Endothelin-1 in Health and Disease.

Discovered almost 40 years ago, the potent vasoconstrictor peptide endothelin-1 (ET-1) has a wide range of roles both physiologically and pathologically. In recent years, there has been a focus on the contribution of ET-1 to disease. This has led to the development of various ET receptor antagonists, some of which are approved for the treatment of pulmonary arterial hypertension, while clinical trials for other diseases have been numerous yet, for the most part, unsuccessful. However, given the vast physiological impact of ET-1, it is both surprising and disappointing that therapeutics targeting the ET-1 pathway remain limited. Strategies aimed at the pathways influencing the synthesis and release of ET-1 could provide new therapeutic avenues, yet research using cultured cells in vitro has had little follow up in intact ex vivo and in vivo preparations. This article summarises what is currently known about the synthesis, storage and release of ET-1 as well as the role of ET-1 in several diseases including cardiovascular diseases, COVID-19 and chronic pain. Unravelling the ET-1 pathway and identifying therapeutic targets has the potential to treat many diseases whether through disease prevention, slowing disease progression or reversing pathology.

Phospholemman Phosphorylation Regulates Vascular Tone, Blood Pressure, and Hypertension in Mice and Humans.

BACKGROUND: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans. METHODS: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting. RESULTS: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men. CONCLUSIONS: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans.

Endothelium-Dependent Hyperpolarization: The Evolution of Myoendothelial Microdomains.

ABSTRACT: Endothelium-derived hyperpolarizing factor (EDHF) was envisaged as a chemical entity causing vasodilation by hyperpolarizing vascular smooth muscle (VSM) cells and distinct from nitric oxide (NO) ([aka endothelium-derived relaxing factor (EDRF)]) and prostacyclin. The search for an identity for EDHF unraveled the complexity of signaling within small arteries. Hyperpolarization originates within endothelial cells (ECs), spreading to the VSM by 2 branches, 1 chemical and 1 electrical, with the relative contribution varying with artery location, branch order, and prevailing profile of VSM activation. Chemical signals vary likewise and can involve potassium ion, lipid mediators, and hydrogen peroxide, whereas electrical signaling depends on physical contacts formed by homocellular and heterocellular (myoendothelial; MEJ) gap junctions, both able to conduct hyperpolarizing current. The discovery that chemical and electrical signals each arise within ECs resulted in an evolution of the single EDHF concept into the more inclusive, EDH signaling. Recognition of the importance of MEJs and particularly the fact they can support bidirectional signaling also informed the discovery that Ca2+ signals can pass from VSM to ECs during vasoconstriction. This signaling activates negative feedback mediated by NO and EDH forming a myoendothelial feedback circuit, which may also be responsible for basal or constitutive release of NO and EDH activity. The MEJs are housed in endothelial projections, and another spin-off from investigating EDH signaling was the discovery these fine structures contain clusters of signaling proteins to regulate both hyperpolarization and NO release. So, these tiny membrane bridges serve as a signaling superhighway or infobahn, which controls vasoreactivity by responding to signals flowing back and forth between the endothelium and VSM. By allowing bidirectional signaling, MEJs enable sinusoidal vasomotion, co-ordinated cycles of widespread vasoconstriction/vasodilation that optimize time-averaged blood flow. Cardiovascular disease disrupts EC signaling and as a result vasomotion changes to vasospasm.

Intrinsic regulation of microvascular tone by myoendothelial feedback circuits.

The endothelium is an important regulator of arterial vascular tone, acting to release nitric oxide (NO) and open Ca2+-activated K+ (KCa) channels to relax vascular smooth muscle cells (VSMCs). While agonists acting at endothelial cell (EC) receptors are widely used to assess the ability of the endothelium to reduce vascular tone, the intrinsic EC-dependent mechanisms are less well characterized. In small resistance arteries and arterioles, the presence of heterocellular gap junctions termed myoendothelial gap junctions (MEGJs) allows the passage of not only current, but small molecules including Ca2+ and inositol trisphosphate (IP3). When stimulated to contract, the increase in VSM Ca2+ and IP3 can therefore potentially pass through MEGJs to activate adjacent ECs. This activation releases NO and opens KCa channels, which act to limit contraction. This myoendothelial feedback (MEF) is amplified by EC Ca2+ influx and release pathways, and is dynamically modulated by processes regulating gap junction conductance. There is a remarkable localization of key signaling and regulatory proteins within the EC projection toward VSM, and the intrinsic EC-dependent signaling pathways occurring with this highly specialized microdomain are reviewed.

Direct visualization of the arterial wall water permeability barrier using CARS microscopy.

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies.

Neuropeptide-Y causes coronary microvascular constriction and is associated with reduced ejection fraction following ST-elevation myocardial infarction.

AIMS: The co-transmitter neuropeptide-Y (NPY) is released during high sympathetic drive, including ST-elevation myocardial infarction (STEMI), and can be a potent vasoconstrictor. We hypothesized that myocardial NPY levels correlate with reperfusion and subsequent recovery following primary percutaneous coronary intervention (PPCI), and sought to determine if and how NPY constricts the coronary microvasculature. METHODS AND RESULTS: Peripheral venous NPY levels were significantly higher in patients with STEMI (n = 45) compared to acute coronary syndromes/stable angina ( n = 48) or with normal coronary arteries (NC, n = 16). Overall coronary sinus (CS) and peripheral venous NPY levels were significantly positively correlated (r = 0.79). STEMI patients with the highest CS NPY levels had significantly lower coronary flow reserve, and higher index of microvascular resistance measured with a coronary flow wire. After 2 days they also had significantly higher levels of myocardial oedema and microvascular obstruction on cardiac magnetic resonance imaging, and significantly lower ejection fractions and ventricular dilatation 6 months later. NPY (100-250 nM) caused significant vasoconstriction of rat microvascular coronary arteries via increasing vascular smooth muscle calcium waves, and also significantly increased coronary vascular resistance and infarct size in Langendorff hearts. These effects were blocked by the Y1 receptor antagonist BIBO3304 (1 µM). Immunohistochemistry of the human coronary microvasculature demonstrated the presence of vascular smooth muscle Y1 receptors. CONCLUSION: High CS NPY levels immediately after reperfusion correlate with microvascular dysfunction, greater myocardial injury, and reduced ejection fraction 6 months after STEMI. NPY constricts the coronary microcirculation via the Y1 receptor, and antagonists may be a useful PPCI adjunct therapy.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

KEY POINTS: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. ABSTRACT: Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

Endothelial-smooth muscle cell interactions in the regulation of vascular tone in skeletal muscle.

The SMCs of skeletal muscle arterioles are intricately sensitive to changes in membrane potential. Upon increasing luminal pressure, the SMCs depolarize, thereby opening VDCCs, which leads to contraction. Mechanisms that oppose this myogenic tone can involve voltage-dependent and independent dilator pathways, and can be endothelium-dependent or independent. Of particular interest are the pathways leading to hyperpolarization of SMCs, as these can potentially evoke both local and conducted dilation. This review focuses on three agonists that cause local and conducted dilation in skeletal muscle: ACh, ATP, and KCl. The mechanisms for the release of these agonists during motor nerve stimulation and/or hypoxia, and their actions to open either Ca2+ -activated K+ channels (KCa ) or inwardly rectifying K+ channels (KIR ) are described. By causing local and conducted dilation, each agonist has the ability to improve skeletal muscle blood flow during exercise and ischemia.

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.

Endothelial cell (EC) Ca(2+)-activated K channels (SK(Ca) and IK(Ca) channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK(Ca) channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca(2+) events (Ca(2+) puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (~2 min(-1)) inositol 1,4,5-trisphosphate receptor-based Ca(2+) events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca(2+) events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca(2+) increases, which on occasion led to intracellular Ca(2+) waves. The concurrent vasodilation associated with increases in Ca(2+) event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK(Ca) channel blocker), but not by apamin (SK(Ca) channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca(2+) event frequency; at low pressures the consequence is activation of EC IK(Ca) channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.

Crucial role for sensory nerves and Na/H exchanger inhibition in dapagliflozin and empagliflozin-induced arterial relaxation.

AIMS: Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) from sensory nerves independent of glucose transport. METHODS AND RESULTS: The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using Wire-Myography. SGLT2, CGRP, TRPV1 and NHE1, expression was determined by Western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin.All SGLT inhibitors (1µM-100µM) and cariporide (30µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. CONCLUSIONS: SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Linking hyperpolarization to endothelial cell calcium events in arterioles.

Our understanding of the relationship between EC membrane potential and Ca(2+) entry has been shaped historically by data from cells in culture. Membrane hyperpolarization was associated with raised cytoplasmic [Ca(2+) ] ascribed to the increase in the inward electrochemical gradient for Ca(2+) , as ECs are generally thought to lack VGCC. Ca(2+) influx was assumed to reflect the presence of an undefined Ca(2+) "leak" channel, although the original research articles with isolated ECs did not elucidate which Ca(2+) influx channel was involved or indeed if a transporter might contribute. Overall, these early studies left many unanswered questions, not least whether a similar mechanism operates in native ECs that are coupled to each other and, in many smaller arteries and arterioles, to the adjacent vascular SMCs via gap junctions. This review discusses whether Ca(2+) leak through constitutively active EC Ca(2+) channels or a more defined, gated pathway might underlie the reported link between enhanced Ca(2+) entry and hyperpolarization. Electrophysiological evidence from ECs in isolation is compared with those in intact arteries and arterioles and the possible physiological relevance of EC Ca(2+) entry driven by hyperpolarization discussed.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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 Gßγ subunit inhibitor gallein and the PLC inhibitor U-73122 attenuated NPY-induced vasoconstriction. Signalling via the Gßγ-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 Gßγ 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. CONCLUSIONS AND IMPLICATIONS: These data suggest that the Gßγ 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.

Tracking endothelium-dependent NO release in pressurized arteries.

Background: Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Methods: Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), Cu2FL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or en face imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1 µM), and NO donor (SNAP, 10 µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR0. Results: In cell-free chambers using either Cu2FL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both Cu2FL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with Cu2FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR0 following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30 min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP. Conclusion: These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.

Crucial role for Sodium Hydrogen Exchangers in SGLT2 inhibitor-induced arterial relaxations.

Introduction: Sodium dependent glucose transporter 2 (SGLT2 or SLC5A2) inhibitors effectively lower blood glucose and are also approved treatments for heart failure independent of raised glucose. One component of the cardioprotective effect is reduced cardiac afterload but the mechanisms underlying peripheral relaxation are ill defined and variable. We speculated that SGLT2 inhibitors promoted arterial relaxation via the release of the potent vasodilator calcitonin gene-related peptide (CGRP) from sensory nerves independent of glucose transport. Experimental approach: The functional effects of SGLT2 inhibitors (dapagliflozin, empagliflozin, ertugliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted mesenteric and renal arteries from male Wistar rats using Wire-Myography. SGLT2, NHE1, CGRP and TRPV1 expression in both arteries was determined by Western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin. Results: All SGLT2 inhibitors produced a concentration dependent relaxation (1µM-100µM) of mesenteric arteries that was considerably greater than in renal arteries. Cariporide relaxed mesenteric arteries but not renal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that was absent in renal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin produced significantly greater relaxations in mesenteric arteries compared to renal arteries. Relaxations to dapagliflozin, empagliflozin and cariporide were attenuated by incubation with the CGRP receptor antagonist BIBN-4096, the Kv7 blocker linopirdine and the TRPV1 antagonist AMG-517 as well as by depletion of neuronal CGRP. Neither dapagliflozin nor empagliflozin directly activated heterologously expressed TRPV1 channels or Kv7 channels. Strikingly, only NHE1 colocalised with TRPV1 in sensory nerves, and cariporide pre-application prevented the relaxant response to SGLT2 inhibitors. Conclusions: SGLT2 inhibitors relax mesenteric arteries by a novel mechanism involving the release of CGRP from sensory nerves following inhibition of the Na + /H + exchanger.

Spatial distribution and mechanical function of elastin in resistance arteries: a role in bearing longitudinal stress.

OBJECTIVE: Despite the role that extracellular matrix (ECM) plays in vascular signaling, little is known of the complex structural arrangement between specific ECM proteins and vascular smooth muscle cells. Our objective was to examine the hypothesis that adventitial elastin fibers are dominant in vessels subject to longitudinal stretch. METHODS AND RESULTS: Cremaster muscle arterioles were isolated, allowed to develop spontaneous tone, and compared with small cerebral arteries. 3D confocal microscopy was used to visualize ECM within the vessel wall. Pressurized arterioles were fixed and stained with Alexa 633 hydrazide (as a nonselective ECM marker), anti-elastin, or anti-type 1 collagen antibody and a fluorescent nuclear stain. Exposure of cremaster muscle arterioles to elastase for 5 minutes caused an irreversible lengthening of the vessel segment that was not observed in cerebral arteries. Longitudinal elastin fibers were demonstrated on cremaster muscle arterioles using 3D imaging but were confirmed to be absent in cerebral vessels. The fibers were also distinct from type I collagen fibers and were degraded by elastase treatment. CONCLUSIONS: These results indicate the importance of elastin in bearing longitudinal stress in the arteriolar wall and that these fibers constrain vascular smooth muscle cells. Differences between skeletal muscle and cerebral small arteries may reflect differences in the local mechanical environment, such as exposure to longitudinal stretch.

Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries.

Background: Adequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries ex vivo using endothelium-dependent and -independent vasodilators. Methods: Human and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM. Results: 3D rendered ultrastructure images of human right atrial (RA-) IMCAs revealed extensive homo-and hetero-cellular contacts, including to longitudinally-arranged smooth muscle cells (l-SMCs) found between the endothelial cells (ECs) and radially-arranged medial SMCs (r-SMCs). Local and conducted vasodilatation followed focal application of bradykinin in both human and porcine RA-IMCAs, and relied on hyperpolarization of SMCs, but not nitric oxide. Bradykinin initiated asynchronous oscillations in endothelial cell Ca2+ in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca2+-activated K+ channels (KCa) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these KCa channels. Extensive electrical coupling was demonstrated between r-SMCs and l-SMCs, providing an additional pathway to facilitate the well-established myoendothelial coupling. Conducted dilation was still evident in a human RA-IMCA with poor myogenic tone, and heterocellular contacts were visible in the 3D reconstructed artery. Hyperpolarization and conducted vasodilation was also observed to adenosine which, in contrast to bradykinin, was sensitive to combined block of ATP-sensitive (KATP) and inwardly rectifying (KIR) K+ channels. Conclusions: These data extend our understanding of the mechanisms that coordinate human coronary microvascular blood flow and the mechanistic overlap with porcine IMCAs. The unusual presence of l-SMCs provides an additional pathway for rapid intercellular signaling between cells of the coronary artery wall. Local and conducted vasodilation follow hyperpolarization of the ECs or SMCs, and contact-coupling between l-SMCs and r-SMCs likely facilitates this vasodilation.