Endothelial Cell Ca2 Research Articles

Inside the USCAP Journals

Inside the USCAP Journals

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.

Quercetin depletes intracellular Ca2+ stores and blunts ATP-triggered Ca2+ signaling in bEnd.3 endothelial cells.

Quercetin is a flavonol polyphenol widely found in many vegetables, grains, and fruits. Quercetin has been shown to inhibit proliferation and invasion of various glioma cells and is regarded as a potential anticancer agent against glioma. However, whether and how this drug could affect brain blood vessels and endothelial cells (EC) are less understood. Further, there is hitherto no report on how quercetin affects brain EC Ca2+ homeostasis. In this report, we investigated the effects of quercetin on Ca2+ homeostasis in mouse brain bEnd.3 EC. We demonstrated that quercetin raised cytosolic Ca2+ level in a concentration-dependent manner. Quercetin-triggered Ca2+ signal composed of both internal Ca2+ release and extracellular Ca2+ influx. Quercetin caused Ca2+ release from the endoplasmic reticulum, and consistently, inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) by xestospongin C (XeC) suppressed quercetin-triggered Ca2+ release. Quercetin also caused Ca2+ release from lysosomes, an observation in concordance with the inhibition of quercetin-triggered Ca2+ release by trans-Ned-19, a blocker of two-pore channels. As quercetin depleted intracellular Ca2+ storage, it suppressed ATP-induced Ca2+ release and thereby blunted ATP-triggered Ca2+ signaling. In addition, quercetin co-treatment significantly suppressed ATP-stimulated nitric oxide release. Our work therefore showed, for the first time, quercetin perturbed intracellular Ca2+ stores and strongly suppressed ATP-triggered response in bEnd.3 cells.

P719Endothelial expression of adenylate cyclase type 9 (Adcy9) regulates endothelial cell signalling and vasodilation

Abstract Background/Introduction The clinical benefits of the cholesterol ester transfer protein (CETP) inhibitor dalcetrapib are dependent on the ADCY9 gene, which encodes adenylate cyclase (AC) type 9 (AC9). AC9 is one of nine membrane-bound isoforms of AC producing cyclic adenosine monophosphate (cAMP). We demonstrated that Adcy9 inactivation in the mouse protects from atherosclerosis, but only in absence of CETP. Adcy9 inactivation is also associated with improved endothelial function, including greater endothelial-dependent vasodilation (EDV) in response to acetylcholine (ACh). This suggests that Adcy9 may control endothelial Ca2+, an essential mediator of endothelial cell (EC) signalling. We hypothesized that Adcy9 is expressed in the endothelium and controls EC signalling. Purpose Our aim was to study AC9 expression in the vascular endothelium and the role of AC9 expression on endothelial cAMP signalling and Ca2+ dynamics. Methods The effects of Adcy9 inactivation were studied using wild-type (WT) and Adcy9-inactivated (Adcy9Gt/Gt) mice and siRNA-mediated inactivation of ADCY9 in human coronary artery endothelial cells (HCAEC). In the mouse, AC9 expression was quantified in whole aorta (± endothelium) and primary cultures of lung EC (LEC) by Western blot. Endothelial Ca2+ pulsars were monitored in mouse femoral arteries in response to acetylcholine (ACh, 10 μM). cAMP accumulation to AC activators VIP and forskolin were measured in LEC and HCAEC. EDV to VIP was studied ex vivo in mouse femoral arteries using pressurized arteriography. Results AC9 is expressed in LEC and the aorta, and its detection in the latter decreased by 33% after mechanical removal of the endothelium (P<0.05). AC activation with forskolin (10–4 M) in LEC led to increased cAMP accumulation in response to Adcy9 inactivation (WT: 1129±138 pmol cAMP/mg of protein, Adcy9Gt/Gt: 1965±169, n=3; P<0.01). ADCY9 inactivation in HCAEC also increased cAMP accumulation to forskolin 10–4M (control siRNA: 85±17, ADCY9 siRNA: 150±6, n=3; P<0.01). In LEC, receptor-dependent accumulation of cAMP to VIP (10–5 M) was higher in Adcy9Gt/Gt (1509±258)) compared to WT (915±170, n=3; P<0.05) mice. In mouse femoral arteries, VIP-induced maximal vasodilation was increased by Adcy9 inactivation in the presence of the endothelium (Adcy9Gt/Gt: 85±4%, n=6; WT: 52±9%, n=5, P<0.05), but not in its absence (Adcy9Gt/Gt: 67±12%, n=6 and WT: 59±15%, n=5). In the femoral artery endothelium, ACh increased Ca2+ pulsar frequency more in Adcy9Gt/Gt (243±32%, n=4) than in WT (178±19%, n=5; P<0.05) mice. Conclusion ADCY9 is expressed in the endothelium, and its inactivation potentiates endothelial cell Ca2+ dynamics, cAMP accumulation and VIP-induced vasodilation. We therefore identify ADCY9 as a new molecular pathway regulating endothelial-dependent vasodilation. This suggests that ADCY9's endothelial biology could be involved in the ADCY9 genotype-dependent clinical effects of dalcetrapib. Acknowledgement/Funding DalCor

IP3‐evoked Ca2+ release in Endothelial Cells is Modulated by the Immunosuppressant FK506‐binding Protein (FKBP12)

The endoplasmic reticulum (ER) is integral to the regulation of cytoplasmic Ca2+ concentration in endothelial cells. Ca2+ release from the ER is controlled by two intracellular receptor/channel complexes, the inositol 1,4,5‐triphosphate receptor (IP3R) and the ryanodine receptor (RyR). These receptors have been shown to be regulated by the accessory FK506‐binding protein (FKBP). FK506 is a potent immunosuppressant, which is routinely used after organ transplant to lower the risk of rejection, and has also been used in drug‐eluting stents. FK506 has been shown to act on smooth muscle cells either directly by binding to the channel, or indirectly via FKBP modulation of two targets, calcineurin or the mammalian target of rapamycin (mTOR). However very little is known on the role FKBPs play in endothelial cell Ca2+ signaling.To investigate this, Ca2+ signals were measured in the endothelium of second order mesenteric arteries using Cal‐520 (5 μM). IP3Rs were activated by localised photo‐uncaging of IP3 (5 μM), and RyR by the application of Caffeine (10 mM). FK506 (10 μM), which displaces FKBP from each receptor to inhibit calceinurin, increased the IP3‐evoked Ca2+ rise. The phosphatase inhibitors cypermethrin and okadaic acid also increased the IP3‐mediated Ca2+ release. Cypermethrin and okadaic acid did not prevent FK506‐induced increase in Ca2+ release. These results suggest that while calceinurin may modulate IP3‐evoked Ca2+ release, the phosphatase is not necessary for the FK506‐mediated potentiation of IP3‐evoked Ca2+ increase.Rapamycin (10 μM), which disrupts the binding of FKBP12 to the receptor and inhibits mTOR, did not significantly alter IP3‐induced Ca2+ release in endothelial cells, indicating that mTOR does not exert a significant role in endothelial IP3‐induced Ca2+ release.Interestingly Caffeine, the activator of RyR‐mediated Ca2+ release, did not activate Ca2+ signals in the endothelium either in intact tissue or in freshly isolated endothelial patches, suggesting that Caffeine‐activated RyRs do not play a role in endothelial Ca2+ release.These results indicate that FKBPs play an integral role in regulating endothelial IP3‐mediated Ca2+ release, and may underlie the severe endothelial dysfunction associated with FK506 immunosuppressants.Support or Funding InformationWellcome TrustBritish Heart FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulation of Muscarinic Receptors Activate Acid Sensing Ion Channel 1 in Isolated Mesenteric Artery Endothelial Tubes

Acid sensing ion channel 1 (ASIC1) belongs to the amiloride‐sensitive degenerin/epithelial sodium channel superfamily and is permeable to both Na+ and Ca2+. ASIC1 is expressed in vascular smooth muscle and/or endothelial cells of various vascular beds and is activated following stimulation of G protein‐coupled receptors (GPCR). In the pulmonary circulation, ASIC1 contributes to GPCR‐induced vasoconstriction and vascular smooth muscle Ca2+ influx via a store‐operated Ca2+ entry (SOCE) mechanism. In small mesenteric arteries, we have recently shown that ASIC1 contributes to acetylcholine (ACh)‐induced vasodilation and endothelial cell Ca2+ entry, however ASIC1‐mediated Ca2+ influx is not SOCE‐dependent in mesenteric artery endothelial cells. Thus, further investigation is needed to understand the mechanism by which ASIC1 is activated by GPCR in small mesenteric artery endothelial cells. Previous studies show ASIC1 is activated by arachidonic acid, therefore we hypothesized that acetylcholine stimulates ASIC1 in mesenteric artery endothelial cells through activation of phospholipase A2 (PLA2) and release of arachidonic acid. To test this hypothesis, we examined endothelial Ca2+ influx via Mn2+ quenching of fura‐2 fluorescence in freshly isolated mesenteric artery endothelial tubes. The PLA inhibitor methyl arachidonyl fluorophosphonate (MAF; 500 nM) attenuated the Ca2+ entry response to ACh, confirming that PLA2‐signaling contributes to the ACh‐induced Ca2+ entry in mesenteric artery endothelial cells. In addition, the selective inhibitor of ASIC1, psalmotoxin 1 (PcTX1; 20 nM), attenuated arachidonic acid‐induced Ca2+‐influx in mesenteric artery endothelial tubes. Although these studies suggest stimulation of muscarinic receptors activate ASIC1 through a PLA2‐arachidonic acid signaling mechanism in mesenteric endothelium, further studies are needed to identify the downstream effector of ASIC1.Support or Funding InformationSupported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), and American Heart Association Grant 18TPA34110281 (to N.L. Jernigan).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neural activity drives dynamic Ca2+ signals in capillary endothelial cells that shape local brain blood flow

Brain capillaries constitute a vast, arborescent network of microscopic tubes composed of endothelial cells (ECs) that lie in close apposition to all neurons. Capillaries send long‐range electrical signals to regulate arteriolar diameter and control blood flowing into the brain, but it is unknown whether higher resolution control of blood flow exists within the capillary bed. Here, we demonstrate that capillary ECs exhibit recurrent, long‐lasting, high‐amplitude, local Ca2+ signals in vivo that depend on neuronal activity. These signals are mediated by Gq‐protein‐coupled receptors, prominently including prostaglandin E2‐induced activation of the EP1 receptor, and reflect Ca2+ release through endoplasmic reticulum IP3 receptors and Ca2+ entry through TRPV4 channels. EC Ca2+ elevation at capillary branch points relaxes overlying pericytes, which is associated with a nitric oxide‐dependent increase in local blood flow. Capillary and arteriolar ECs respond robustly to neural stimulation, suggesting coordinated Ca2+ signaling in these compartments directs blood flow to satisfy neuronal metabolic needs.Support or Funding InformationSupported by American Heart Association postdoctoral fellowships and a Scientist Development Grant (14POST20480144; 17SDG33670237 to T.A.L.; 17POST33650030 to O.F.H), the Totman Medical Research Trust (to M.T.N.), Fondation Leducq (to M.T.N.), EC Horizon 2020 (to M.T.N.), and National Institutes of Health (NIH 4P20 GM103644/4‐5 to the Vermont Centre on Behavior and Health (T.A.L); P30‐GM‐103498 to the COBRE imaging facility at the University of Vermont College of Medicine; R24‐HL‐120847 to M.I.K; P01‐HL‐095488, R01‐HL‐121706, R37‐DK‐053832 and R01‐HL‐131181 to M.T.N., and FOR2372 to G.M.K and E.K).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

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

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. 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.

Valproic acid inhibits ATP-triggered Ca2+ release via a p38-dependent mechanism in bEND.3 endothelial cells.

Valproic acid (VA) is currently used to treat epilepsy and bipolar disorder. It has also been demonstrated to promote neuroprotection and neurogenesis. Although beneficial actions of VA on brain blood vessels have also been demonstrated, the effects of VA on brain endothelial cell (EC) Ca2+ signaling are hitherto unreported. In this report, we examined the effects of VA on agonist-triggered Ca2+ signaling in mouse cortical bEND.3 EC. While VA (100μm) did not cause an acute inhibition of ATP-triggered Ca2+ signaling, a 30-min VA treatment strongly suppressed ATP-triggered intracellular Ca2+ release; however, such treatment did not affect Ca2+ release triggered by cyclopiazonic acid, an inhibitor of SERCA Ca2+ pump, suggesting there was no reduction in Ca2+ store size. VA-activated p38 signaling, and VA-induced inhibition of ATP-triggered Ca2+ release was prevented by SB203580, a p38 inhibitor, suggesting VA caused the inhibition by activating p38. Remarkably, VA treatment did not affect acetylcholine-triggered Ca2+ release, suggesting VA may not inhibit inositol 1,4,5-trisphosphate-induced Ca2+ release per se, and may not act directly on Gq or phospholipase C. Taken together, our results suggest VA treatment, via a p38-dependent mechanism, led to an inhibition of purinergic receptor-effector coupling.

Acid Sensing Ion Channel 1 Contributes to Endothelium‐Derived Hyperpolarizing Factor Induced Vasodilation in Small Mesenteric Arteries.

Acid sensing ion channel 1 (ASIC1) belongs to the amiloride‐sensitive degenerin/epithelial sodium channel superfamily, and is permeable to both Na+ and Ca2+. ASIC channels have been found to regulate blood pressure through mechanotransduction in arterial baroreceptors and are also pH sensors in carotid body chemoreceptors. ASIC1 is additionally expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds; yet little is known regarding the role of ASIC1 in the regulation of local vascular resistance. Although extracellular H+ is the recognized activator of ASIC1, we have previously shown this channel can be activated following G protein‐coupled receptor stimulation. In small mesenteric arteries, we found that ASIC1 contributes to acetylcholine (ACh)‐induced endothelial cell Ca2+ entry and vasodilation. However, the mechanisms involved in activating ASIC1 and its downstream effects on vasodilation are unknown. We hypothesized that ACh activates ASIC1‐mediated endothelial cell Ca2+ entry to mediate endothelium‐derived hyperpolarizing factor (EDHF)‐type vasodilation through activation of small (SK) and intermittent (IK) conductance Ca2+‐activated K+ channels. To test this hypothesis, we used pressure myography to study the effects of ASIC1 on ACh‐induced vasodilation (10−9 – 10−5 M) of freshly isolated mesenteric arteries (100–200 μm inner diameter), in the presence of Nω‐nitro‐L‐arginine (nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). ACh‐induced vasodilation was abolished by specific inhibitors of SK and IK channels (apamin and TRAM‐34, respectively), confirming that the ACh‐induced dilation is EDHF‐dependent. The specific ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM) similarly blocks the EDHF‐mediated vasodilatory response, suggesting ASIC1 contributes to ACh‐induced vasodilation through an EDHF‐mediated pathway. Future studies will examine the upstream mechanisms involved in activation of endothelial ASIC1 and subsequent downstream effects to mediate EDHF‐dependent vasodilation. These studies suggest a role for ASIC1 as a novel contributor to agonist‐induced mesenteric vasodilation and cardiovascular homeostasis.Support or Funding InformationSupported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), and R01 HL132883 (to T.C. Resta).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extracellular Histones Trigger Endothelial Calcium Signals that Paradoxically do not Cause Vasodilation, and Instead, Compromise Endothelial Function

Elevated levels of histone proteins are found in the circulation of patients following traumatic injury and are associated with vascular dysfunction, coagulopathy, sepsis, and poor patient outcome, yet the mechanism(s) involved are not known. One possible mechanism to affect vascular function is through alteration in endothelial cell (EC) calcium, as suggested by effects on cultured endothelial cells (Nat Med, 2009). We examined the spatial and temporal characteristics of histone‐induced EC Ca2+ signals in en face resistance‐sized mesenteric arteries (MAs) from endothelial cell‐specific genetically encoded Ca2+ indicator mice (cx40‐GCaMP5‐mCherry), using a spinning confocal microscope. Addition of histone protein to the bath solution (10 mg/ml, unfractionated histone protein mixture) induced large Ca2+ signals within seconds of application that spread within and between ECs. Histone‐induced signals are approximately twice as bright and spread 7 times farther than typical spontaneous Ca2+ signals in MA ECs. Contrary to the expectation that histone‐induced Ca2+ signaling would elicit vasodilation, luminal application of histones had no effect on diameter, and instead reduced dilations to endothelial‐dependent vasodilators. After 30 minutes of histone exposure, global cytosolic Ca2+ increased to greater than 500 nM, and appeared to cause cell death. To test this, we performed live cell imaging experiments in the presence of propidium iodide (PI, 100 ng/ml) as a real‐time marker of cell death. After 30 minutes of exposure to histone protein, 25% of EC nuclei per field were labeled with PI. To determine the route of Ca2+ increase, we used pharmacologic and genetic approaches to test known Ca2+ pathways such as IP3R mediated release, and TRPV4 and P2XR channel‐mediated influx. Removal of extracellular Ca2+, but not depletion of intracellular Ca2+ stores, prevented histone‐induced Ca2+ signals. Histone induced signals and cell death was not suppressed by inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels. However, Ca2+ events were suppressed by non‐selective P2X/YR antagonists (50 mM suramin and 10 mM PPADS), P2XR antagonists (1 mM TNPATP), and a P2X7R antagonist (100 mM amiloride). The response to histones was reduced, but not eliminated, in MA ECs from P2X7R‐knockouts, suggesting a possible involvement of P2X7Rs as well as an unknown pathway. However, P2X7R is sufficient to generate histone induced cation current in Xenopus ooctyes. The data demonstrate that histones are robust activators Ca2+ signaling and EC death in native resistance‐sized arteries.Support or Funding InformationDMC is supported by NIH (5K99HL133451‐02); KF by NIH (1R01GM123010‐01); and MTN by NIH (5R01HL131181‐02, 5R01HL121706‐04, 5R37DK053832‐19, 7UM1HL120877‐04), The Fondation Leducq, Horizon 2020, and the Totman Medical Research Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperglycaemia disrupts conducted vasodilation in the resistance vasculature of db/db mice

Vascular dysfunction in small resistance arteries is observed during chronic elevations in blood glucose. Hyperglycaemia-associated effects on endothelium-dependent vasodilation have been well characterized, but effects on conducted vasodilation in the resistance vasculature are not known. Small mesenteric arteries were isolated from healthy and diabetic db/db mice, which were used as a model of chronic hyperglycaemia. Endothelium-dependent vasodilation via the Gq/11-coupled proteinase activated receptor 2 (PAR2) was stimulated with the selective agonist SLIGRL. The Ca2+-sensitive fluorescent indicator fluo-8 reported changes in endothelial cell (EC) [Ca2+]i, and triple cannulated bifurcating mesenteric arteries were used to study conducted vasodilation. Chronic hyperglycaemia did not affect either EC Ca2+ or local vasodilation to SLIGRL. However, both acute and chronic exposure to high glucose or the mannitol osmotic control attenuated conducted vasodilation to 10μM SLIGRL. This investigation demonstrates for the first time that a hypertonic solution containing glucose or mannitol can interfere with the spread of a hyperpolarizing current along the endothelium in a physiological setting. Our findings reiterate the importance of studying the effects of hyperglycaemia in the vasculature, and provide the basis for further studies regarding the modulation of junctional proteins involved in cell to cell communication by diseases such as diabetes.

Pregnancy-adapted uterine artery endothelial cell Ca2+ signaling and its relationship with membrane potential.

Pregnancy‐derived uterine artery endothelial cells (P‐UAEC) express P2Y2 receptors and at high cell density show sustained and synchronous [Ca2+]i burst responses in response to ATP. Bursts in turn require coupling of transient receptor potential canonical type3 channel (TRPC3) and inositol 1,4,5‐triphosphate receptor type 2 (IP3R2), which is upregulated in P‐UAEC in a manner dependent on connexin 43 (Cx43) gap junctions. While there is no known direct interaction of TRPC3 with Cx43, early descriptions of TRPC3 function showed it may also be influenced by altered membrane potential (V m). Herein, we ask if enhanced TRPC3 Ca2+ bursting due to enhanced Cx43 coupling may be coupled via dynamic alterations in V m in P‐UAEC, as reported in some (HUVEC) but not all endothelial cells. Following basic electrical characterization of UAEC, we employed a high sensitivity cell imaging system to simultaneously monitor cell V m and [Ca2+]i in real time in continuous monolayers of UAEC. Our findings show that while acute and sustained phase [Ca2+]i bursting occur dose‐dependently in response to ATP, V m is not coregulated with any periodicity related to [Ca2+]i bursting. Only a small but significant progressive change in V m is seen, and this is more closely related to overall mobilization of Ca2+. Surprisingly, this is also most apparent in NP‐UAEC > P‐UAEC. In contrast [Ca2+]i bursting is more synchronous in P‐UAEC and even achieves [Ca2+]i waves passing through the P‐UAEC monolayer. The relevance of these findings to mechanisms of pregnancy adaptation and its failure in hypertensive pregnancy are discussed.

Molecular and functional characterization of the endothelial ATP-sensitive potassium channel

ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system, where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells, but their presence and function in this context are less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch clamp, ex vivo coronary perfusion Langendorff heart experiments, and endothelial cell Ca2+ imaging comparing eKO and wild-type mice show that Kir6.1-containing KATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca2+], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the KATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation, and this resulted in greater cardiac injury during ischemia-reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial KATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation, and has a protective role in ischemia reperfusion.

Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function.

The integral role of the endothelium during the coordination of blood flow throughout vascular resistance networks has been recognized for several decades now. Early examination of the distinct anatomy and physiology of the endothelium as a signaling conduit along the vascular wall has prompted development and application of an intact endothelial "tube" study model isolated from rodent skeletal muscle resistance arteries. Vasodilatory signals such as increased endothelial cell (EC) Ca2+ ([Ca2+ ]i ) and hyperpolarization take place in single ECs while shared between electrically coupled ECs through gap junctions up to distances of millimeters (≥2mm). The small- and intermediate-conductance Ca2+ activated K+ (SKCa /IKCa or KCa 2.3/KCa 3.1) channels function at the interface of Ca2+ signaling and hyperpolarization; a bidirectional relationship whereby increases in [Ca2+ ]i activate SKCa /IKCa channels to produce hyperpolarization and vice versa. Further, the spatial domain of hyperpolarization among electrically coupled ECs can be finely tuned via incremental modulation of SKCa /IKCa channels to balance the strength of local and conducted electrical signals underlying vasomotor activity. Multifunctional properties of the voltage-insensitive SKCa /IKCa channels of resistance artery endothelium may be employed for therapy during the aging process and development of vascular disease.

Endothelial Cell Reactive Oxygen Species and Ca2+ Signaling in Pulmonary Hypertension.

Pulmonary hypertension (PH) refers to a disorder characterized by elevated pulmonary arterial pressure, leading to right ventricular overload and eventually right ventricular failure, which results in high morbidity and mortality. PH is associated with heterogeneous etiologies and distinct molecular mechanisms, including abnormal migration and proliferation of endothelial and smooth muscle cells. Although the exact details are not fully elucidated, reactive oxygen species (ROS) have been shown to play a key role in promoting abnormal function in pulmonary arterial smooth muscle and endothelial cells in PH. In endothelial cells, ROS can be generated from sources such as NADPH oxidase and mitochondria, which in turn can serve as signaling molecules in a wide variety of processes including posttranslational modification of proteins involved in Ca2+ homeostasis. In this chapter, we discuss the role of ROS in promoting abnormal vasoreactivity and endothelial migration and proliferation in various models of PH. Furthermore, we draw particular attention to the role of ROS-induced increases in intracellular Ca2+ concentration in the pathobiology of PH.

Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca2+ release

There is abundant evidence that H2O2 can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca2+]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3µM) and l-NAME (300µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5µM) and TRAM-34 (10µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100Uml−1 each) was present. S&C also reduced the carbachol-induced EC [Ca2+]i increase by 74%. When applied in Ca2+-free external medium, carbachol caused a transient increase in EC [Ca2+]i. This was reduced by catalase, and was enhanced when 1µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca2+]i and vasodilatation, but adding 10µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe2+ chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca2+ store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca2+ influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca2+ release.

Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/.

Extracellular matrix fibronectin initiates endothelium-dependent arteriolar dilatation via the heparin-binding, matricryptic RWRPK sequence of the first type III repeat of fibrillar fibronectin.

The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms. In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. This vasodilatory signal requires the heparin-binding matricryptic RWRPK sequence in the first type III repeat of fibrillar fibronectin. The fibronectin-dependent component of the integrated muscle contraction-dependent arteriolar vasodilatation is coupled through an endothelial cell-dependent signalling pathway. Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin- and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg(-1)) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 μm, n = 12 and 17.2 ± 2.4 μm, n = 14, respectively) whereas mutation of the active sequence (R(613) WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 μm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 μm, n = 12) that was significantly decreased (to 7.0 ± 2.7 μm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,8-10 and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca(2+) (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.