Pressure Myography Research Articles

Abstract 058: Novel Mechanisms at Immune-Vascular Interfaces Regulate Myogenic Tone of Resistance Arteries and Induce Hypertension in Mice

Multiple reports recognized that immunity participates in hypertension (HTN), although direct mechanisms that orchestrate the immune-vascular interaction are still unknown. Mice infused with AngiotensinII (AngII) typically increase blood pressure (BP), concomitantly recruiting immune cells in the vasculature, with CD8 T cells playing a crucial role. To investigate the molecular regulators of vascular-immune interface, we established a system of vessel organ culture, enabling the study of physiological properties of resistance arteries via a modified pressure myograph which keeps vessels alive and functional for several days, while co-culturing immune cells of interest. We tested potential direct effects of CD8 T cells isolated from AngII-HTN or vehicle mice on resistance arteries from naïve mice. After co-incubation in the organ culture, CD8 T cells from HTN mice significantly increased myogenic tone (MT) of arteries, while no effect was induced by CD8 T of vehicle mice (%MT=CD8 AngII :27±2; CD8 veh :16±2, p<0.01). Our previous studies showed that an intracellular signaling involved in the acquisition of CD8 effectors functions, phosphatidylinositol-3-kinase-γ (PI3Kγ), was also involved in AngII-induced HTN, being its inhibition protective toward increase in BP and in vascular resistance. Here we took advantage of a mouse model expressing constitutively active PI3Kγ (PI3Kγ CX ), to test immune functions of PI3Kγ in HTN. PI3Kγ CX showed a spontaneous hypertensive phenotype (SBP mmHg :129±1 vs WT:104±2, p<0.001) accompanied by infiltration of activated CD8 T cells in renal vasculature. Then, CD8 T cells isolated from PI3Kγ CX mice and placed in co-culture with WT arteries increased their MT. Conversely, the CD8 T cells from WT mice had no effect when cultured with vessels (%MT=CD8 CX :31±1; CD8 WT :19±1, p<0.001). To test the in vivo relevance, we performed an adoptive transfer of CD8 CX in WT mice, finding that they developed spontaneous HTN, while no effect was induced by CD8 WT (SBP mmHg : CD8 CX 128±2 vs CD8 WT 103±2, p<0.001). Taken together these data show that CD8 T cells from AngII-HTN mice are able to directly increase MT of resistance arteries and that PI3Kγ signaling is a crucial modulator of this effector function, likely contributing to BP increase.

Propofol-induced vasodilation of mesenteric arterioles via BKCa channel and gap junction.

The present study aimed to investigate the role of propofol in mediating the vasomotor activity of the mesenteric arteriole (MA) of Sprague Dawley (SD) rats, and to elucidate the underlying mechanisms. The pressure myograph technique was used to examine the effect of different concentrations of propofol on the relaxation of blood vessels in the 2–3 mm MA segments freshly separated from the SD rats. The whole-cell patch-clamp technique was applied to observe the outward current of single vascular smooth muscle cells (VSMCs) obtained from the MAs of the SD rats. Furthermore, immunofluorescence was utilized to assess the expression of connexin (Cx) in the MAs of SD rats. The results indicated the following: i) Propofol relaxed the MA of SD rats in a concentration-dependent manner from 1×10−7 to 3×10−4 mol/l; ii) in the acutely dissociated VSMCs, propofol (1×10−7 to 3×10−4 mol/l) enhanced the outward current of VSMCs in a concentration-dependent manner; iii) the enhanced outward currents induced by propofol (1×10−5 mol/l) may be reversed by tetraethylammonium (TEA; 1 mmol/l), a calcium-activated K+ channel inhibitor; iv) the effect of propofol on the relaxation of the vasculature wAS reduced after perfusion with 1 mmol/l TEA; v) Cx40, Cx43 and Cx45 were expressed on the MA; 6) 18β-glycyrrhetintic acid and 2-aminoethoxydiphenyl borate, two types of gap junction blocker, inhibited the propofol-induced relaxation. The present study provides evidence that propofol relaxes the MA, which may be associated with its effect of enhancing the channel current of large-conductance calcium voltage-activated potassium channels, contributing to the K+ outflow and leading to VSMC hyperpolarization; the gap junction may facilitate the hyperpolarization, which may lead to vascular synchronized relaxation and thereby reduce the blood pressure.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for <em>Ex Vivo</em> Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Sustained conduction of vasomotor responses in rat mesenteric arteries in a two-compartment invitro set-up.

AimConduction of vasomotor responses may contribute to long‐term regulation of resistance artery function and structure. Most previous studies have addressed conduction of vasoactivity only during very brief stimulations. We developed a novel set‐up that allows the local pharmacological stimulation of arteries in vitro for extended periods of time and studied the conduction of vasomotor responses in rat mesenteric arteries under those conditions.MethodsThe new in vitro set‐up was based on the pressure myograph. The superfusion chamber was divided halfway along the vessel into two compartments, allowing an independent superfusion of the arterial segment in each compartment. Local and remote cumulative concentration‐response curves were obtained for a range of vasoactive agents. Additional experiments were performed with the gap junction inhibitor 18β‐glycyrrhetinic acid and in absence of the endothelium.ResultsPhenylephrine‐induced constriction and acetylcholine‐induced dilation were conducted over a measured distance up to 2.84 mm, and this conduction was maintained for 5 minutes. Conduction of acetylcholine‐induced dilation was inhibited by 18β‐glycyrrhetinic acid, and conduction of phenylephrine‐induced constriction was abolished in absence of the endothelium. Constriction in response to high K+ was not conducted. Absence of remote stimulation dampened the local response to phenylephrine.ConclusionThis study demonstrates maintained conduction of vasoactive responses to physiological agonists in rat mesenteric small arteries likely via gap junctions and endothelial cells, providing a possible mechanism for the sustained functional and structural control of arterial networks.

Mineralocorticoid receptor antagonism prevents obesity-induced cerebral artery remodeling and reduces white matter injury in rats.

Midlife obesity is a risk factor for dementia development. Obesity has also been linked to hyperaldosteronism, and this can be modeled in rats by high fat (HF) feeding from weaning. Aldosterone, or activation of the mineralocorticoid receptor (MR) causes cerebrovascular injury in lean hypertensive rats. We hypothesized that rats fed a HF diet would show inward middle cerebral artery (MCA) remodeling that could be prevented by MR antagonism. We further proposed that the cerebral artery remodeling would be associated with white mater injury. Three-week-old male Sprague-Dawley rats were fed a HF diet±the MR antagonist canrenoic acid (Canr) for 17weeks. Control rats received normal chow (control NC). MCA structure was assessed by pressure myography. The MCAs from HF fed rats had smaller lumens and thicker walls when compared to arteries from control NC rats; Canr prevented the MCA remodeling associated with HF feeding. HF feeding increased the mRNA expression of markers of cell proliferation and vascular inflammation in cerebral arteries and Canr treatment prevented this. White mater injury was increased in the rats fed the HF diet and this was reduced by Canr treatment. The expression of doublecortin, a marker of new and immature neurons was reduced in HF fed rats, and MR antagonism normalized this. These data suggest that HF feeding leads to MR dependent remodeling of the MCA and this is associated with markers of dementia development.

Angiotensin receptor blockade in juvenile male rat offspring: Implications for long-term cardio-renal health

Inhibition of the renin-angiotensin system in early postnatal life is a potential therapeutic approach to prevent long-term cardiovascular and kidney diseases in individuals born small. We determined the long-term effects of juvenile losartan treatment on cardiovascular and kidney function in control male rat offspring and those exposed to uteroplacental insufficiency and born small. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed in late gestation in Wistar Kyoto rats. At weaning, male offspring were randomly assigned to receive losartan in their drinking water or drinking water alone from 5 to 8 weeks of age, and followed to 26 weeks of age. Systolic blood pressure and kidney function were assessed throughout the study. Pressure myography was used to assess passive mechanical wall properties in mesenteric and femoral arteries from 26-week-old offspring. Losartan treatment for three weeks lowered systolic blood pressure in both Control and Restricted groups but this difference was not sustained after the cessation of treatment. Losartan, irrespective of birth weight, mildly increased renal tubulointerstitial fibrosis when assessed at 26 weeks of age. Mesenteric artery stiffness was increased by the early losartan treatment, and was associated with increased collagen and decreased elastin content. Losartan also exerted long-term increases in fat mass and decreases in skeletal muscle mass. In this study, untreated Restricted offspring did not develop hypertension, vascular dysfunction or kidney changes as anticipated. Regardless, we demonstrate that short-term losartan treatment in the juvenile period negatively affects postnatal growth, and kidney and vascular parameters in adulthood, irrespective of birth weight. The long-term effects of early-life losartan treatment warrant further consideration in settings where the potential benefits may outweigh the risks; i.e. when programmed adulthood diseases are apparent and in childhood cardiovascular and kidney diseases.

VASCULAR NO AND SUPROXIDE PRODUCTION IS INFLUENCED BY HIGH DIETARY SALT INTAKE IN TFF3−/− MICE

Objective: Arachidonic acid metabolism and products are shown to affect vascular reactivity mechanism. TFF3−/− mice have protective phenotype of favorable ratio of ω6/ω-3 fatty acids and modified metabolism of arachidonic acid. However, the role of TFF3 peptide in hypertension and vascular function is still underinvestigated. The aim of this study was to asses the effect of high salt diet on flow-induced endothelial NO and superoxide production in carotid arteries of wild type mice of mixed background C57Bl6/Sv/129 (WT) and TFF3−/− knock out mice with the same background. Design and method: Eleven weeks old male TFF3−/− or WT mice were divided in 2 groups (N = 4 per group): low salt (LS) and high salt (HS; 4% NaCl for 7 days). Following dietary protocol, mice were anaesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg). Carotid arteries were isolated and cannulated on pressure myograph with or without flow (at delta 80 mmHg), in the absence/presence of the NOS inhibitor L-NAME. NO production was determined by DAF-2DA to DAF-2T conversion fluorescence assay. To assess the production of superoxide radicals, the carotid arteries were loaded with dihydroethidine (DHE, 20 μM). All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by institutional Ethical Committee. p < 0.05 was significant. Results: Basal NO production in no-flow condition was similar among groups. L-NAME blocked the production of NO in all groups. Flow-induced NO production was decreased in HS-WT group compared to no-flow conditions in HS-WT group, and compared to LS-WT group and LS-TFF3−/− group (p < 0.05). There were no changes in endothelial NO production in TFF3−/− mice. Superoxide production was increased in LS-TFF3−/− no-flow group compared to LS-WT in no-flow condition Flow-induced superoxide production was increased in both HS-WT and HS-TFF3−/− groups compared to corresponding LS groups. Conclusions: HS intake decreases flow-induced vascular NO production in WT mice probably due to increased oxidative stress. However, HS intake does not affect NO production in TFF3−/− mice eventhough superoxide levels are increased in basal levels and in flow conditions, suggesting protective role of this phenotype on NO bioavailability.

ROSTAFUROXIN AMELIORATES THE INWARD REMODELING AND STIFFNESS IN RESISTANCE ARTERIES FROM DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSIVE RATS

Objective: We have shown that rostafuroxin (ROSTA), an antagonist of endogenous ouabain (OUA), block the biding of OUA to Na+ K+ -ATPase and inhibits the cSRC downstream activation in mesenteric resistance arteries (MRA), which ameliorates endothelial dysfunction and oxidative stress, as reduces hypertension in deoxycorticosterone acetate (DOCA)-salt rats. In addition, MRA of DOCA-salt rats also present the hypertrophic inward remodeling with stiffness. Moreover, the chronic treatment with OUA induces hypotrophic inward remodeling with stiffness in MRA. In line with these results we investigate the role of endogenous OUA on remodeling and stiffness in MRA of DOCA-salt rats. Design and method: A hypertensive model was established in uninephrectomized Wistar rats using DOCA-salt. After 5 weeks DOCA-salt treatment, with high systolic blood pressure (SBP) stabilization, this hypertensive model was divided into two groups: DOCA-salt and DOCA-salt co-treated with ROSTA (1 mg/kg per day gavage, 3 weeks). The SBP was measured using the tail-cuff method, and vascular remodeling was assessed in MRA using a pressure myograph. Histological and western blot analysis were performed in MRA. Results: Our results reinforce that ROSTA treatment reduces SBP in DOCA-salt rats, but did not restore it to control levels. For the first time, the results suggest that ROSTA ameliorates the remodeling and stiffness observed in MRA of DOCA-salt rats, enhancing the luminal diameter and distensibility, reducing the cross-section area and wall/lumen ratio as well as shifting to the right the stress-strain curve in MRA. ROSTA restores collagen type I/III ratio, internal elastic lamina thickness, tyrosine kinase Src phosphorylation (Tyr418), as well EGFR, cRaf, ERK, p38MAPK, TGFβ1, CTGF, and collagen I protein expression in MRA from DOCA-salt rats. Conclusions: The results suggested that ROSTA reduces hypertension, the hypertrophic inward remodeling and the augmented wall/lumen ratio in MRA of DOCA-salt rats. In addition, endogenous OUA emerges as an important mechanism to induce vascular stiffness via Na+ K+ -ATPase/ cSRC/ EGFR/ cRas/ ERK/ p38MAPK/ CTGF/ TGFβ1 pathway in MRA. These results reinforce the idea that endogenous OUA is a putative target for the treatment of vascular adjustments present in hypertension.

Shear stress sensitizes TRPV4 in endothelium-dependent vasodilatation

The aim of this study was to better understand the role of TRPV4 in the regulation of blood vessel dilatation by blood flow and activation of GPCRs. Using pressure myography, the dilator responses to the TRPV4 agonist GSK1016790A and to acetylcholine, were examined in rat cremaster arterioles exposed to either no shear stress or to 200 μl/min flow for 6 min. In control vessels GSK1016709A caused vasodilatation (pEC50 7.73 ± 0.12 M, ΔDmax 97 ± 3%) which was significantly attenuated by the TRPV4 antagonists GSK2193874 (100 nM) (pEC50 6.19 ± 0.11 M, p < 0.05) and HC067047 (300 nM) (pEC50 6.44 ± 0.12 M) and abolished by removal of the endothelium. Shear conditioned arterioles were significantly more sensitive to GSK1016790A (pEC50 8.34 ± 0.11, p < 0.05). Acetylcholine-induced vasodilatation (pEC50 7.02 ± 0.07 M, ΔDmax 93 ± 2%) was not affected by shear forces (pEC50 7.08 ± 0.07 M, ΔDmax 95 ± 1%). The dilator response to acetylcholine was unaffected by the TRPV4 antagonist GSK2193874 in control arterioles (pEC50 7.24 ± 0.07 M, ΔDmax 97 ± 2%). However, in shear treated arterioles, the acetylcholine-response was significantly attenuated by GSK2193874 (pEC50 6.25 ± 0.12 M, p < 0.05) indicating an induced interaction between TRPV4 and muscarinic receptors. TRPV4 antibodies localized TRPV4 to the endothelium and shear stress had no effect on its localisation. Finally, agonist activation of the M3 muscarinic receptor opened TRPV4 in HEK293 cells.We concluded that shear stress increases endothelial TRPV4 agonist sensitivity and links TRPV4 activation to muscarinic receptor mediated endothelium-dependent vasodilatation, providing strong evidence that blood flow modulates downstream signalling from at least one but not all GPCRs expressed in the endothelium.

SKA-31, an activator of endothelial Ca2+-activated K+ channels evokes robust vasodilation in rat mesenteric arteries

It is now well recognized that endothelial KCa2.3 and KCa3.1 channel activities contribute to dilation of resistance arteries via endothelium-mediated hyperpolarization and vascular smooth muscle relaxation. In this study, we have investigated the functional effect of the KCa channel activator SKA-31 in third order rat mesenteric arteries using arterial pressure myography. Isolated arteries were cannulated, pressurized intraluminally to 70 mmHg at 36 °C and then constricted with 1 μM phenylephrine. Acute bath exposure to SKA-31 evoked a robust and reversible inhibition of developed tone (IC50 = 0.22 μM). The vasodilatory effects of SKA-31 and acetylcholine were blunted in the presence of KCa2.3 and KCa3.1 channel antagonists, and were largely prevented following endothelial denudation. Western blot and q-PCR analyses of isolated mesenteric arteries revealed KCa2.3 and KCa3.1 channel expression at the protein and mRNA levels, respectively. Penitrem-A, an inhibitor of KCa1.1 channels, decreased vasodilatory responses to acetylcholine, sodium nitroprusside and NS-1619, but had little effect on SKA-31. Similarly, bath exposure to the eNOS inhibitor L-NAME did not alter SKA-31 and acetylcholine-mediated vasodilation. Collectively, these data highlight the major cellular mechanisms by which the endothelial KCa channel activator SKA-31 inhibits agonist-evoked vasoconstriction in rat small mesenteric arteries.

Uteroplacental insufficiency temporally exacerbates salt-induced hypertension associated with a reduced natriuretic response in male rat offspring.

Low weight at birth increases the risk of developing chronic diseases in adulthood A diet that is high in salt is known to elevate blood pressure, which is a major risk factor for cardiovascular and kidney diseases The present study demonstrates that growth restricted male rats have a heightened sensitivity to high dietary salt, in the context of raised systolic blood pressure, reduced urinary sodium excretion and stiffer mesenteric resistance vessels Other salt-induced effects, such as kidney hyperfiltration, albuminuria and glomerular damage, were not exacerbated by being born small The present study demonstrates that male offspring born small have an increased cardiovascular susceptibility to high dietary salt, such that that minimizing salt intake is probably of particular benefit to this at-risk population ABSTRACT: Intrauterine growth restriction increases the risk of developing chronic diseases in adulthood. Lifestyle factors, such as poor dietary choices, may elevate this risk. We determined whether being born small increases the sensitivity to a dietary salt challenge, in the context of hypertension, kidney disease and arterial stiffness. Bilateral uterine vessel ligation or sham surgery (offspring termed Restricted and Control, respectively) was performed on 18-day pregnant Wistar Kyoto rats. Male offspring were allocated to receive a diet high in salt (8% sodium chloride) or remain on standard rat chow (0.52% sodium chloride) from 20 to 26weeks of age for 6weeks. Systolic blood pressure (tail-cuff), renal function (24h urine excretions) and vascular stiffness (pressure myography) were assessed. Restricted males were born 15% lighter than Controls and remained smaller throughout the study. Salt-induced hypertension was exacerbated in Restricted offspring, reaching a peak systolic pressure of ∼175mmHg earlier than normal weight counterparts. The natriuretic response to high dietary salt in Restricted animals was less than in Controls and may explain the early rise in arterial pressure. Growth restricted males allocated to a high salt diet also had increased passive arterial stiffness of mesenteric resistance arteries. Other aspects of renal function, including salt-induced hyperfiltration, albuminuria and glomerular damage, were not exacerbated by uteroplacental insufficiency. The present study demonstrates that male offspring exposed to uteroplacental insufficiency and born small have an increased sensitivity to salt-induced hypertension and arterial remodelling.

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Aging and Endothelial Dysfunction: The Role of NADPH Oxidase and the Insufficient Inhibition by PKG

BackgroundNicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐induced oxidative stress plays a critical role in the age‐related impairment of endothelium‐dependent dilation. However, the role of NADPH oxidase (i.e. which nitrogen oxides (NOX) may be involved) and the potential role of protein kinase G (PKG) inhibition of NADPH in human skeletal muscle feed arteries (SMFAs) has yet to be clarified. Therefore, this study sought to determine, in vitro, if treatment with NOX1 inhibitor (ML090), NOX2 inhibitor (gp91ds‐tat), and NOX4 inhibitor (plumbagin) could restore the age‐related endothelial dysfunction in human SMFAs.MethodsPKG, NOX1, NOX2, and NOX4 protein expression and cyclic guanosine monophosphate (cGMP) activity were analyzed in young (24±6 yrs, n=7) and old (77±8 yrs, n=8) subjects via Western blot analysis and an enzyme‐linked immunosorbent assay (ELISA). Using pressure myography, endothelium‐dependent and ‐independent vasodilation were assessed with and without ML090, gp91ds‐tat, and plumbagin in SMFAs obtained from 13 young and 16 old subjects in response to three stimuli 1) flow‐induced shear stress, 2) acetylcholine (ACh), and 3) sodium nitroprusside (SNP). Additionally, L‐NG‐monomethyl arginine citrate (L‐NMMA) was utilized to identify the impact of blocking nitric oxide (NO) synthase on these responses.ResultsNOX2 and NOX4, but not NOX1, protein expression were significantly increased in the old compared to the young SMFAs. In addition, cGMP activity and PKG protein expression were significantly lower in the old compared to the young SMFAs. Endothelial‐dependent vasodilatory dysfunction was evident in response to intraluminal flow (Young: 81 ± 15; Old: 27 ± 14%, P &lt; 0.05) and ACh (Young: 95 ± 17, Old: 34 ± 15 %, P &lt; 0.05). The NOX2 inhibitor, gp91ds‐tat, restored the vasodilation response in the old SMFAs to both flow and Ach (70 ± 11 and 89 ± 13 %, respectively, P &lt; 0.05), while the NOX4 inhibitor, plumbagin, partially restored the vasodilation response in the old SMFAs to flow and ACh (45 ± 15 %, P=0.08; 59 ± 13 %, P=0.07). However, the NOX1 inhibitor, ML090, had no effect. Endothelium‐independent vasodilation (SNP) was not affected by age and, therefore, all NOX inhibitors had no effect on SNP‐induced vasodilation. L‐NMMA negated the restorative effects of NOX2 and NOX4 blockade.ConclusionsBoth NOX2 and to a lesser extent NOX4 appear to play an important role in the age‐associated endothelial dysfunction exhibited by SMFAs. Furthermore, in combination, these findings suggest that there is likely an age‐related weakening of the NO‐initiated link between cGMP, PKG, and NADPH oxidase, with PKG demonstrating insufficient NADPH oxidase inhibition with advancing age and, ultimately, attenuated vascular function.Support or Funding Informationthe National Heart, Lung, and Blood Institute at the National Institute of Health (PO1 HL1091830) and the Veteran's Administration Rehabilitation Research and Development Service (E6910‐R, E1697‐R, E1433‐P, E9275‐L and E1572‐P).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Absence of Endothelial Estrogen Receptor Alpha Decreases Arterial Stiffness and Induces Hypertrophic Remodeling in Angiotensin II infused Female Mice

Vascular stiffening increases the risk of stroke, diastolic heart failure and chronic kidney disease, and is an early biomarker of cardiovascular disease (CVD), the leading cause of death worldwide. Although stiffening of the arteries is a naturally occurring phenomenon that appears with aging, conditions such as obesity and type 2 diabetes accelerate its appearance, particularly in women. Under physiological conditions, estrogen signaling via estrogen receptor alpha (ERα) increases nitric oxide in the endothelium and decreases vascular stiffness. However, estrogen therapy has failed to demonstrate beneficial cardiovascular effects in large clinical trials. We have previously demonstrated that under conditions of over‐nutrition, knockout (KO) of endothelial cell (EC) ERα in obese insulin‐resistant female mice results in reduced arterial stiffness. Given the central role that activation of the Renin‐Angiotensin‐System (RAS) has in the development of CVD, in the present study we studied the effects of Angiotensin II (AngII) infusion in EC‐ERαKO female mice on vascular stiffness. KO and wild type (control) littermates (n=8 per group) were treated with AngII (500 ng/kg/min) via osmotic mini‐pumps for 4 weeks. At the end of the intervention, the mice underwent in‐vivo assessment of vascular stiffness via aortic pulse wave velocity (PWV) and body weight was recorded. The mice were sacrificed at 28 weeks of age and vasomotor function determined in aortic rings using wire myography, while vascular mechanics were assessed in femoral arteries using pressure myography. No differences in body weight, aortic PWV were observed between groups (n=8 per group). No differences were observed either on the functional responses of aortic rings to acetylcholine or sodium nitroprusside between the groups (n=5 per group). However, with regard to the remodeling of femoral arteries, mice lacking ERα had increased wall cross‐sectional area (maximal difference at 120 mmHg, 34.632±4.960 vs. 20.793±1.827 × 103 μm2, p&lt;0.05) and wall‐to‐lumen ratio (maximal difference at 5 mmHg, 0.559±0.026 vs. 0.457±0.028, p&lt;0.05) vs. controls (n=7–8 per group). Furthermore, the mechanical properties of femoral arteries of EC‐ERαKO mice had decreased modulus of elasticity (maximal difference at 120 mmHg, 6.428±0.552 vs. 9.807±1.182 × 106 dynes/cm2, p&lt;0.05) and increased cross‐sectional compliance (maximal difference at 40 mmHg, 8.893±1.130 vs. 3.974±0.825182 × 10−9 cm4/dynes, p&lt;0.05) vs. controls (n=7–8 per group). We did not observe any differences in lumen diameter between the groups. These results suggest that in obese, insulin‐resistant female mice, ablation of EC‐ERα signaling in the setting of RAS activation confers beneficial effects to vascular health by decreasing the stiffness and inducing hypertrophic remodeling of mid‐sized arteries.Support or Funding InformationThis work was supported by National Institutes of Health Grants K08HL129074 (to C.M.), K08HL132012 (to G.L) and NIH R01 HL‐088105 (to L.A.M.‐L.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased Hydrogen Peroxide After Traumatic Brain Injury Disrupts Phosphatidylinositol 4,5‐Bisphosphate Metabolism Causing Impaired Inward Rectifier Potassium Channel Function

Severe trauma, including traumatic brain injury (TBI), can result in several deleterious outcomes including coagulopathy, multi‐organ failure and death. We previously showed that TBI in rodents causes uncoupling of mesenteric artery (MA) nitric oxide production, resulting in excessive reactive oxygen species (ROS) generation and impaired endothelial‐dependent vasodilation. The inward‐rectifier potassium channel (KIR) is an essential component of agonist‐ and flow‐induced vasodilation and signal transduction in mesenteric arteries (MAs); however, its function following trauma has not been assessed. Phosphatidylinositol 4,5‐bisphosphate (PIP2) is a membrane phospholipid which is required for normal KIR channel function in MAs. We hypothesized that oxidative stress, specifically hydrogen peroxide (H2O2), was driving down PIP2 levels, causing KIR channelopathies. We studied male rats 24 hours after fluid percussion TBI, compared to controls. Plasma oxidative‐reduction potential (ORP), an indicator of oxidative stress, was significantly elevated in TBI rats compared to controls. In TBI but not control plasma, catalase application significantly reduced the ORP signal to control values, suggesting that H2O2 contributes to global oxidative stress following TBI. We then measured KIR currents in isolated endothelial cells (ECs) from MAs, using the whole‐cell patch‐clamp technique from control and TBI rats. KIR channel currents were significantly diminished in ECs from TBI rats compared to controls. KIR currents in ECs from TBI MAs were rescued with the addition of PIP2 to the pipette solution when compared to untreated TBI cells, and showed no difference from their control, treated counterparts, implying depletion of PIP2 following TBI is a contributing factor to KIR channel dysfunction. Next, diameter responses of arteries using pressure myography were measured. In control arteries, increasing concentrations of extracellular potassium from 6 to 10 mM K+ caused dilations; concentrations between 14 to 60 mM K+ caused constrictions. Dilations to 10 mM K+ were significantly reduced following TBI, consistent with an impaired KIR channel response. Next, basal KIR function was assessed using the KIR and KIR2.1 antagonists BaCl2 (100 μM) and ML 133 (20 μM). Following TBI, arteries constricted significantly less to both BaCl2 and ML 133. Furthermore, dilations to 10 mM K+ and constrictions to ML 133 were restored in TBI arteries in the presence of catalase. Lastly, To further quantify cellular pathology following TBI, the metabolomic phenotype was measured by liquid chromatography‐mass spectrometry (LC‐MS) in plasma obtained from control and TBI rats. Rats exhibited a profound metabolopathy following TBI; within the lipidome: arachidonate metabolism, glycerophospholipid synthesis, and fatty acid mobilization were significantly altered. These data suggest H2O2 production serves an antagonistic role on KIR channel function through changes in second messenger and lipid signaling pathways in the mesenteric circulation following TBI.Support or Funding InformationThis work was supported by the Totman Medical Research Trust and the National Institute of Health (K08‐GM‐098795 and UM1‐HL‐120877).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of function RGS2 mutations augment vascular contractility ex vivo

ObjectiveRegulator of G protein signaling 2 (RGS2) plays a significant role in alleviating vascular contraction and promoting vascular relaxation due to its GTPase accelerating protein activity toward Gαq. Through a cell‐based Ca2+ mobilization assay, we identified 4 loss‐of‐function (LOF) mutations (Q2L, D40Y, R44H and R188H) out of 16 rare, missense mutations in RGS2 selected from various human exome sequencing projects. This study is to investigate whether these LOF RGS2 mutations disrupt the protein function in regulating vascular contractility.Approach and resultWe confirmed that RGS2‐deficient mice expressed a hypertensive phenotype by telemetric femoral artery blood pressure measurement in conscious mice (WT SBP 120 ± 3 vs RGS2−/−SBP 128 ± 2 mmHg, p&lt;0.01). Isolated mesenteric arteries from RGS2−/− mice showed higher contractile response to 10 nM angiotensin II (AngII) stimulation in pressure myograph while isolated aortic rings from those animals generated more tension upon phenylephrine (PE) stimulation (Emax 238 mg vs 68 mg, p = 0.01) compared to aortic rings from RGS2+/+ mice. Reintroduction of wild‐type RGS2‐GFP plasmids into RGS2−/− mesenteric arteries by reversible permeabilization suppressed the vasoconstrictor response to 50 nM AngII (RGS2 WT plasmid −4% vs pcDNA control −35% of basal diameter, p = 0.01). Transfection with RGS2 LOF mutation plasmidss did not suppress Ang II constriction compared to those expressing WT‐RGS2 (Q2L −20%, D40Y, R44H and R188H −30% of basal diameter, p &lt; 0.05).ConclusionsThis study is the first to investigate the function of human mutant RGS2 in resistance arteries in a near physiological condition. It demonstrates that RGS2 attenuates vasoconstriction in the mesenteric arteries and that RGS2 mutations disrupt this effect. Although these mutations are rare, knowledge about their function may provide insights into pathophysiology and treatment.Support or Funding InformationThis work was supported by the American Heart Association predoctoral fellowship [15PRE24680004] to H.P).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The influence of connexin 45 on renal autoregulation

The kidney maintains constant renal blood flow and glomerular filtration rate despite acute changes in renal perfusion pressure (RPP) via renal autoregulation. Renal autoregulation is made up of the myogenic response and the tubuloglomerular feedback (TGF) mechanism, both acting on the afferent arteriole. Both mechanisms have a conducted component, believed to proceed via gap junctions (GJ). These fluid‐filled pores are made up of two connexons, one from each cell, with connexins (Cx) as building blocks. Several Cx isoforms are expressed in the renal vessel wall, including connexin 45 (Cx45), making it a functional syncytium. Disruption of Cx expression and function in the vasculature reduces myogenic response and causes TGF dysregulation, and is associated with hypertension development. The aim of the study was to explore the influence Cx45 has on renal autoregulation.To investigate the TGF mechanism, experiments were performed using the isolated perfused juxtamedullary nephron preparation. Kidneys were isolated from wild type (WT) and smooth muscle cell knockout (KO) Cx45 mice and perfused with a Tyrode's buffer containing BSA and amino acids mixture, at pH 7.4. Local afferent vasoconstriction was induced by electrical pulse stimulation administered via microelectrode, at the glomerular entrance. RPP was kept constant at 95 mmHg and inner afferent arteriolar diameter was measured locally and at upstream sites to evaluate vascular conduction. Responses between WT and KO were compared.To investigate the myogenic response, arteriolar diameter was measured in response to changes in RPP. Experiments were performed in the same setup as previously mentioned, using a stepwise increase in RPP. Perfusion was initiated at 95 mmHg and increased in steps of 20 mmHg until 195 mmHg is reached. Inner afferent arteriolar diameter was measured 100 μm upstream from the glomerulus, and responses between WT and KO were compared. To eliminate the TGF mechanism and isolate only the myogenic response in the kidneys, papillectomy were performed before repeating the same experimental protocol. Responses between WT and KO were compared.To further investigate the influence of Cx45 in the renal vascular function, interlobar arteries from WT and KO mice were isolated and mounted in a pressure myograph. An experimental protocol consisted of stepwise pressure increases of 25 mmHg, starting at 25 mmHg until 175 mmHg is reached. Inner diameter from both WT and KO were measured and compared. In this set‐up no TGF is present.In both WT and KO mice, electrical stimulation at the glomerular pole of the afferent arteriole reduces the diameter at the local site. The conducted vasoconstriction at upstream sites was attenuated in KO mice. The conducted response is observed at 400 μm in the WT mice and 250 μm in the KO. Preliminary myogenic data shows that pressure increases induce a greater afferent vasoconstriction in WT mice and less so in the Cx45 KO. These results suggest that Cx45 may play a role in TGF signaling and myogenic response in the preglomerular vasculature and thus renal autoregulation.Support or Funding InformationThe work is part of the Dynamical Systems Interdisciplinary Network, University of CopenhagenThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of c‐Kit/sGC signaling axis in vascular reactivity and hypertension

A. IntroductionThe receptor tyrosine kinase c‐Kit has been recently found to have a role in vasculoprotection. Genetic deletion of c‐Kit in smooth muscle cells increases aortic plaque accumulation and atherosclerosis burden in conditional hyperlipidemic mice. The present study tests whether a c‐Kit deficiency in mutant mice (KitW/Wv) leads to hypertension and impaired vascular relaxation.B. MethodsFemale and male c‐Kit mutant (KitW/Wv) and littermate (Kit+/+) control mice were commercially available (Jackson lab). Blood pressure was determined in conscious mice using the tail‐cuff method. The first‐order branch of mesenteric artery was mounted into a pressure myograph (DMT, Aarhus, Denmark) to assess endothelium‐dependent and ‐independent vasoreactivity under isobaric conditions. A search for differentially expressed genes between c‐Kit mutant and control SMC was performed using the Mouse MI‐Ready microarray (Ocean Ridge Biosciences).C. ResultsThe deficiency of c‐Kit causes hypertension in mutant mice with respect to their control littermates (mean arterial pressure: 137.5 ± 5.0 vs. 113.06 ± 0.0, p=0.007). Acetylcholine (ACh) induced a concentration‐dependent relaxation of norepinephrine pre‐contracted vessels in both mutant and littermate mice. The Cox inhibitor indomethacin compromised ACh‐induced vasorelaxation only in c‐Kit deficient vessels but not in those from control mice, which highlighted the importance of endothelium‐derived prostaglandins for vasorelaxation in the absence of c‐Kit. Conversely, arteries of mutant mice were poor responders to a NO agonist (SNP). The secondary messenger, cyclic guanosine monophosphate, vasodilated both types of vessels in a similar manner, which indicated that defective NO activity in mutants was due to deficiencies in soluble guanylyl cyclase (sGC) or NO synthesis. A significant downregulation of smooth muscle sGC beta 1 subunit was further found in c‐Kit mutant cells using transcriptomics analysis.D. ConclusionThese results suggest the existence of a novel c‐Kit/sGC signaling axis in SMC that may be relevant for the control of vascular reactivity and hypertension.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transient Increase in Sympathetic Constriction of Mesenteric Arteries from High Fat Diet‐Fed Dahl Salt‐sensitive Female Rats

Obesity (BMI ≥ 30) is a risk factor for hypertension. Obesity‐associated hypertension is a risk factor for cardiovascular disease, stroke, type 2 diabetes and kidney failure. Hypertension is less common in premenopausal women compared to age‐matched men. This disparity fades away after the onset of menopause. Increased sympathoexcitation in the neuroeffector junction leads to increase in constriction of blood vessels. Differences in the mechanism/s neurogenic arterial constriction in male and female are not well understood. We hypothesized that sympathetic nerve activity in high fat diet‐induced hypertension is greater in male than female rat mesenteric arteries (MA). Rats were fed either control (C) or high fat (HF) diet beginning at 3 weeks of age, and used at 10, 17 and 24 weeks on diet. Body weight and systolic blood pressure (SBP) were measured weekly. HF males gained more weight than HF females at all time points. SBP was higher in HF rats compared to C rats in both sexes and across all time points. The estrus cycle stage was determined cytologically by vaginal lavage prior to euthanasia. Mesenteric arteries (Inner diameter; male=275–350 μm, female=245–315 μm) were harvested and mounted in a pressure (60 mmHg) myograph and inner diameter was measured continuously. MA contractile responses to electrical field stimulation (30 stimuli at 0.2–30 Hz) and drug applications were measured. Neurogenic constriction of MA from HF females was greater than HF males at 5 and 10 Hz at 10 wks, and at 5–30 Hz at 17 wks. At 24 wks the difference disappears. Constrictions caused by norepinephrine (NE) and ATP were similar in MA from HF male and female rats. Immunostaining for tyrosine hydroxylase (sympathetic nerve marker) and vesicular nucleotide transporter (ATP marker) revealed greater sympathetic nerve intensity in HF male than female rats at 10 and 24 wks. There was also greater intensity in MA from HF males than HF females at 10 and 17 wks. Moreover, HF males have higher NE and ATP nerve density than HF females at 10 and 17 but not for ATP at 24 wks. MA NE levels were similar in male and female rats. Prejunctional α2‐AR and NE transporter functions were not impaired in MA from HF Dahl salt‐sensitive rats. Taken together, despite sex differences in sympathetic nerve density in MA, we detected only transient sex differences in sympathetic neurogenic arterial constrictions. Contrary to our hypothesis, we found that HF females transiently have an upregulation of neurogenic sympathetic activity and this warrants further investigation into the mechanism.Support or Funding InformationResearch supported by PPG NIH Grant P01HL070687This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.