular Adipose Tissue of Renal Artery Depends on Severity of Arterial Dysfunction to Nitric Oxide and Severity of Metabolic Parameters.

Perivascular adipose tissue (PVAT) enhances vascular relaxation of mesenteric arteries in SHRSP.Z-Leprfa/IzmDmcr rats (SPZF), a metabolic syndrome model. We investigated and compared the effects of PVAT on the renal artery in SPZF with those on SHR/NDmcr-cp rats (CP). Renal arteries with and without PVAT were isolated from 23-week-old SPZF and CP. The effects of PVAT on acetylcholine- and nitroprusside-induced relaxation were examined using bioassays with phenylephrine-contracted arterial rings. Acetylcholine-induced relaxations without PVAT in SPZF and CP were 0.7- and 0.5-times lower in females than in males, respectively. In the presence of PVAT, acetylcholine-induced relaxations increased 1.4- and 2-times in male and female CP, respectively, but did not differ in SPZF. Nitroprusside-induced relaxation with and without PVAT was 0.7-times lower in female than in male SPZF but did not differ in CP. Angiotensin-II type-1 receptor (AT1R)/AT1R-associated protein mRNA ratios were lower in CP than in the SPZF and negatively correlated with the difference in arterial relaxation with and without PVAT. The effects of renal artery PVAT differed between the SPZF and CP groups. Higher levels of enhanced AT1R activity in SPZF PVAT may drive these differences by impairing the vascular smooth muscle responses to nitric oxide.

17β-Estradiol Treatment Improves Acetylcholine-Induced Relaxation of Mesenteric Arteries in Ovariectomized UC Davis Type 2 Diabetes Mellitus Rats in Prediabetic State.

We recently reported sex differences in mesenteric arterial function of the UC Davis type-2 diabetes mellitus (UCD-T2DM) rats as early as the prediabetic state. We reported that mesenteric arteries (MA) from prediabetic male rats exhibited a greater impairment compared to that in prediabetic females. However, when females became diabetic, they exhibited a greater vascular dysfunction than males. Thus, the aim of this study was to investigate whether the female sex hormone, estrogen preserves mesenteric arterial vasorelaxation in UCD-T2DM female rats at an early prediabetic state. Age-matched female Sprague Dawley and prediabetic (PD) UCD-T2DM rats were ovariectomized (OVX) and subcutaneously implanted with either placebo or 17β-estradiol (E2, 1.5 mg) pellets for 45 days. We assessed the contribution of endothelium-derived relaxing factors (EDRF) to acetylcholine (ACh)-induced vasorelaxation, using pharmacological inhibitors. Responses to sodium nitroprusside (SNP) and phenylephrine (PE) were also measured. Additionally, metabolic parameters and expression of some targets associated with vascular and insulin signaling were determined. We demonstrated that the responses to ACh and SNP were severely impaired in the prediabetic state (PD OVX) rats, while E2 treatment restored vasorelaxation in the PD OVX + E2. Moreover, the responses to PE was significantly enhanced in MA of PD OVX groups, regardless of placebo or E2 treatment. Overall, our data suggest that 1) the impairment of ACh responses in PD OVX rats may, in part, result from the elevated contractile responses to PE, loss of contribution of endothelium-dependent hyperpolarization (EDH) to vasorelaxation, and a decreased sensitivity of MA to nitric oxide (NO), and 2) the basis for the protective effects of E2 may be partly attributed to the elevation of the NO contribution to vasorelaxation and its interaction with MA as well as potential improvement of insulin signaling. Here, we provide the first evidence of the role of E2 in protecting MA from early vascular dysfunction in prediabetic female rats.

Interaction of alcohol & phosphatidic acid in maternal rat uterine artery function

Alcohol has been demonstrated to impair maternal uterine arterial adaptations in Fetal Alcohol Spectrum Disorder (FASD) animal models. However, the exact mechanism remains inconclusive. We hypothesized that phosphatidic acid (PA), a direct target of alcohol metabolism, would alleviate alcohol-induced vascular dysfunction of the maternal uterine artery. Mean fetal weight, and crown-rump length of the alcohol administered rats were ~9 % and 7.6 % lower than the pair-fed control pups, respectively. Acetylcholine (Ach)-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P < 0.05). Supplementation of 10−5 M PA reversed alcohol-induced vasodilatory deficit; no difference was detected after PA treatment between pair-fed control and alcohol groups (P = 0.37). There was a significant interaction between PA concentrations and alcohol exposure (PA X Alcohol effect, P < 0.0001). Pair-wise comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats, with % relaxation significantly improved at PA concentrations > 10−7 M (P < 0.05). Alcohol significantly reduced vasodilatory P-Ser1177 endothelial nitric oxide synthase (eNOS) levels in the uterine artery (↓90.7 %; P = 0.0029). PA treatment significantly reversed P-Ser1177 eNOS level in alcohol uterine arteries (153.7 %↑; P = 0.005); following ex vivo PA, there was no difference in P-Ser1177 eNOS levels between Control and Alcohol. Neither alcohol treatment nor PA affected total eNOS levels. Our data provide the first evidence of the interaction of alcohol and PA in rat maternal uterine artery vascular function and demonstrates PA’s relationship with the eNOS system. Overall, the current study demonstrates that PA may be a promising therapeutic molecule of interest in alcohol-related gestational vascular dysfunction.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Small extracellular vesicles from plasma of women with preeclampsia increase myogenic tone and decrease endothelium-dependent relaxation of mouse mesenteric arteries.

Preeclampsia (PE) is a common syndrome of pregnancy, characterized by new-onset hypertension and proteinuria after gestational week 20, or new onset of hypertension and significant end-organ dysfunction. In the worst cases, it can threaten the survival of both mother and baby. Extracellular vesicles (EVs) are lipid-bilayer nanoparticles released from cells. They are involved in cell-cell communication and transport of diverse cargo molecules. Small extracellular vesicles (sEVs, exosomes) are defined by their size and biogenesis within the endocytic compartment of the cell or reverse budding of the plasma membrane. The function of circulating gestational EVs, released from maternal organs or the placenta, remains to be explored. Here, we focused on sEVs that circulate in the maternal blood in the third trimester of human pregnancy and hypothesized that sEVs from pregnant women with PE play a role in regulation of vessel tone. When compared to sEVs from women with uncomplicated pregnancies, ex vivo exposure of isolated mouse mesenteric arteries to sEVs purified from the plasma of pregnant women with PE led to constriction in response to intraluminal pressure. This effect was not observed using microvesicles from the plasma of women with PE or using PE plasma that was depleted of EVs. Blood vessels exposed to sEVs from women with PE were also more resistant to methacholine-stimulated relaxation. Immunofluorescence microscopy confirmed the presence of sEVs within the vessel wall. Together, these data support the notion that circulating sEVs from pregnant women play a role in the regulation of arterial tone.

SEX SPECIFIC CHANGES OF CAROTID ARTERIAL FUNCTION IN RESPONSE TO VITAMIN D DEFICIENCY

Objective: Male sex and Vitamin D deficiency is associated with increased ischemic stroke risk that may be partially explained by local changes of circulatory control. Our aim was to examine the vascular reactivity and histological changes of isolated carotid artery of female and male rats in Vitamin D deficient and Vitamin D supplemented state. Design and method: Female and male Wistar rats were fed with vitamin D reduced rat chow for eight weeks. Another group received normal chow with further supplementation of vitamin D. Phenylephrine induced contraction, acetylcholine and estrogen induced relaxation of carotid arteries was examined by wire myography. Elastic fiber density was evaluated on resorcin-fuchsin stained tissue sections. Smooth muscle cell actin (SMA), thromboxane A2 receptor and endothelial nitric oxide synthase (eNOS) density and protein tyrosine nitration (NT) was measured by immunohistochemistry. Results: Both male sex and vitamin D deficiency resulted in increased phenylephrine induced contraction. Inhibition of cyclooxygenases by indomethacin increased the degree of contraction only in vitamin D supplemented females. Male gender was associated with reduced acetylcholine induced relaxation, and it was not altered by vitamin D deficiency. Indomethacin increased relaxation in male vessels. Estrogen induced relaxation was significantly reduced by vitamin D deficiency in female but not in male animals. Vitamin D deficiency resulted in reduced elastic fiber density in female, but decreased smooth muscle cell actin density in male rats. Thromboxane A2 receptor density was increased in vitamin D deficient males. NT immunohistochemistry was low in both male group, whereas eNOS was reduced in vitamin D deficient males. Conclusions: In rats male gender was associated with increased phenylephrine induced contraction and reduced acetylcholine induced relaxation of the carotid artery. Vitamin D deficiency caused increased contraction in both sexes; did not change acetylcholine induced relaxation, but reduced the estrogen induced relaxation in female arteries. These functional differences were accompanied with sex specific histological changes of the vessel. The observed sex specific properties and responses of carotid artery to vitamin D deficiency may play a role in the gender difference of cardiovascular and stroke risk.

Targeting Piezo1 channels to reverse pulmonary artery vasoconstriction in rat model of pulmonary hypertension

Piezo1, a stretch-activated calcium permeable channel is a sensor of mechanical stress present in endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Our previous studies have shown that endothelial Piezo1 contributes to intrapulmonary artery (IPA) relaxation in mice. The present study was undertaken to investigate the implication of Piezo1 in both rat ECs and SMCs in pulmonary hypertension (PH). Chronically hypoxic rats, an animal model of PH, were exposed to chronic hypoxia (CH, −50% O 2 for 1 week), whereas normoxic rats were housed in normoxic condition (NX). NX or CH IPA tone variation induced by YODA1 was quantified by myograph. Intracellular calcium concentration ([Ca2+]i) was determined with Cal520-AM probes. Protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), Akt and pAkt were quantified by Western blot analysis. Immunofluorescence labeling and patch-clamp revealed the presence of Piezo1 in both ECs and SMCs. In IPAs, YODA1 induced a contraction in NX condition. By contrast, YODA1 induced an endothelium and nitric oxide-dependent relaxation in CH IPAs. Activation of Piezo1 channels by its agonist YODA1 increased [Ca2+]i. However, the [Ca2+]i increase in ECs was greater in CH as compared to NX condition. In addition, CH condition increased level of eNOS proteins as compared to NX condition and YODA1 induced a phosphorylation of Akt (Ser 473) and eNOS (Ser 1177) in NX and CH conditions. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i and Akt-eNOS- pathway activation that could counteract PH.

Raspberry Supplementation Attenuates Blood Pressure and Improves Mesenteric Artery Relaxation in a Nitric Oxide-Independent Mechanism in Angiotensin II-Treated Rats

ObjectivesBerries are rich in polyphenols with strong antioxidant capacity. They have been suggested to exert antihypertensive properties, which may be due to an increase in the levels of endothelial nitric oxide (NO), a potent vasodilator. Thus, the objective of this study was to evaluate the effects of raspberry supplementation on vascular function in angiotensin (Ang) II-treated rats. MethodsEight-week-old Sprague-Dawley rats (n = 8/group) were fed an AIN-93M diet (control, Ang II, and Losartan (LOS; 20 mg/kg/BW) groups) or AIN-93M diet supplemented with 10% w/w freeze-dried raspberry (RB + Ang II) for 7 weeks. At week 5, rats were implanted with subcutaneous osmotic minipumps that delivered 0.9% saline (CON) or Ang II (270 ng/kg BW/min) for an additional 3 weeks. Blood pressure (BP) was measured using tail-cuff plethysmography (n = 7–8/group). First order mesenteric arteries were isolated for vascular relaxation measurements following constriction by U46619 (1 μM) in a wire myograph (n = 3–8/group). Data were found to be normal and analyzed using ANOVA or mixed-model where appropriate. ResultsAng II group showed a significant increase in systolic BP (SBP) (+26.8%, p = 0.002) 2 weeks after implantation as compared to baseline, while CON, RB + Ang II, and LOS groups showed no differences. Final SBP was significantly lower in the CON (–26.4%, p = 0.01), LOS (–28.3%, p = 0.0005), and RB + Ang II (–20.8%, p = 0.03) groups compared to Ang II group. Relaxation was significantly inhibited in the Ang II group in the presence of acetylcholine after incubation with L-NAME (100 μM), a NO synthase inhibitor, when compared to LOS (p = 0.0003) and RB + Ang II (p = 0.0004) groups. Similarly, relaxation in the Ang II group was significantly inhibited after incubation with ODQ (10 μM), a sGC inhibitor, when compared to CON (p = 0.0002), LOS (p = 0.0004), and RB + Ang II (p = 0.0001) group. No significant changes in vessel relaxation were observed after incubation with LY294002 (20 μM), a PI3K inhibitor. ConclusionsWe demonstrate that RB supplementation attenuated Ang II-induced hypertension and abolished the inhibitory effects of L-NAME and ODQ. This suggests that RB may exert its vasodilatory effects in a NO-independent manner. Funding SourcesThe Agriculture and Food Research Initiative (grant no. 2019–67,017-29,257/project accession no. 1,018,642) from the USDA National Institute of Food and Agriculture.

Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.

T18/S19 diphosphorylation of myosin regulatory Light Chain is Responsible for the impaired relaxation of pulmonary arteries in pulmonary hypertensive rats

Pulmonary arteries (PA) are exposed to hemodynamic environment and contractile stimuli different from those of systemic arteries. Previous studies reported that in situapplication of sodium nitroprusside (SNP, NO donor) lowered the PA pressure, which was less prominent than the response of systemic arteries. Here, we investigated the relaxation speed and the in vitro effects of NO‐dependent signaling modulators to compare between isolated PA and mesenteric arteries (MA) of rats. In the dual‐wire myography study, PA showed significantly slower relaxation after the response to 80 mM KCl (80K) or to pharmacological agonists (U46619 and PGF2a). Surprisingly, addition of 30 mM SNP could not prevent the 80K contraction in PA while significantly attenuated the contraction of MA. The pretreatment with soluble guanylate cyclase (sGC) inhibitor (ODQ, 10 mM) markedly further slowed the relaxation of PA after 80K or agonist‐induced contraction. Perplexingly, immunoblot assay revealed higher expression of sGC‐a with similar level of sGC‐b1 in PA than MA. The expression of RhoA‐kinase (ROCK) was higher while myosin light chain phosphatase (MYPT) appeared lower in PA than MA. The treatment with ROCK inhibitor (Y27632, 10 mM) could abolish the different of relaxation response between PA and MA. Taken together, we speculate that despite the higher expression and the functional importance of NO/sGC pathway in PA, the higher level of ROCK and relatively low MYPT in PA are responsible for the characteristic slow relaxation after removal of stimuli.

Mice Deficient in Endothelial Cell‐Selective Adhesion Molecule Develop Vascular Endothelial and Left Ventricle Diastolic Dysfunction

The Endothelial Cell‐Selective Adhesion Molecule (ESAM) mediates inflammatory cell transmigration and also involved in pathological angiogenesis. Here we investigated the role of ESAM in affecting vasodilator and cardiac contractile function. To that end ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed left ventricle (LV) diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail‐cuff plethysmography, was not different between ESAM knockout and WT mice, whereas the heart to body weight ratio is increased in ESAM knockout mice. Using an unbiased automated tracing of the microvasculature we found a decreased vascularization (total vascular length) of the myocardium in ESAM knockout mice. In addition, vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium‐dependent, acetylcholine‐induced relaxation of the pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium‐independent, sodium nitroprusside‐induced relaxation was similar in two groups. Thus, we conclude that ESAM deficiency causes vasodilator dysfunction, impaired angiogenesis underlying the development LV diastolic dysfunction.

Endothelial Cell Mineralocorticoid Receptor (ECMR) deletion improves fetal growth and vascular function in the RUPP mouse model of preeclampsia

Preeclampsia (PE), a hypertensive disorder of pregnancy, induces adverse pregnancy outcomes including fetal growth restriction (FGR) and afflicts ~5‐10% of pregnancies. Compelling clinical data indicate that PE risk and severity in pregnant women is strongly associated with endothelial dysfunction. Previous work by our group shows that deletion of endothelial cell mineralocorticoid receptors (ECMR) improves endothelial function in premenopausal hypertensive female mice, however whether ECMR promotes endothelial dysfunction in PE is unknown. We tested the hypothesis that ECMR deletion improves FGR and endothelial function in a mouse model of PE, the reduced uterine perfusion pressure (RUPP) mouse. Pregnant ECMR‐intact (WT) and ECMR‐deficient (KO) mice were randomized to RUPP or sham surgery on GD13 and sacrificed on GD18 (WT Sham=6, WT RUPP=5, KO Sham=7, KO RUPP=6). Vascular function was measured via wire myography on 2nd order mesenteric arteries. Conscious blood pressure was measured by radiotelemetry throughout pregnancy in a subset of mice. RUPP surgery did not decrease maternal weights in either WT or KO mice (P&gt;0.05) at GD18. RUPP surgery significantly decreased GD18 pup weight, a measure of FGR, (726±20mg WT+Sham vs 660±11mg WT+RUPP, *P&lt;0.05) and trended to decrease placental efficiency (pup/placenta weight) (8.23±0.23 WT+Sham vs 7.45±0.22 WT+RUPP P=0.13) in WT pregnant mice. ECMR deletion protected pregnant mice both from RUPP‐induced reductions in pup weight (710±20mg KO+Sham vs 690±20mg KO+RUPP) and decreases in placental efficiency (7.63±0.19 KO+Sham vs 7.76±0.31 KO+RUPP). RUPP reduced acetylcholine‐mediated relaxation (10‐9‐3x10‐5 M) in mesenteric arteries of WT pregnant mice but not KO pregnant mice (*P&lt;0.05, 2‐way ANOVA, repeated measures), indicating RUPP induced endothelial dysfunction in WT mice only. Preincubation with nitric oxide (NO) synthase inhibitor LNAME ablated differences in Ach‐mediated relaxation between WT+sham and WT+RUPP mice (P&gt;0.05), indicating that reductions in endothelial function in WT+RUPP mice were mediated by reduced NO. However, ECMR deletion as a variable increased Ach‐mediated relaxation in the presence of LNAME in both Sham and RUPP mice (*P&lt;0.05). Therefore, ECMR deletion may have protected pregnant mice from endothelial dysfunction via a non‐NO mechanism. RUPP did not increase a‐receptor agonist phenylephrine (Phe)‐induced constriction in WT mice, however, ECMR deletion as a variable decreased Phe‐mediated constriction independent of RUPP surgery indicating that ECMR deletion reduces smooth muscle‐mediated vasoconstriction in pregnant sham and RUPP mice. These data indicate that specific deletion of ECMR improves vascular function to increase vasodilation and prevent constriction in PE pregnancy. Preliminary data further indicates that systolic blood pressure increased from GD8‐18 (pre‐post RUPP) in WT+RUPP but decreased in KO+RUPP pregnant mice (2.7 vs ‐8.6 DmmHg, respectively, N=1). Lastly RUPP surgery trended to increase plasma levels of the ECMR agonist corticosterone in WT pregnant mice (2.03±0.4pg/ml WT+Sham vs 3.33±0.95pg/ml WT+RUPP). Collectively, these data indicate that ECMR activation promotes FGR, vascular dysfunction and hypertension in the RUPP model of PE which may lead to clinical studies for the consideration of specific MR antagonists as therapeutic options for PE pregnancies.

Sex‐Dependent Changes in Vascular Function in Adult Rats Following Prenatal Exposure to Methamphetamine

BackgroundPrenatal exposure to central nervous system stimulants such as cocaine, nicotine, and caffeine alters cardiovascular function in adult offspring. Methamphetamine use during pregnancy is associated with negative consequences in the offspring. However, most studies of methamphetamine use during pregnancy have focused on behavioral and neurological outcomes. Relatively little is known regarding the impact of prenatal methamphetamine exposure on the adult cardiovascular system. This study examined the hypothesis that methamphetamine use during pregnancy alters contractile function of the vasculature in the adult offspring. We also investigated the role of perivascular adipose tissue (PVAT) in methamphetamine‐induced changes in vascular function.MethodsPregnant female rats received daily injections of saline or methamphetamine (5 mg/kg) throughout gestation. Agonist‐induced contraction and relaxation responses were measured in aortas of 5 month old offspring in the presence and absence of perivascular adipose tissue.ResultsPrenatal methamphetamine significantly attenuated acetylcholine‐induced relaxation in male (but not female) aortas when PVAT remained intact. However, prenatal methamphetamine had no impact on acetylcholine‐induced relaxation when PVAT was removed. Nitroprusside‐induced relaxation was unaffected by prenatal methamphetamine. Prenatal exposure to methamphetamine had no impact on acetylcholine‐induced relaxation in third order mesenteric arteries of male or female offspring regardless of the presence of PVAT. Angiotensin II‐induced contractile responses were significantly potentiated in male (but not female) aortas regardless of the presence of PVAT. This effect was abolished by L‐Nitro arginine methyl ester (L‐NAME). Contractile responses to phenylephrine and serotonin were unaffected in both male and female aortas. Basal blood pressure in adult male and female offspring was also unaffected by prenatal exposure to methamphetamine.ConclusionsPrenatal exposure to methamphetamine sex‐dependently alters PVAT function in the aorta. Methamphetamine also enhances angiotensin II‐induced contraction through a mechanism that involves suppression of nitric oxide signaling. These data provide evidence that methamphetamine use during pregnancy induces sex‐dependent changes in the vasculature that may increase the risk of cardiovascular disease in adult offspring.

Chronic Unpredictable Stress Impairs the Reactivity of the Pudendal Artery and Corpus Cavernosum in Male Mice

Erectile dysfunction (ED) can be associated with psychological disorders as well as vascular disorders. It is known that stress is associated with erectile dysfunction, however, it is not clear if psychological stressors could lead to a vascular and cavernosal dysfunction that could contribute to a vasculogenic erectile dysfunction. A recent study reported a positive correlation between hyperprolactinemia, stress and erectile dysfunction, and interestingly, prolactin was shown to decrease endothelium nitric oxide and endothelium‐ dependent vasorelaxation of aorta and coronary arteries. In this study, we aimed to evaluate the relaxation of the corpus cavernosum and pudendal artery of mice subject to chronic unpredictable stress (CUS). We hypothesize that CUS will lead to an impairment of the relaxation of the pudendal artery and corpus cavernosum leading to erectile dysfunction.MethodsMale C57Bl/6 mice were submitted to 28 days of chronic unpredictable stress (CUS). The pudendal artery and corpus cavernosum were removed and mounted in a myograph to evaluate reactivity. Tissues were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2 and concentration‐response curves to acetylcholine (ACh; 1 nM – 30 uM)) were obtained. Using non‐linear regression, we obtained the maximal response (Emax) and potency (pEC50) of ACh. Data are expressed as mean ± S.E.M. P&lt;0.05 was considered statistically different.ResultsNeither the potency nor the maximal response to acetylcholine was changed in the pudendal artery of stressed animals (Fig. 1A) whereas in the corpus cavernosum, there was a significant decrease in the maximal relaxation induced by acetylcholine (control: 88 ± 4% vs stress: 75 ± 4%, n=7 each group; Fig. 1B).ConclusionOur findings demonstrate that CUS for 4 weeks impairs acetylcholine‐induced relaxation of the corpus cavernosum. This may contribute to the development of erectile dysfunction in stressed males.

Mice with Brain‐Specific Deletion of Hsd11b2 have an impaired renal vascular and tubular response to high salt intake

Loss‐of‐function mutations in Hsd11b2, the gene encoding the cortisol‐metabolising enzyme 11βHSD2, causes the hypertensive syndrome of Apparent Mineralocorticoid Excess. In affected individuals, overactivation of the mineralocorticoid receptor by cortisol is causative. Hypertension is salt‐sensitive, attributed to enhanced sodium reabsorption in the distal nephron and renal sodium retention. However, in the brain, 11βHSD2‐expressing neurons regulate salt appetite. We previously generated mice with conditional knockout of Hsd11b2 in the brain (Hsd11b2.BKO), which displayed enhanced salt preference and salt‐sensitive hypertension (DOI: 10.1161/CIRCULATIONAHA.115.019341). The mechanisms of salt‐sensitivity are unknown. In the current study we examined renal artery vasoreactivity and the in vivo acute pressure natriuresis response in male Hsd11b2.BKO mice and control littermates.Mice (n=10 per genotype/diet) were fed either high salt (3% Na) or 0.3% Na diet for 7 days. Animals were humanely killed, the renal arteries were isolated and mounted on a wire‐myograph to generate cumulative concentration‐response curves to phenylephrine and the nitric oxide donor, sodium nitroprusside. High salt diet significantly increased the sensitivity of the renal artery to phenylephrine‐induced vasoconstriction in Hsd11b2.BKO mice (LogEC50 0.3% diet ‐6.41±0.07 vs high salt ‐6.91±0.11; p=0.0013) but not in control mice. Sodium nitroprusside induced concentration‐dependent relaxation of renal arteries: the maximum relaxation was significantly reduced from 88±3% to 65±4% in Hsd11b2.BKO after high salt feeding (p&lt;0.01); this was not seen in control mice. In other experiments, mice (n=5‐7 per genotype/diet) were anesthetised (thiobutabarbital sodium, 120mg/kg IP) and fractional renal sodium excretion was measured at baseline and following acutely increased blood pressure, achieved by sequential arterial ligation. In all mice, sodium excretion increased with blood pressure. In the high salt group, the pressure natriuresis response was significantly attenuated in Hsd11b2.BKO mice, compared to control animals.We find that genetic amplification of mineralocorticoid signalling in the brain attenuates the normal renal vascular and tubular adaptation to high salt intake. This regulation of renal salt excretion by the central nervous system may have implications for salt‐sensitive hypertension in humans.

Perivascular Adipose Tissue Compensation for Endothelial Dysfunction in the Superior Mesenteric Artery of Female SHRSP.Z-Leprfa/IzmDmcr Rats

Regulation of arterial tone by perivascular adipose tissue (PVAT) differs between sexes. In male SHRSP.Z-Lepr^fa/IzmDmcr rats (SHRSP.ZF), PVAT exerts a compensatory relaxation effect for the loss of endothelium-mediated vasorelaxation, which occurs during the early stages of metabolic syndrome. However, this effect deteriorates by 23 weeks of age. Here, therefore, we compared the effects of PVAT in female and male SHRSP.ZF. Acetylcholine-induced relaxation in superior mesenteric artery without PVAT did not differ between 23-week-old females and males. However, the presence of PVAT enhanced relaxation in 23-week-old females, but not in males. The mRNA levels of angiotensin II type 1 receptor (AT1R) in PVAT did not differ between sexes, but AT1R-associated protein (ATRAP) and apelin levels were higher in females than in males. We observed a positive relationship between differences in artery relaxation with and without PVAT and ATRAP or apelin mRNA levels. In 30-week-old females, PVAT-enhanced relaxation disappeared, and mRNA levels of AT1R increased, while apelin levels decreased compared to 23-week-old females. These results demonstrated that in SHRSP.ZF, PVAT compensation for endothelium dysfunction extended to older ages in females than in males. Apelin and AT1R/ATRAP expression in PVAT may be predictors of favorable effects.

Endothelial Cullin3 Mutation Impairs Nitric Oxide-Mediated Vasodilation and Promotes Salt-Induced Hypertension.

Human hypertension caused by in-frame deletion of CULLIN3 exon-9 (Cul3∆9) is driven by renal and vascular mechanisms. We bred conditionally activatable Cul3∆9 transgenic mice with tamoxifen-inducible Tie2-CREERT2 mice to test the importance of endothelial Cul3. The resultant mice (E-Cul3∆9) trended towards elevated nighttime blood pressure (BP) correlated with increased nighttime activity, but displayed no difference in daytime BP or activity. Male and female E-Cul3∆9 mice together exhibited a decline in endothelial-dependent relaxation in carotid artery. Male but not female E-Cul3∆9 mice displayed severe endothelial dysfunction in cerebral basilar artery. There was no impairment in mesenteric artery and no difference in smooth muscle function, suggesting the effects of Cul3∆9 are arterial bed-specific and sex-dependent. Expression of Cul3∆9 in primary mouse aortic endothelial cells decreased endogenous Cul3 protein, phosphorylated (S1177) endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Protein phosphatase (PP) 2A, a known Cul3 substrate, dephosphorylates eNOS. Cul3∆9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor okadaic acid, but not by a PP1 inhibitor tautomycetin. Because NO deficiency contributes to salt-induced hypertension, we tested the salt-sensitivity of E-Cul3∆9 mice. While both male and female E-Cul3∆9 mice developed salt-induced hypertension and renal injury, the pressor effect of salt was greater in female mutants. The increased salt-sensitivity in female E-Cul3∆9 mice was associated with decreased renovascular relaxation and impaired natriuresis in response to a sodium load. Thus, CUL3 mutations in the endothelium may contribute to human hypertension in part through decreased endothelial NO bioavailability, renovascular dysfunction, and increased salt-sensitivity of BP.

High Salt Upregulates Ca2+-Sensing Receptor Expression and Ca2+-Induced Relaxation of Contracted Mesenteric Arteries from Dahl Salt-Sensitive Rats.

High Ca2+ lowers blood pressure in hypertension, but the mechanism is not clear. The missing link may be the perivascular sensory nerve Ca2+-sensing receptor (CaSR) that mediates a vasodilator system after activation by interstitial Ca2+ Our results show that high salt increased CaSR expression in mesenteric arteries as well as Ca2+ relaxation of contracted mesenteric arteries from salt-sensitive (SS) rats. The CaSR was expressed as a doublet (≈120-150 kDa) in arteries from animals fed a high-salt diet for 1-4 weeks. The higher molecular weight glycosylated protein increased in arteries from SS animals; however, expression of the low molecular mass high-mannose protein decreased over 4 weeks of feeding the diet. In tissues from salt-resistant (SR) rats, the diet decreased CaSR expression after 4 weeks. Ca2+ relaxation of mesenteric arteries under phenylephrine tone increased in SS rats but decreased in arteries from SR rats fed the high-salt diet. Ca2+-activated K+ channels have a larger role in Ca2+ relaxation of arteries in SR than SS rats. The data suggest that high salt epigenetically regulates the receptor at the translational level in vivo and that the in vitro effect of Ca2+ is on receptor trafficking and signaling. In conclusion, upregulated expression of the CaSR in salt sensitivity increased receptor-mediated vascular relaxation. These findings show that CaSR signaling may compensate for changes in the vasculature in salt-sensitive hypertension. SIGNIFICANCE STATEMENT: The perivascular sensory nerve Ca2+-sensing receptor (CaSR) mediates Ca2+ relaxation of isolated mesenteric arteries under tension. This receptor may therefore play a significant role in relaxation of resistance arteries in vivo, thus explaining the blood pressure-lowering effect of dietary Ca2+. The present studies describe the effect of high salt-induced upregulation of the CaSR in salt-sensitive rats and the roles played by Ca2+-activated K+ channels and nitric oxide in Ca2+ responses.

Kv7 Channels in Cyclic-Nucleotide Dependent Relaxation of Rat Intra-Pulmonary Artery.

Pulmonary hypertension is treated with drugs that stimulate cGMP or cAMP signalling. Both nucleotides can activate Kv7 channels, leading to smooth muscle hyperpolarisation, reduced Ca2+ influx and relaxation. Kv7 activation by cGMP contributes to the pulmonary vasodilator action of nitric oxide, but its contribution when dilation is evoked by the atrial natriuretic peptide (ANP) sensitive guanylate cyclase, or cAMP, is unknown. Small vessel myography was used to investigate the ability of Kv7 channel blockers to interfere with pulmonary artery relaxation when cyclic nucleotide pathways were stimulated in different ways. The pan-Kv7 blockers, linopirdine and XE991, caused substantial inhibition of relaxation evoked by NO donors and ANP, as well as endothelium-dependent dilators, the guanylate cyclase stimulator, riociguat, and the phosphodiesterase-5 inhibitor, sildenafil. Maximum relaxation was reduced without a change in sensitivity. The blockers had relatively little effect on cAMP-mediated relaxation evoked by forskolin, isoprenaline or treprostinil. The Kv7.1-selective blocker, HMR1556, had no effect on cGMP or cAMP-dependent relaxation. Western blot analysis demonstrated the presence of Kv7.1 and Kv7.4 proteins, while selective activators of Kv7.1 and Kv7.4 homomeric channels, but not Kv7.5, caused pulmonary artery relaxation. It is concluded that Kv7.4 channels contribute to endothelium-dependent dilation and the effects of drugs that act by stimulating cGMP, but not cAMP, signalling.

The Origin of Vasomotion and Stochastic Resonance in Vasomotion.

Vasomotion is the spontaneous time-dependent contraction and relaxation of micro arteries and the oscillating frequency is about 0.01–0.1 Hz. The physiological mechanism of vasomotion has not been thoroughly understood. From the dynamics point of view, the heartbeat is the only external loading exerted on the vascular system. We speculate that the nonlinear vascular system and the variable period of the heartbeat might induce the low-frequency vasomotion. In this study, the laser Doppler flowmeter is used to measure the time series of radial artery blood flow and reconstructed modified time series that has the same period as the measured time series but different heartbeat curves. We measured the time series of radial artery blood flow in different conditions by adding different noise disturbances on the forearm, and we decomposed the experiment pulse signal by Hilbert–Huang transform. The wavelet spectral analyses showed that the low-frequency components were induced by the variable period but independent of the shape of the heartbeat curve. Furthermore, we simulated the linear flow in a single pipe and the nonlinear flow in a piping network and found that the nonlinear flow would generate low-frequency components. From the results, we could deduce that the variable period of heartbeat and the nonlinearity of the vascular system induce vasomotion. The noise has effects on the blood signals related to the respiratory activities (∼0.3 Hz) but little influence on that related to the cardiac activities (∼1 Hz). Adding white noise and then stopping would induce an SNR increase in the frequency band related to vasomotion (∼0.1 Hz).