Third-order Mesenteric Arteries Research Articles

Is there a role for biogenic amine receptors in mediating β-phenylethylamine and RO5256390-induced vascular contraction?

BackgroundSubstantial evidence indicates trace amines can induce vasoconstriction independently of noradrenaline release. However, the mechanism underlying noradrenaline-independent vasoconstrictor responses to trace amines has not yet been established. This study evaluates the role of trace amine-associated receptor 1 (TAAR1) and other biogenic amine receptors in mediating β-phenylethylamine and the TAAR-1 selective agonist RO5256390-induced vasoconstriction. MethodsVasoconstrictor responses to β-PEA and the TAAR1-selective agonist, RO5256390 were assessed in vitro in endothelium-denuded aortic rings and third-order mesenteric arteries of male Sprague Dawley rats. Resultsβ-PEA and RO5256390 induced concentration-dependent vasoconstriction of aortic rings but not third-order mesenteric arteries. Vasoconstrictor responses in aortic rings were insensitive to antagonists of 5-HT. The murine-selective TAAR1 antagonist, EPPTB, had no effect on either β-PEA or RO5256390-induced vasoconstriction. The α1-adrenoceptor antagonist, prazosin, and the α2-adrenoceptor antagonist, yohimbine, induced a shift of the β-PEA concentration response curve too small to be ascribed to antagonism of α1-or α2-adrenoceptors, respectively. The α2-adrenoceptor antagonist atipamezole had no effect on β-PEA or RO5256390-induced vasoconstriction. ConclusionVasoconstrictor responses to trace amines are not mediated by classical biogenic amine neurotransmitter receptors. Insensitivity of β-PEA vasoconstrictor responses to EPPTB, may be explained by its low affinity for rat rather than murine TAAR1. Therefore, TAAR1 remains the most likely candidate receptor mediating vasoconstrictor responses to trace amines and that prazosin and yohimbine have low affinity for TAAR1.

Fibulin-5 Is Required for Small Artery Development and Function

Introduction: Fibulins are a family of eight extracellular matrix proteins with diverse functions. Fibulin-4 and -5 (FBLN-4 and -5), in particular, have been shown to be important for elastic fiber assembly. Mutations in each of the genes lead to cutis laxa, a syndrome characterized by loose skin with variable organ involvement, particularly in the vasculature, lungs and skeleton. We recently characterized a mouse model carrying a disease-causing mutation in Fbln4 (E57K) and, contrary to conduit arteries that had elastic fiber fragmentation and stiffness, Fbln4E57K resistance arteries were structurally intact. Functionally however, Fbln4E57K mesenteric arteries exhibited endothelial dysfunction secondary to increased shear stress from large artery stiffness. Loss of FBLN5 has been shown to lead to elastic fiber fragmentation in conduit arteries, but the consequences of its loss on resistance arteries are unknown. Therefore, the objectives of this study were to determine the structural and functional consequences of FBLN5 loss on resistance arteries. Methods: We utilized adult Fbln5 knock-out (KO) mice and examined the ultrastructure of renal and mesenteric arteries via transmission electron microscopy. Functionally, using pressure myography, we characterized the myogenic tone and reactivity of Fbln5 KO and littermate control third-order mesenteric arteries to various vasoactive substances including acetylcholine, papaverine, angiotensin II, phenylephrine and potassium chloride. Results: While the internal elastic lamina (IEL) of Fbln5KO mice resistance arteries was largely intact, the external elastic lamina (EEL) was nearly absent. Similar to Fbln4E57K mice, Fbln5KO mesenteric arteries exhibited impaired endothelial-dependent vasorelaxation, likely secondary to proximal large artery stiffness. Unlike Fbln4E57K mesenteric arteries however, Fbln5 KO mesenteric arteries exhibited increased myogenic tone as well as a hypercontractile response to angiotensin II and potassium chloride, but not to phenylephrine. Conclusions: In addition to highlighting differences in the requirements for elastic fiber assembly along the arterial tree, these data emphasize the differential contribution of FBLN4 and FBLN5 to IEL vs. EEL in resistance arteries and suggest pathway-specific changes in smooth muscle cell contractility secondary to FBLN5 deficiency. This work was supported by the Washington University Pediatric Nephrology Research Core Pilot Grant Program to CMH. Funds were also provided by The Department of Pediatrics at Washington University in St. Louis School of Medicine to CMH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of vasoactive substances on biomechanics of small resistance arteries of male and female Dahl salt-sensitive rats.

Changes in vascular biomechanics leading to increase in arterial stiffness play a pivotal role in circulatory dysfunction. Our objectives were to examine sex-specific pharmacological changes related to the biomechanics and any structural modifications in small resistance arteries of Dahl salt-sensitive male and female rats. The composite Young modulus (CYM) was determined using pressure myograph recordings, and immunohistochemistry was used for the evaluation of any structural changes in the third-order mesenteric arteries (n = 6). Animals on high-salt diet developed hypertension with significant elevation in central and peripheral blood pressures and pulse wave velocity compared to those on regular diet. There were no significant differences observed in the CYM between any of the groups (i.e., males and females) in vehicle-treated time-control studies. The presence of verapamil (0.3 μM) significantly reduced CYM in hypertensive males without changes within females compared to vehicle. This effect was abolished by phenylephrine (0.3 μM). BaCl2 (100 μM), ouabain (100 μM), and L-NAME (0.3 μM) combined significantly increased CYM in vessels from in normotensive males and females but not in hypertensive males compared to vehicle. The increase in CYM was abolished in the presence of phenylephrine. Sodium nitroprusside (0.3 μM), in the presence of phenylephrine, significantly reduced CYM in male normotensive versus hypertensive, with no differences within females. Significant differences were observed in immunohistochemical assessment of biomechanical markers of arterial stiffness between males and females. Our findings suggest sex possibly due to pressure differences to be responsible for adaptive changes in biomechanics, and varied pharmacological responses in hypertensive state.

Gβγ subunit signalling underlies neuropeptide Y-stimulated vasoconstriction in rat mesenteric and coronary arteries.

Raised serum concentrations of the sympathetic co-transmitter neuropeptide Y (NPY) are linked to cardiovascular diseases. However, the signalling mechanism for vascular smooth muscle (VSM) constriction to NPY is poorly understood. Therefore, the present study investigated the mechanisms of NPY-induced vasoconstriction in rat small mesenteric (RMA) and coronary (RCA) arteries. Third-order mesenteric or intra-septal arteries from male Wistar rats were assessed in wire myographs for isometric tension, VSM membrane potential and VSM intracellular Ca2+ events. NPY stimulated concentration-dependent vasoconstriction in both RMA and RCA, which was augmented by blocking NO synthase or endothelial denudation in RMA. NPY-mediated vasoconstriction was blocked by the selective Y1 receptor antagonist BIBO 3304 and Y1 receptor protein expression was detected in both the VSM and endothelial cells in RMA and RCA. The selective Gβγ subunit inhibitor gallein and the PLC inhibitor U-73122 attenuated NPY-induced vasoconstriction. Signalling via the Gβγ-PLC pathway stimulated VSM Ca2+ waves and whole-field synchronised Ca2+ flashes in RMA and increased the frequency of Ca2+ flashes in myogenically active RCA. Furthermore, in RMA, the Gβγ pathway linked NPY to VSM depolarization and generation of action potential-like spikes associated with intense vasoconstriction. This depolarization activated L-type voltage-gated Ca2+ channels, as nifedipine abolished NPY-mediated vasoconstriction. These data suggest that the Gβγ subunit, which dissociates upon Y1 receptor activation, initiates VSM membrane depolarization and Ca2+ mobilisation to cause vasoconstriction. This model may help explain the development of microvascular vasospasm during raised sympathetic nerve activity.

PS-B04-3: SHORT-TERM LOW IODINE DIET EXACERBATES VASCULAR REACTIVITY WITHOUT ALTERING THYROID HORMONE PROFILES IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Iodine is an essential microelement required for thyroid hormone (TH) synthesis and regulation. Recent evidence links iodine to direct cardiovascular functional changes independent of TH. We aimed to determine the short-term effect of a low iodine diet on cardiovascular parameters in young Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. Design and method: 5-week-old WKY and SHRSP males and females (n = 5–7 per group) were assigned a normal (NID) or low iodine (LID) diet for 4 weeks. Iodine content was 1.2 mg/kg for NID and 0.211 mg/kg for LID. Tail cuff blood pressure (BP) and body weight were measured pre-diet and weekly. Transthoracic echocardiography, food intake, water consumption, and urine production were determined pre-diet and fortnightly. At sacrifice, thyroid tissue was weighed and snap-frozen for sodium iodide symporter (NIS) gene expression. Plasma was collected for thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), and free thyroxine (fT4) measurement. Third-order mesenteric arteries were assessed for vascular function using wire myography. Results: Baseline TSH, fT3, and fT4 levels were not significantly different between strains. LID did not influence food intake, body weight, thyroid weight, left ventricular (LV) mass, relative wall thickness (RWT) or BP. TSH levels, but not fT3 or fT4, significantly increased by 63.3% in SHRSP males (p = 0.0002) and 74.5% in females fed LID (p = 0.018) but not in WKY when compared to strain, sex- and age-matched rats fed NID. NIS expression significantly increased by 3.27-fold in WKY males fed LID when compared to WKY males fed NID (p = 0.03) but not in SHRSP males. A similar trend in NIS expression was observed in WKY females fed LID. Water intake/urine output ratio significantly increased by 53.62% in SHRSP females fed LID compared to SHRSP females fed NID (p = 0.04). Trends towards reduced stroke volume (SV) and cardiac output (CO) were observed in males fed LID compared to those fed NID irrespective of strain. Vascular reactivity to U46619 in third-order mesenteric resistance arteries was significantly increased by 24.9% in SHRSP males fed LID compared to SHRSP males fed NID (p = 0.03). A similar trend was evident in SHRSP females fed LID when compared to SHRSP females fed NID. Conclusions: Our results demonstrate that short-term LID increases vascular reactivity in SHRSP male rats and is associated with altered expression of genes that encode iodine transport proteins yet independent of thyroid hormone levels.

Sex hormones and vascular reactivity: a temporal evaluation in resistance arteries of male rats

The role of androgens in vascular reactivity is controversial, particularly regarding their age-related actions. The objective of this study was to conduct a temporal evaluation of the vascular reactivity of resistance arteries of young male rats, as well as to understand how male sex hormones can influence the vascular function of these animals. Endothelium-mediated relaxation was characterized in third-order mesenteric arteries of 10-, 12-, 16-, and 18w (week-old) male rats. Concentration-response curves to acetylcholine (ACh, 0.1 nmol/L-10 µmol/L) were constructed in arteries previously contracted with phenylephrine (PE, 3 µmol/L), before and after the use of nitric oxide synthase or cyclooxygenase inhibitors. PE concentration-response curves (1 nmol/L-100 μmol/L) were also built. The levels of vascular nitric oxide, superoxide anion, and hydrogen peroxide were assessed and histomorphometry analysis was performed. The 18w group had impaired endothelium-dependent relaxation. All groups showed prostanoid-independent and nitric oxide-dependent vasodilatory response, although this dependence seems to be smaller in the 18w group. The 18w group had the lowest nitric oxide and hydrogen peroxide production, in addition to the highest superoxide anion levels. Besides functional impairment, 18w animals showed morphological differences in third-order mesenteric arteries compared with the other groups. Our data show that time-dependent exposure to male sex hormones appears to play an important role in the development of vascular changes that can lead to impaired vascular reactivity in mesenteric arteries, which could be related to the onset of age-related cardiovascular changes in males.

Dynein Coordinates β2-Adrenoceptor-Mediated Relaxation in Normotensive and Hypertensive Rat Mesenteric Arteries.

The voltage-gated potassium channel (Kv)7.4 and Kv7.5 channels contribute to the β-adrenoceptor-mediated vasodilatation. In arteries from hypertensive rodents, the Kv7.4 channel is downregulated and function attenuated, which contributes to the reduced β-adrenoceptor-mediated vasodilatation observed in these arteries. Recently, we showed that disruption of the microtubule network, with colchicine, or inhibition of the microtubule motor protein, dynein, with ciliobrevin D, enhanced the membrane abundance and function of Kv7.4 channels in rat mesenteric arteries. This study aimed to determine whether these pharmacological compounds can improve Kv7.4 function in third-order mesenteric arteries from the spontaneously hypertensive rat, thereby restoring the β-adrenoceptor-mediated vasodilatation. Wire and intravital myography was performed on normotensive and hypertensive male rat mesenteric arteries and immunostaining was performed on isolated smooth muscle cells from the same arteries. Using wire and intravital microscopy, we show that ciliobrevin D enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline. This effect was inhibited partially by the Kv7 channel blocker linopirdine and was dependent on an increased functional contribution of the β2-adrenoceptor to the isoprenaline-mediated relaxation. In mesenteric arteries from the spontaneously hypertensive rat, ciliobrevin D and colchicine both improved the isoprenaline-mediated vasorelaxation and relaxation to the Kv7.2 -7.5 activator, ML213. Immunostaining confirmed ciliobrevin D enhanced the membrane abundance of Kv7.4. As well as an increase in the function of Kv7.4, the functional changes were associated with an increase in the contribution of β2-adrenoceptor following isoprenaline treatment. Immunostaining experiments showed ciliobrevin D prevented isoprenaline-mediated internalizationof the β2-adrenoceptor. Overall, these data show that colchicine and ciliobrevin D can induce a β2-adrenoceptor-mediated vasodilatation in arteries from the spontaneously hypertensive rat as well as reinstating Kv7.4 channel function.

Sex differences in the participation of endothelial mediators and signaling pathways involved in the vasodilator effect of a selective GPER agonist in resistance arteries of gonadectomized Wistar rats

AimEndothelial mechanisms underlying the vascular effects of estrogen modulated by the G protein-coupled estrogen receptor (GPER) are not well understood, especially in gonadal sex hormone deprivation. Thus, we investigated vascular function and endothelial signaling pathways involved in the selective activation of GPER in resistance arteries of gonadectomized rats. MethodsGonadectomy was performed in Wistar rats of both sexes. After 21 days, the animals were euthanized. Concentration-response curves were obtained by cumulative additions of G-1 in third-order mesenteric arteries. The vasodilatory effects of G-1 were evaluated before and after endothelium removal or incubation with pharmacological inhibitors. Tissue protein expression was measured by western blotting. Assays with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) and 2′,7′ dichlorodihydrofluorescein-diacetate (H2DCF-DA) were performed in the arteries investigated. Immunolocalization was assessed by immunofluorescence. ResultsG-1 induced partially endothelium-dependent relaxation in both sexes. The three isoforms of the enzyme nitric oxide synthase contributed to the production and release of nitric oxide in both gonadectomized groups, but the role of inducible nitric oxide synthase is more expressive in males. The mechanistic pathway by which endothelial nitric oxide synthase is phosphorylated appears to differ between sexes, with the rapid signaling pathway phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3k-Akt-eNOS) being identified for males and mitogen-activated protein kinase/extracellular signal-regulated kinase/endothelial nitric oxide synthase (MEK-ERK-eNOS) for females. The contribution of hydrogen peroxide as an endothelial relaxation mediator seems to be greater in females. ConclusionThese results provide new insights into the effects of estrogen-induced responses via GPER on vascular function in gonadal sex hormone deprivation.

Gabapentinoid-induced peripheral edema and acute heart failure: A translational approach combining a nationwide pharmacovigilance study with preclinical experimental data

Gabapentinoids are ligands of the α2-δ subunit of voltage-gated calcium channels (Cav) that have been associated with a risk of peripheral edema and acute heart failure of unknown clinical and pathophysiological characteristics. Characterize the onset patterns of gabapentinoid-induced peripheral edema and investigate the pharmacological mechanisms. All cases of peripheral edema or heart failure [MedDRA Hight-Level Term ‘total volume fluid increased’ or High-Level Group Term ‘heart failures’] involving gabapentin or pregabalin reported to the French Pharmacovigilance Centers between January 1, 1994 and April 30, 2020 were considered to describe their onset patterns (e.g., time to onset, patient comorbidities). Based on these data, we investigated the impact of gabapentinoids on the myogenic tone of rat third-order mesenteric arteries (Halpern's arteriography) and on the electrophysiological properties of rat ventricular cardiomyocytes (whole-cell voltage-clamp). A total of 58 reports were included (gabapentin n = 5, pregabalin n = 53). The female-to-male ratio was 4:1 and the median age was 77 years (IQR 57–85, range 32–95). The median time to onset were 23 days (IQR 10–54) and 17 days (IQR 3–30) for non-cardiogenic edema and acute heart failure, respectively. Whatever the type of peripheral edema, the main patient comorbidity was hypertension (17/50, 34%), they frequently occurred after a dose escalation (27/45, 60%) and the evolution was rapidly favorable after gabapentinoid discontinuation (median 7 days, IQR 5–13). On rat mesenteric arteries, gabapentinoids significantly decreased the myogenic tone to the same extent as verapamil and nifedipine. Gabapentinoids had no significant effect on Cav1.2 currents of ventricular cardiomyocytes. Gabapentinoids can cause concentration-dependent peripheral edema of early onset. While the pharmacological substrate of gabapentinoid-induced cardiac decompensation deserves further investigations, the primary mechanism of non-cardiogenic peripheral edema is vasodilatory edema secondary to altered myogenic tone, independent of Cav1.2 blockade. Clinicians should be alert to this risk and to the management of gabapentinoid-induced peripheral edema in order to limit any risk of prescribing cascade with diuretics.

G-Protein-Coupled Estrogen Receptor Expression in Rat Uterine Artery Is Increased by Pregnancy and Induces Dilation in a Ca2+ and ERK1/2 Dependent Manner.

Increasing levels of estrogens across gestation are partly responsible for the physiological adaptations of the maternal vasculature to pregnancy. The G protein-coupled estrogen receptor (GPER) mediates acute vasorelaxing effects in the uterine vasculature, which may contribute to the regulation of uteroplacental blood flow. The aim of this study was to investigate whether GPER expression and vasorelaxation may occur following pregnancy. Elucidation of the functional signalling involved was also investigated. Radial uterine and third-order mesenteric arteries were isolated from non-pregnant (NP) and pregnant rats (P). GPER mRNA levels were determined and—concentration–response curve to the GPER-specific agonist, G1 (10−10–10−6 M), was assessed in arteries pre-constricted with phenylephrine. In uterine arteries, GPER mRNA expression was significantly increased and vasorelaxation to G1 was significantly enhanced in P compared with NP rats. Meanwhile, in mesenteric arteries, there was a similar order of magnitude in NP and P rats. Inhibition of L-type calcium channels and extracellular signal-regulated kinases 1/2 significantly reduced vasorelaxation triggered by G1 in uterine arteries. Increased GPER expression and GPER-mediated vasorelaxation are associated with the advancement of gestation in uterine arteries. The modulation of GPER is exclusive to uterine arteries, thus suggesting a physiological contribution of GPER toward the regulation of uteroplacental blood flow during pregnancy.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Gabapentinoid-induced peripheral edema and acute heart failure: A translational study combining pharmacovigilance data and in vitro animal experiments

IntroductionGabapentinoids are ligands of the α2-δ subunit of voltage-gated calcium channels (Cav) that have been associated with a risk of peripheral edema and acute heart failure in connection with a potentially dual mechanism, vascular and cardiac. Objectives & methodsAll cases of peripheral edema or heart failure involving gabapentin or pregabalin reported to the French Pharmacovigilance Centers between January 1, 1994 and April 30, 2020 were included to describe their onset patterns (e.g., time to onset). Based on these data, we investigated the impact of gabapentinoids on the myogenic tone of rat third-order mesenteric arteries and on the electrophysiological properties of rat ventricular cardiomyocytes. ResultsA total of 58 reports were included (gabapentin n = 5, pregabalin n = 53). The female-to-male ratio was 4:1 and the median age was 77 years (IQR 57–85, range 32–95). The median time to onset were 23 days (IQR 10–54) and 17 days (IQR 3–30) for non-cardiogenic edema and acute heart failure, respectively. Cardiogenic and non-cardiogenic peripheral edema occurred frequently after a dose escalation (27/45, 60%), and the course was rapidly favorable after discontinuation of gabapentinoid (median 7 days, IQR 5–13). On rat mesenteric arteries, gabapentinoids significantly decreased the myogenic tone to the same extent as verapamil and nifedipine. Acute application of gabapentinoids had no significant effect on Cav1.2 currents of ventricular cardiomyocytes. ConclusionGabapentinoids can cause concentration-dependent peripheral edema of early onset. The primary mechanism of non-cardiogenic peripheral edema is vasodilatory edema secondary to altered myogenic tone, independent of Cav1.2 blockade under the experimental conditions tested.

Perivascular adipose tissue (PVAT)‐mediated buffering of vasoconstriction in placental ischemia‐induced hypertensive rats

Preeclampsia is a pregnancy-specific hypertensive disorder diagnosed along with systemic endothelial and vascular dysfunction. A novel regulator of vascular tone is the perivascular adipose tissue (PVAT) that surrounds most blood vessels and functions to blunt vasoconstriction. However, it is unknown whether PVAT becomes dysfunctional in preeclampsia. Preeclampsia arises from placental ischemia to elicit release of pro-vasoconstrictive factors into the maternal circulation. In this study, it was hypothesized that placental ischemic rats have attenuated PVAT-mediated buffering of vasoconstriction. Placental ischemia was induced on gestational day 14 in Sprague-Dawley rats by conducting the Reduced Uterine Perfusion Pressure (RUPP) protocol. Conscious blood pressure measurements (carotid catheter) and wire myography were conducted on day 19. Mean arterial blood pressure was significantly elevated (P<0.05) in RUPP (N=6, 117±3 mmHg) vs. Pregnant (N=6, 97±3 mmHg) rats. In aortas, the maximum response (as % increase in force from baseline tension) to KCl-induced depolarization was not affected (P>0.05) by RUPP or the presence of PVAT: Pregnant+PVAT (43±9%) vs. Pregnant-PVAT (42±5%) and RUPP+PVAT (42±%7) vs. RUPP-PVAT (48±7%). Furthermore, phenylephrine (Phe, alpha1-adrenergic receptor agonist)-induced vasoconstriction was similar (P>0.05) in Pregnant+PVAT (69±11%) vs. Pregnant-PVAT (70±12%) and RUPP+PVAT (38±12%) vs. RUPP-PVAT (34±9%), with the 2-way ANOVA detecting a main effect only for RUPP (P<0.05). It was then assessed whether the endothelium controls PVAT function whereby denuding the endothelium did not impact (P>0.05) any of the respective Phe responses in Pregnant+PVAT-Endo (60±12%), Pregnant-PVAT-Endo (72±11%), RUPP+PVAT-Endo (30±15%), or RUPP-PVAT-Endo (22±7%). In smaller, third-order mesenteric arteries, direct depolarization by KCl responses were similar (P>0.05) between Pregnant+PVAT (176±19%), Pregnant-PVAT (149±17%), RUPP+PVAT (148±30%), and RUPP-PVAT (167±27%). As for Phe, the 2-way ANOVA identified a main effect (P<0.05) only for PVAT to blunt Phe-induced vasoconstriction in Pregnant+PVAT (259±38%) vs. Pregnant-PVAT (337±26%) and RUPP+PVAT (176±39%) vs. RUPP-PVAT (379±47%), with no difference for the influence of PVAT between RUPP and Pregnant groups. Removal of the endothelium further attenuated Phe responses in the PVAT groups: Pregnant+PVAT-Endo (174±66%) and RUPP+PVAT-Endo (-3±3%) vs. their respective groups without PVAT: Pregnant-PVAT-Endo (379±39%) and RUPP-PVAT-Endo (339±167%). In fact, the degree of this attenuation to Phe-induced vasoconstriction was the greatest (P<0.05) in the RUPP+PVAT-Endo (-104±3%) group compared all other groups: RUPP-PVAT-Endo (19±59%), Pregnant+PVAT-Endo (10±11%), and Pregnant-PVAT-Endo (10±11%). In conclusion, as it was found that removal of the endothelium allows for enhanced PVAT blunting of vasoconstriction in RUPP rats, these data indicate that placental ischemia drives endothelial dysfunction to subsequently disrupt PVAT function in small arteries during pregnancy.

Pathophysiological adaptations of resistance arteries in rat offspring exposed in utero to maternal obesity is associated with sex-specific epigenetic alterations

Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little is known about resistance arteries. They are of particular interest because of their specific role in the regulation of local blood flow. Then our study aims to determine if maternal obesity can directly program fetal vascular dysfunction of resistance arteries, independently of metabolic disorders. With a model of rats exposed in utero to mild maternal diet-induced obesity (OMO), we investigated third-order mesenteric arteries of 4-month old rats in absence of metabolic disorders. The methylation profile of these vessels was determined by reduced representation bisulfite sequencing (RRBS). Vascular structure and reactivity were investigated using histomorphometry analysis and wire-myography. The metabolic function was evaluated by insulin and glucose tolerance tests, plasma lipid profile, and adipose tissue analysis. At 4 months of age, small mesenteric arteries of OMO presented specific epigenetic modulations of matrix metalloproteinases (MMPs), collagens, and potassium channels genes in association with an outward remodeling and perturbations in the endothelium-dependent vasodilation pathways (greater contribution of EDHFs pathway in OMO males compared to control rats, and greater implication of PGI2 in OMO females compared to control rats). These vascular modifications were detected in absence of metabolic disorders. Our study reports a specific methylation profile of resistance arteries associated with vascular remodeling and vasodilation balance perturbations in offspring exposed in utero to maternal obesity, in absence of metabolic dysfunctions.

Resistance mesenteric arteries display hypercontractility in the resolution time of Strongyloides venezuelensis infection

The blood flow in the mesenteric region is crucial for nutrient absorption and immune response in the gastrointestinal tract. The presence of nematodes or their excreted/secreted products seems to provoke vascular dysfunction. However, it is unclear whether and how the intestinal nematodes with habitat in the intestinal niche could affect the mesenteric vascular resistance. In this study, male Wistar rats were infected with 2000 larvae of S. venezuelensis, and experiments were conducted at 0 (non-infected control), 10 or 30 days post-infection (DPI). Eggs were counted in rats’ feces and adult worms recovered from the small intestine. Second- or third-order mesenteric arteries were extracted for concentration-response curves (CRC) to phenylephrine [PE; in the presence or absence of L-NAME or indomethacin] and acetylcholine. The number of eggs and adult worms were significantly higher in the 10 DPI group than those of 30 DPI group. Augmented PE-induced contraction was seen after 30 DPI compared to 10 DPI or control group. Hypercontractility to PE was partially prevented by L-NAME and wholly abolished by indomethacin incubation. Endothelium-dependent relaxation and endothelial nitric oxide synthase expression were unchanged among groups. COX-1 and COX-2 display a different pattern of expression over the infection. Hypercontractility observed in mesenteric resistance arteries in the resolution time of S. venezuelensis infection may represent systemic damage, which can generate significant cardiovascular and gastrointestinal repercussions.

Comparison of Adrenergic and Purinergic Receptor Contributions to Vasomotor Responses in Mesenteric Arteries of C57BL/6J Mice and Wistar Rats

Introduction: The sympathetic nervous system can modulate arteriolar tone through release of adenosine triphosphate and norepinephrine, which bind to purinergic and adrenergic receptors (ARs), respectively. The expression pattern of these receptors, as well as the composition of neurotransmitters released from perivascular nerves (PVNs), can vary both in organ systems within and across species, such as mice and rats. Objective: This study explores the function of α_1A subtypes in mouse and rat third-order mesenteric arteries and investigates PVN-mediated vasoconstriction to identify which neurotransmitters are released from sympathetic PVNs. Methods: Third-order mesenteric arteries from male C57BL/6J mice and Wistar rats were isolated and mounted on a wire myograph for functional assessment. Arteries were exposed to phenylephrine (PE) and then incubated with either α_1A antagonist RS100329 (RS) or α_1D antagonist BMY7378, before reexposure to PE. Electrical field stimulation was performed by passing current through platinum electrodes positioned adjacent to arteries in the absence and presence of a nonspecific alpha AR blocker phentolamine and/or P2X₁-specific purinergic receptor blocker NF449. Results: Inhibition of α₁ ARs by RS revealed that PE-induced vasoconstriction is primarily mediated through α_1A and that the contribution of the α_1A AR is greater in rats than in mice. In the mouse model, sympathetic nerve-mediated vasoconstriction is mediated by both ARs and purinergic receptors, whereas in rats, vasoconstriction appeared to only be mediated by ARs and a nonpurinergic neurotransmitter. Further, neither model demonstrated that α_1D ARs play a significant role in PE-mediated vasoconstriction. Conclusions: The mesenteric arteries of male C57BL/6J mice and Wistar rats have subtle differences in the signaling mechanisms used to mediate vasoconstriction. As signaling pathways in humans under physiological and pathophysiological conditions become better defined, the current study may inform animal model selection for preclinical studies.

Polarized Intracellular Endothelial Pannexin 3 Can Regulate Vascular Function

Pannexin 3 (Panx3) expression is generally thought to be restricted to skin, cartilage and bone where it is routinely observed on intracellular compartments. However, multiple RNAseq studies have demonstrated consistent expression of Panx3 in endothelium throughout the vasculature. A physiological role for endothelial Panx3 has yet to be described. To assess the expression of Panx3 in intact endothelium, we first performed immunogold labelling on high pressure frozen third‐order mesenteric arteries. Panx3 preferentially localized to internal membranes in transverse sections of the endothelium. Next, we used multiple Panx3 antibodies raised against different topographical locations on third‐order mesenteric arteries prepared en face. We found that endothelial Panx3 was polarized downstream of blood flow, co‐localizing with a subset of calnexin‐positive perinuclear membranes, indicating association with the endoplasmic reticulum (ER). To test whether Panx3 was flow‐sensitive, primary cultures of human endothelial cells were seeded in parallel plate flow chambers and exposed to laminar shear stress (10 dynes/cm2 for 18 hours). Panx3 was polarized in the direction of flow similar to that seen in intact resistance arteries. To interrogate the physiological role of endothelial Panx3, we generated an inducible, cell‐specific Panx3 knockout mouse (Panx3fl/fl/Cdh5‐CreERT2+), which reduces both Panx3 mRNA and protein following tamoxifen injection. Panx3fl/fl/Cdh5‐CreERT2+ mice had normal blood cell parameters, heart function, body length, body weight, and fat content. Because we identified Panx3 in the ER perinuclear region, we began by assessing intracellular calcium on intact third‐order mesenteric arteries. Loss of endothelial Panx3 reduced intracellular calcium activity in endothelium under unstimulated conditions. Because calcium is vital to arterial dilation, we performed pressure myography. We found that resistance arteries from Panx3fl/fl/Cdh5‐CreERT2+ mice exhibit significantly enhanced vasoconstriction in response to phenylephrine, indicating impaired endothelial negative feedback on the smooth muscle cells. Due to the role of resistance arteries in blood pressure regulation, we hypothesized there may be a change in the blood pressure of these mice. We used radiotelemetry and found Panx3fl/fl/Cdh5‐CreERT2+ mice were hypertensive compared to tamoxifen‐injected littermate controls. The sum of this data suggests that endothelial Panx3 is polarized on the ER where it can regulate calcium activity in the endothelium, independent of ATP release, and may be important for vascular function.Support or Funding InformationHL149221 (AGW) and HL120840 (BEI)

Impact of chronic hyperleptinemia on placental ischemia‐induced hypertension in pregnant rats

Obesity is a risk factor for preeclampsia, which is new‐onset hypertension during pregnancy. The mechanisms linking obesity and preeclampsia are poorly understood. The adipokine, leptin, is elevated in the circulation in proportion to fat mass and elicits hypertension when infused into non‐pregnant animals. However, less is studied about how hyperleptinemia impacts blood pressure regulation during pregnancy. A contributing factor to hypertension during preeclampsia is placental ischemia. Therefore, we tested the hypothesis that chronic hyperleptinemia exaggerates placental ischemia‐induced hypertension. On gestational day 14, timed‐pregnant Sprague‐Dawley rats were implanted with osmotic pumps to intravenously deliver recombinant rat leptin at 1.0 ug/kg per min or vehicle and placental ischemia was produced by the reduced uterine perfusion pressure (RUPP) procedure or Sham surgery. By day 19, leptin levels were elevated (P&lt;0.05) in Sham (leptin‐infused, N=9: 41.0±2.8 ng/mL vs vehicle, N=23: 0.8±0.1 ng/mL) and RUPP (leptin‐infused, N=9: 29.7±7.2 ng/mL vs vehicle, N=17: 1.0±0.1 ng/mL). RUPP elicited hypertension (Sham+vehicle: 100±1 mmHg vs RUPP+vehicle: 122±3 mmHg mean arterial blood pressure, P&lt;0.05). In contrast to our hypothesis, hyperleptinemia attenuated RUPP hypertension (RUPP+leptin: 109±4 mmHg, P&lt;0.05 vs RUPP+vehicle) and did not alter blood pressure in Sham rats (Sham+leptin: 100±6 mmHg vs Sham+vehicle). Therefore, we examined whether hyperleptinemia was vasoprotective by promoting vasorelaxation during pregnancy in isolated third‐order mesenteric arteries. Although endothelial‐dependent vasorelaxation to acetylcholine (measured as logEC50 sensitivity) was similar within Sham (vehicle: −6.8±0.1 M vs leptin: −6.9±0.2 M) and RUPP (vehicle: −7.0±0.2 M vs leptin: −7.0±0.3 M) groups, endothelial‐independent vasorelaxation to the nitric oxide (NO)‐donor, sodium nitroprusside, was increased (P&lt;0.05) in both Sham (vehicle: −6.0±0.2 M vs leptin: −6.8±0.3 M) and RUPP (vehicle: −6.9±0.2 M vs leptin: −7.6±0.01 M) groups. This suggested that NO‐cyclic guanosine monophosphate (cGMP) signaling was increased. Plasma levels of cGMP were elevated (P&lt;0.05) within Sham (vehicle: 1.6±0.3 pmol/mL vs leptin: 3.9±0.6 pmol/mL) and RUPP (vehicle: 3.6±0.8 pmol/mL vs leptin: 5.8±1.1 pmol/mL) groups. In contrast to our original hypothesis, chronic hyperleptinemia does not produce hypertension during pregnancy likely via promoting vascular NO‐cGMP signaling.Support or Funding InformationHL130577

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

Mesenchymal stem cells promote mesenteric vasodilation through hydrogen sulfide and endothelial nitric oxide.

Mesenteric ischemia is a devastating process that can result in intestinal necrosis. Mesenchymal stem cells (MSCs) are becoming a promising treatment modality. We hypothesized that 1) MSCs would promote vasodilation of mesenteric arterioles, 2) hydrogen sulfide (H2S) would be a critical paracrine factor of stem cell-mediated vasodilation, 3) mesenteric vasodilation would be impaired in the absence of endothelial nitric oxide synthase (eNOS) within the host tissue, and 4) MSCs would improve the resistin-to-adiponectin ratio in mesenteric vessels. H2S was measured with a specific fluorophore (7-azido-3-methylcoumarin) in intact MSCs and in cells with the H2S-producing enzyme cystathionine β synthase (CBS) knocked down with siRNA. Mechanical responses of isolated second- and third-order mesenteric arteries (MAs) from wild-type and eNOS knockout (eNOSKO) mice were monitored with pressure myography, after which the vessels were snap frozen and later analyzed for resistin and adiponectin via multiplex beaded assay. Addition of MSCs to the myograph bath significantly increased vasodilation of norepinephrine-precontracted MAs. Knockdown of CBS in MSCs decreased H2S production by MSCs and also decreased MSC-initiated MA dilation. MSC-initiated vasodilation was further reduced in eNOSKO vessels. The MA resistin-to-adiponectin ratio was higher in eNOSKO vessels compared with wild-type. These results show that MSC treatment promotes dilation of MAs by an H2S-dependent mechanism. Furthermore, functional eNOS within the host mesenteric bed appears to be essential for maximum stem cell therapeutic benefit, which may be attributable, in part, to modifications in the resistin-to-adiponectin ratio.NEW & NOTEWORTHY Stem cells have been shown to improve survival, mesenteric perfusion, and histological injury scores following intestinal ischemia. These benefits may be due to the paracrine release of hydrogen sulfide. In an ex vivo pressure myography model, we observed that mesenteric arterial dilation improved with stem cell treatment. Hydrogen sulfide release from stem cells and endothelial nitric oxide synthase within the vessels were critical components of optimizing stem cell-mediated mesenteric artery dilation.