Isolated Resistance Arteries Research Articles

Targeting endothelial KCa channels in vivo restores arterial and endothelial function in type 2 diabetic rats

ObjectiveThis study tested the hypothesis that administration of the KCa channel activator SKA-31 restores endothelium-dependent vasodilation in vivo in Type 2 Diabetic (T2D) rats. BackgroundAcute treatment of isolated resistance arteries from T2D rats and humans with SKA-31 significantly improved endothelium-dependent vasodilation. However, it is unknown whether these in situ actions translate to intact vascular beds in vivo. MethodsMale Sprague Dawley (SD) and T2D Goto-Kakizaki (GK) rats (26–32 weeks of age) were injected intraperitoneally with either drug vehicle or 10 mg/kg SKA-31. Doppler ultrasound imaging was used to record reactive hyperemia/flow-mediated dilation (FMD) in the femoral artery following release of an occlusion cuff on the distal hind limb, along with diameter changes in the left main coronary artery in response to inhaled isoflurane (2 % → 5 %). ResultsVehicle treated SD rats exhibited a robust and reversible FMD response, the magnitude and time course of which did not differ in SD rats treated with SKA-31. In contrast, only a weak FMD response was observed in vehicle-treated T2D GK rats, whereas prior SKA-31 administration restored FMD to the level observed in control SD rats. Exposure of SD rats to 5 % isoflurane caused robust coronary artery dilation, which was not altered by prior treatment with SKA-31. In T2D GK rats, 5 % isoflurane inhalation alone did not increase coronary artery diameter, however, a strong vasodilatory response was observed following SKA-31 treatment. SKA-31 administration did not modify intrinsic heart rate responses in either protocol. ConclusionsEnhancement of KCa channel activity in vivo restores endothelium-dependent vasodilation in T2D rats that exhibit peripheral endothelial dysfunction.

A Common Alpha Globin Gene Deletion is Associated with Decreased Arterial Alpha Globin Gene Expression and Altered Vasoreactivity among Black Americans

Alpha globin binds to endothelial nitric oxide (NO) synthase (eNOS) and limits the diffusion of NO from endothelial cells into vascular smooth muscle. Previously, we demonstrated that pharmacologic disruption of alpha globin‐eNOS binding enhanced NOS‐dependent vasodilation in human arteries. Here, we explore the hypothesis that individuals with alpha globin gene deletions will have reduced alpha globin gene expression in resistance arteries and altered arterial vasoreactivity.To test this hypothesis, we screened healthy Black Americans (clinical trial # 02692872) for a common 3.7 kb gene deletion in the alpha globin gene locus (HBA) that reduces the number of functional gene copies. We excluded people with risk factors for cardiovascular disease and created a nested case control study that included 15 people with four copies, 8 people with three copies, and 2 people with two copies of HBA. Each individual underwent an incisional subcutaneous adipose biopsy (clinical trial # 03937817), from which we isolated resistance arteries less than 200 μm in diameter. Resistance arteries were cannulated and perfused free of blood before undergoing multiphoton imaging, gene expression analysis, or pressure myography.Multiphoton imaging of adipose resistance arteries revealed alpha globin to co‐localize with eNOS near the myoendothelial junction between endothelial and smooth muscle cells. Arteries from individuals with three functional copies of HBA had lower expression of HBA compared to arteries from individuals with four functional copies of HBA(1715 copies/ng cDNA vs 1176 copies/ng cDNA, p = 0.03). The concentration of phenylephrine required to constrict the human adipose arteries to 50% of baseline was 8 times greater for arteries from subjects with three copies of HBA compared to arteries from subjects with four copies of HBA (log10[EC50]: ‐4.78 (95% CI: ‐4.29 to ‐5.22) vs ‐5.68 ± (95%CI: ‐5.33 to ‐5.98) p = 0.003). Arteries from individuals with three copies of HBA also had reduced maximal constriction to phenylephrine compared to subjects with four copies of HBA(p < 0.01).These results offer compelling new genetic evidence from humans supporting the role of alpha globin as a regulator of NO signaling in resistance arteries. We demonstrate that HBA deletions reduce arterial expression of HBA and change arterial vasoreactivity. This clinical investigation defines a novel vascular phenotype for individuals with alpha thalassemia and provides insight into a vascular mechanism by which alpha globin gene deletions protect against severe malaria, chronic kidney disease, and vascular complications of sickle cell disease.

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species.

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar-Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

Biochemical characterization of AeD7L2 and its physiological relevance in blood feeding in the dengue mosquito vector, Aedes aegypti.

Aedes aegypti saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis. Among these salivary proteins are the D7s, which are highly abundant and have been shown to act as scavengers of biogenic amines and eicosanoids. In this work, we performed comparative structural modeling, characterized the binding capabilities, and assessed the physiological functions of the Ae. aegypti salivary protein AeD7L2 compared to the well-characterized AeD7L1. AeD7L1 and AeD7L2 show different binding affinities to several biogenic amines and biolipids involved in host hemostasis. Interestingly, AeD7L2 tightly binds U-46619, the stable analog of thromboxane A2 (KD =69.4nm), which is an important platelet aggregation mediator, while AeD7L1 shows no binding. We tested the ability of these proteins to interfere with the three branches of hemostasis: vasoconstriction, platelet aggregation, and blood coagulation. Pressure myography experiments showed these two proteins reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U-46619. These proteins also inhibited platelet aggregation induced by low doses of collagen or U-46619. However, D7 long proteins did not affect blood coagulation. The different ligand specificity and affinities of AeD7L1 and AeD7L2 matched our experimental observations from studying their effects on vasoconstriction and platelet aggregation, which confirm their role in preventing host hemostasis. This work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding.

β‐Hydroxybutyrate (βOHB) Activates Gpr109a to Contribute to the Anti‐vascular Aging Effect of Autophagy

The aged phenotype is classically viewed as the accumulation of cellular debris and dysfunctional organelles. Although waste products are an inevitable consequence of normal cellular metabolism, multiple systems are devoted to its repair, clearance, or recycling. Autophagy is the constitutively active catabolic process responsible for cellular degradation. Autophagic activity has also been implicated as a modulator of longevity, as well as inversely related with chronological vascular aging and premature vascular aging associated with hypertension. However, the mechanisms by which autophagy exerts anti‐vascular aging effects are still unknown. Evolutionarily, autophagy functions to mobilize nutrients in times of starvation. Specifically, hepatic autophagy releases free fatty acids from triglycerides, which are oxidized to generate ketone bodies. Therefore, we hypothesized that fasting would increase the systemic expression of ketone body β‐hydroxybutyrate (βOHB) in Dahl salt‐sensitive rats fed a low salt‐diet, and this would be prevented by inhibition of autophagy with chloroquine (CQ). As hypothesized, fasting increased serum βOHB, and treatment with CQ blocked this increase (mmol/L, non‐fasting: 0.38±0.01 vs. fasting: 1.01±0.06* vs. fasting+CQ: 0.45±0.04, *p<0.05) (Figure 1). Next we questioned whether βOHB could potentially contribute to the anti‐vascular aging effect of autophagy. Previously, we have observed that βOHB is a potent vasodilator via endothelial potassium channels. However, it is not known if βOHB acts as an agonist or antagonist to any specific receptor to mediate endothelium‐dependent relaxation. To help answer this question we needed to switch to a genetic knockout model, as pharmacological antagonists for the βOHB receptors, Gpr109a and Gpr41, do not exist. βOHB activates Gpr109a, whereas it is generally recognized as an antagonist of Gpr41. Therefore, we hypothesized that vascular relaxation to βOHB would be significantly attenuated in isolated mesenteric resistance arteries from Gpr109a−/− mice, but not Gpr41−/− mice. As hypothesized, vasodilatory effect of βOHB was significantly blunted in Gpr109a−/− mice, but not Gpr41−/− mice [Emax (%relaxation), WT: 75±2 vs. Gpr109a−/−: 53±3* vs. Gpr41−/−: 73±1, *p<0.05] (Figure 2). Overall, these data reveal that ketone body βOHB is stimulated by autophagy and that activation of Gpr109a by βOHB can cause vasodilation of isolated resistance arteries. Significantly, our data define a novel mechanism that underlies the anti‐vascular aging effect of autophagy.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (R00GM118885 and R01HL143082).Fasting (24 h) stimulates serum βOHB expression and this is prevented by autophagy inhibition (chloroquine 50 mg/kg i.p.; CQ). n=6–10. One way ANOVA: *p<0.05.Figure 1βOHB mediates vasodilation partially via Gpr109a, and not Gpr41. Concentration response curves to βOHB. n=2–4. Two‐way ANOVA: *p<0.05. vs. wild type (WT).Figure 2

Vasoresponsiveness in patients with heart failure (VASOR): protocol for a prospective observational study

BackgroundVasoplegia is a severe complication which may occur after cardiac surgery, particularly in patients with heart failure. It is a result of activation of vasodilator pathways, inactivation of vasoconstrictor pathways and the resistance to vasopressors. However, the precise etiology remains unclear. The aim of the Vasoresponsiveness in patients with heart failure (VASOR) study is to objectify and characterize the altered vasoresponsiveness in patients with heart failure, before, during and after heart failure surgery and to identify the etiological factors involved.MethodsThis is a prospective, observational study conducted at Leiden University Medical Center. Patients with and patients without heart failure undergoing cardiac surgery on cardiopulmonary bypass are enrolled. The study is divided in two inclusion phases. During phase 1, 18 patients with and 18 patients without heart failure are enrolled. The vascular reactivity in response to a vasoconstrictor (phenylephrine) and a vasodilator (nitroglycerin) is assessed in vivo on different timepoints. The response to phenylephrine is assessed on t1 (before induction), t2 (before induction, after start of cardiotropic drugs and/or vasopressors), t3 (after induction), t4 (15 min after cessation of cardiopulmonary bypass) and t5 (1 day post-operatively). The response to nitroglycerin is assessed on t1 and t5. Furthermore, a sample of pre-pericardial fat tissue, containing resistance arteries, is collected intraoperatively. The ex vivo vascular reactivity is assessed by constructing concentrations response curves to various vasoactive substances using isolated resistance arteries. Next, expression of signaling proteins and receptors is assessed using immunohistochemistry and mRNA analysis. Furthermore, the groups are compared with respect to levels of organic compounds that can influence the cardiovascular system (e.g. copeptin, (nor)epinephrine, ANP, BNP, NTproBNP, angiotensin II, cortisol, aldosterone, renin and VMA levels). During inclusion phase 2, only the ex vivo vascular reactivity test is performed in patients with (N = 12) and without heart failure (N = 12).DiscussionUnderstanding the difference in vascular responsiveness between patients with and without heart failure in detail, might yield therapeutic options or development of preventive strategies for vasoplegia, leading to safer surgical interventions and improvement in outcome.Trial registrationThe Netherlands Trial Register (NTR), NTR5647. Registered 26 January 2016.

Persistent insulin signaling coupled with restricted PI3K activation causes insulin-induced vasoconstriction.

Insulin modulates vasomotor tone through vasodilator and vasoconstrictor signaling pathways. The purpose of the present work was to determine whether insulin-stimulated vasoconstriction is a pathophysiological phenomenon that can result from a combination of persistent insulin signaling, suppressed phosphatidylinositol-3 kinase (PI3K) activation, and an ensuing relative increase in MAPK/endothelin-1 (ET-1) activity. First, we examined previously published work from our group where we assessed changes in lower-limb blood flow in response to an oral glucose tolerance test (endogenous insulin stimulation) in lean and obese subjects. The new analyses showed that the peak rise in vascular resistance during the postprandial state was greater in obese compared with lean subjects. We next extended on these findings by demonstrating that insulin-induced vasoconstriction in isolated resistance arteries from obese subjects was attenuated with ET-1 receptor antagonism, thus implicating ET-1 signaling in this constriction response. Last, we examined in isolated resistance arteries from pigs the dual roles of persistent insulin signaling and blunted PI3K activation in modulating vasomotor responses to insulin. We found that prolonged insulin stimulation did not alter vasomotor responses to insulin when insulin-signaling pathways remained unrestricted. However, prolonged insulinization along with pharmacological suppression of PI3K activity resulted in insulin-induced vasoconstriction, rather than vasodilation. Notably, such aberrant vascular response was rescued with either MAPK inhibition or ET-1 receptor antagonism. In summary, we demonstrate that insulin-induced vasoconstriction is a pathophysiological phenomenon that can be recapitulated when sustained insulin signaling is coupled with depressed PI3K activation and the concomitant relative increase in MAPK/ET-1 activity.NEW & NOTEWORTHY This study reveals that insulin-induced vasoconstriction is a pathophysiological phenomenon. We also provide evidence that in the setting of persistent insulin signaling, impaired phosphatidylinositol-3 kinase activation appears to be a requisite feature precipitating MAPK/endothelin 1-dependent insulin-induced vasoconstriction.

ADAM17 Cleaves the Insulin Receptor α‐Subunit on Endothelial Cells and Induces Vascular Insulin Resistance in Type 2 Diabetes

Microvascular insulin resistance is a hallmark of type 2 diabetes (T2D) and an underlying event in the pathophysiology of endothelial dysfunction and cardiovascular disease. However, the molecular mechanisms by which T2D causes vascular insulin resistance remain largely unknown. Hyperglycemia and the proinflammatory state associated with T2D are characterized, in part, by an increased activity of ADAM17, an enzyme that cleaves the ectodomain of various transmembrane proteins. Here, we hypothesized that increased expression and activation of ADAM17 sheds the insulin receptor α‐subunit (IRα) from endothelial cells causing vascular insulin resistance in T2D. We tested this hypothesis in isolated resistance arteries from T2D and non‐T2D subjects undergoing bariatric surgery and in cultured human umbilical vein endothelial cells (HUVECs). Resistance arteries from T2D subjects exhibited increased ADAM17 expression, reduced presence of endothelial IRα as well as the endogenous ADAM17 inhibitor, TIMP3, and impaired insulin‐induced vasodilation, relative to arteries from non‐T2D subjects (P<0.05). Exposure of HUVECs to high glucose (30mM, 24h) increased the expression and activity of ADAM17 (P<0.05), led to increased shedding of IRα detected in the cell culture media supernatant (P<0.05) and decreased IRα on cell surfaces as observed using super‐resolution microscopy. Moreover, pharmacological inhibition of ADAM17 led to reduced shedding of IRα and improved insulin signaling in cells exposed to high glucose or the ADAM17 activator, PMA (P<0.05). Collectively, these findings suggest that T2D, and in particular hyperglycemia, leads to ADAM17 shedding of IRα from the endothelial surface and reduced insulin‐dependent vasodilation, all of which supports ADAM17 activity as a new therapeutic target to ameliorate vascular insulin resistance in T2D.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of protein kinase C impairs endothelium‐dependent vasorelaxation in isolated human omental resistance arteries

Chronic diseases such as obesity and diabetes are associated with endothelial dysfunction and increased cardiovascular disease risk. Notably, protein kinase C (PKC) has been identified as a major molecular contributor to chronic disease‐associated microvascular impairments. However, the molecular mechanisms by which PKC activation causes microvascular endothelial dysfunction are not fully understood. Herein, we tested the hypothesis that PKC activation impairs microvascular vasorelaxation through an endothelial nitric oxide synthase (eNOS)‐dependent mechanism in human isolated resistance arteries and cultured endothelial cells. Accordingly, resistance arteries were isolated from the omentum of 32 patients (52±3 years of age, 43.3±1.7 BMI, 76% women) that underwent abdominal surgery and analyzed for vasomotor function ex vivo using wire myography. We found that pharmacological inhibition of PKC (ruboxistaurin, 1μM) for 30 minutes enhanced endothelium‐dependent vasorelaxation in response to bradykinin relative to untreated arteries (p<0.05). This effect of ruboxistaurin was abrogated by NOS inhibition (L‐NAME, 300μM), suggesting that improved endothelium‐dependent relaxation with PKC antagonism is nitric oxide‐dependent (p<0.05). Next, we found that treatment with the PKC activator, phorbol 12‐myristate 13‐acetate (PMA, 0.5μM), for 30 minutes impaired endothelium‐dependent and ‐independent vasorelaxation, which was abolished with concomitant incubation of ruboxistaurin (p<0.05). Furthermore, we found that PMA treatment reduced Akt and eNOS activation in cultured endothelial cells (HUVEC; p<0.05). In conclusion, these findings suggest that in isolated human abdominal adipose tissue resistance arteries, PKC activation impairs microvascular endothelial function likely through an eNOS‐dependent mechanism. Follow‐up experiments are ongoing to determine the involvement of specific PKC isoforms and upstream molecular regulators contributing to PKC‐mediated impairments in endothelial function.Support or Funding InformationResearch support: National Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prolonged Administration of the KCa Channel Activator SKA‐31 Improves Cardiac Function and Blood Pressure in Type 2 Diabetic Goto‐Kakizaki Rats

Endothelial dysfunction represents an independent risk factor contributing to the onset and progression of cardiovascular complications (e.g. hypertension, cardiomyopathy, stroke) in patients with Type 2 Diabetes (T2D). It is further predicted that reduction/reversal of endothelial dysfunction will mitigate T2D‐associated cardiovascular deficits. Small‐ and intermediate‐conductance, Ca2+‐activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacologic activators of these channels (e.g. SKA‐31) induce robust vasodilation upon acute application to isolated resistance arteries and intact animals. However, the effects of prolonged, in vivo administration of a KCa activator on the cardiovascular system have not been examined to date. In our current study, we have hypothesized that daily administration of SKA‐31 (10 and 30 mg/kg, I.P. injection) to male, non‐obese Type 2 Diabetic Goto‐Kakizaki rats (T2D GK, 14 weeks of age) for 12 weeks would oppose the development of diabetes‐related cardiac dysfunction. Echocardiographic analyses revealed that left ventricular (LV) ejection fraction, stroke volume and fractional shortening all significantly declined over time in vehicle‐treated rats, but remained at pre‐trial levels in animals administered either 10 or 30 mg/kg SKA‐31 for 12 weeks (n = 5–6/group). Chronic blood pressure measurements over the same period via radiotelemetry further revealed that SKA‐31 treatment lowered mean arterial pressure, compared with vehicle treated animals, with effects observed on both systolic and diastolic pressures (n = 5–6/group). To determine if the observed improvements in cardiac performance may be due to direct changes in the responsiveness of the coronary vasculature, isolated hearts from vehicle and SKA‐31 treated T2D GK rats were mounted in a Langendorff perfusion system under constant pressure (n ≥ 3/group). We observed that endothelium‐dependent, agonist‐evoked increases in coronary flow were similar in hearts from vehicle and SKA‐31 treated animals, as was baseline LV developed pressure. Histological staining of isolated hearts revealed differing levels of LV collagen content in the 3 treatment groups. In summary, these results indicate that prolonged SKA‐31 administration in T2D GK rats can improve cardiac function, which likely occur via both direct and indirect effects.Support or Funding InformationThis study was supported by research funding to APB from the Canadian Institutes of Health Research (MOP‐142467) and the Natural Sciences and Engineering Research Council of Canada (RGPIN‐2017‐04116), and to HW from the National Institutes of Health (R21 NS101876).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Omental Arteries from Diabetic Hypertensive Subjects are Larger and Stiffer than those from Non‐Diabetic Normotensives

Diabetes and hypertension are associated with structural and mechanical changes of the resistance vasculature that contribute to cardiovascular disease development and progression. We have previously reported that in mouse isolated resistance arteries prolonged vasoconstriction leads to vascular inward remodeling and stiffening, through a process that requires smooth muscle actin polymerization. We hypothesized that human arteries from diabetic hypertensive subjects would be inwardly remodeled and stiffer than those from non‐diabetic normotensives. We also hypothesized that actin stress fiber disruption would dilate and soften the arteries from diabetic hypertensives. To test these hypotheses, omental samples were obtained from patients undergoing abdominal surgery at The University of Missouri Hospital. Small arteries were isolated, cannulated and analyzed using pressure myography. Vascular luminal diameters and wall‐thicknesses were monitored by videomicroscopy at different levels of intraluminal pressure (5–120mmHg) under passive conditions (calcium‐free buffer with 100μM adenosine and 2mM EGTA) in the absence or presence of Mycalolide‐B (2uM) to disrupt the actin cytoskeleton. Investigators were blinded to the subject condition throughout the experimental procedures. Data were used for assessment of vascular remodeling and characterization of vascular wall mechanical properties. Subsequently, the arterial parameters obtained were compared across groups according to diabetic and hypertensive status retrieved from medical records. Results indicate that small omental arteries from diabetic hypertensive (N=10) subjects have larger diameters and are stiffer than those from non‐diabetic normotensives (N=22, P<0.05). Data also show that disruption of the actin cytoskeleton had no significant effects on their mechanical properties. We conclude that small omental arteries from diabetic and hypertensive subjects undergo a process of outward remodeling that results in them becoming larger and stiffer and that, at the single time point of our sampling, this is not dependent on vascular cell actin stress fibers.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 485: Novel Mechanisms at Immune-vascular Interfaces Regulates Myogenic Tone of Resistance Arteries and Induce Hypertension in Mice

In the search for mechanisms regulating the pathophysiological relationship existing between hypertension and immune system, CD8 effector T cells emerge as possible mediators of target organ colonization. However, how the crosstalk at vascular interfaces is established still remains unidentified. Phosphatidylinositol-3-kinase gamma (PI3Kγ) is an intracellular signaling involved in the acquisition of CD8 effectors functions and was previously showed by our group as involved in hypertension. The hypothesis here tested rely on the possibility that PI3Kγ could regulate trafficking of immune cells at vascular interfaces relevant for hypertension. By taking advantage of a knock-in mouse model, expressing a constitutively active PI3Kγ isoform (PI3Kγ CX/CX ), we conceived an experimental setting allowing to assess the direct crosstalk between immune cells and vasculature, obtained by co-culturing CD8 T cells with isolated resistance arteries mounted in pressurized systems. Thus, CD8 isolated from PI3Kγ CX/CX mice were co-cultured for 3 days with WT resistance arteries and, after the incubation period, vascular function was tested by challenging arteries to increasing intraluminal pressure to detect myogenic tone (MT). CD8 T cells with constitutively active PI3Kγ spontaneously increased MT of mesenteric arteries, a behavior typically associated with hypertension. Conversely, the same T cell population from WT mice, had no effect when cultured with vessels. In addition, PI3Kγ CX/CX CD8 infiltrated naïve vessels upon co-culture suggesting an intrinsic ability to colonize resistance districts relevant for hypertension. To test the in vivo relevance of the observed phenotype, we performed an adoptive transfer of PI3Kγ CX/CX CD8 in WT mice, finding that they developed spontaneous hypertension, accompanied by infiltration of effector CD8 in perivascular spaces of resistance districts as kidneys. Taken together these data show that PI3Kγ signaling in CD8 T cells is crucial for their effector functions in target vasculature of hypertension, likely contributing to BP increase by increasing peripheral resistance. These results also suggest translational potential as PI3Kγ inhibitors could modulate the immune response involved in hypertension.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

Acute Pressor Response to Psychosocial Stress Is Dependent on Endothelium-Derived Endothelin-1.

BackgroundAcute psychosocial stress provokes increases in circulating endothelin‐1 (ET‐1) levels in humans and animal models. However, key questions about the physiological function and cellular source of stress‐induced ET‐1 remain unanswered. We hypothesized that endothelium‐derived ET‐1 contributes to the acute pressor response to stress via activation of the endothelin A receptor.Methods and ResultsAdult male vascular endothelium‐specific ET‐1 knockout mice and control mice that were homozygous for the floxed allele were exposed to acute psychosocial stress in the form of cage switch stress (CSS), with blood pressure measured by telemetry. An acute pressor response was elicited by CSS in both genotypes; however, this response was significantly blunted in vascular endothelium‐specific ET‐1 knockout mice compared with control mice that were homozygous for the floxed allele. In mice pretreated for 3 days with the endothelin A antagonist, ABT‐627, or the dual endothelin A/B receptor antagonist, A‐182086, the pressor response to CSS was similar between genotypes. CSS significantly increased plasma ET‐1 levels in control mice that were homozygous for the floxed allele. CSS failed to elicit an increase in plasma ET‐1 in vascular endothelium‐specific ET‐1 knockout mice. Telemetry frequency domain analyses suggested similar autonomic responses to stress between genotypes, and isolated resistance arteries demonstrated similar sensitivity to α1‐adrenergic receptor‐mediated vasoconstriction.ConclusionsThese findings specify that acute stress‐induced activation of endothelium‐derived ET‐1 and subsequent endothelin A receptor activation is a novel mediator of the blood pressure response to acute psychosocial stress.

Small molecule activators of Ca2+-activated K+ channels can enhance endothelium-dependent responses in isolated resistance arteries

Small molecule activators of Ca2+-activated K+ channels can enhance endothelium-dependent responses in isolated resistance arteries

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered "designer" rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

New selective inhibitors of calcium-activated chloride channels - T16A(inh) -A01, CaCC(inh) -A01 and MONNA - what do they inhibit?

T16A(inh)-A01, CaCC(inh)-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Isolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. Rodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16A(inh) -A01 (0.1-10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16A(inh) -A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration-response curves ±chloride, with 10 μM T16A(inh) -A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16A(inh)-A01 ±chloride. Moreover, T16A(inh) -A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCC(inh) -A01 and MONNA (0.1-10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. T16A(inh) -A01, CaCC(inh) -A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A and inhibition of CaCC in vascular tissue in the concentration range that inhibits the isolated conductance.

The Effect of Creatine Kinase Inhibition on Contractile Properties of Human Resistance Arteries.

Creatine kinase (CK) is a main predictor of blood pressure, and this is thought to largely depend on high resistance artery contractility. We previously reported an association between vascular contractility and CK in normotensive pregnancy, but pregnancy is a strong CK inducer, and data on human hypertension are lacking. Therefore, we further explored CK-dependency of vascular contractility outside the context of pregnancy in normotensive and hypertensive women. Nineteen consecutive women, mean age 42 years (SE 1.3), mean systolic/diastolic blood pressure respectively 142.6 (SE 5.9)/85.6 (3.4) mm Hg (9 hypertensive), donated an omental fat sample during abdominal surgery. We compared vasodilation after the specific CK inhibitor 2,4-dinitro-1-fluorobenzene (DNFB; 10(-6) mol/l) to sodium nitroprusside (10(-6) mol/l) in isolated resistance arteries using a wire myograph. Additionally, we assessed predictors of vasoconstrictive force. DNFB reduced vascular contractility to 24.3% (SE 4.4), P < 0.001, compared to baseline. Sodium nitroprusside reduced contractility to 89.8% (SE 2.3). Maximum contractile force correlated with DNFB effect as a measure of CK (r = 0.8), and with vessel diameter (r = 0.7). The increase in contractile force was 16.5 mN [9.1-23.9] per unit DNFB effect in univariable and 10.35 mN [2.10-18.60] in multivariable regression analysis. This study extends on our previous findings in pregnant normotensive women of CK-dependent microvascular contractility, indicating that CK contributes significantly to resistance artery contractility across human normotension and primary hypertension outside the context of pregnancy. Further studies should explore the effect of CK inhibitors on clinical blood pressure.