Endothelial Ca2 Research Articles

Ablation of Endothelial TRPV4 Channels Alters the Dynamic Ca2+ Signaling Profile in Mouse Carotid Arteries.

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca2+ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca2+ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca2+ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca2+ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4−/−) to identify and characterize TRPV4-dependent endothelial Ca2+ dynamics. We find that carotid arteries from both ecTRPV4−/− and WT mice exhibit a range of basal and acetylcholine (ACh)-induced Ca2+ dynamics, similar in net frequency. Analysis of discrete Ca2+ event parameters (amplitude, duration, and spread) and event composite values reveals that while ecTRPV4−/− artery endothelium predominantly produces large Ca2+ events comparable to and in excess of those produced by WT endothelium, they are deficient in a particular population of small events, under both basal and ACh-stimulated conditions. These findings support the concept that TRPV4 channels are responsible for generating a distinct population of focal Ca2+ transients in the intact arterial endothelium, likely underlying their essential role in vascular homeostasis.

Targeting the Endothelial Ca2+ Toolkit to Rescue Endothelial Dysfunction in Obesity Associated-Hypertension.

Obesity is a major cardiovascular risk factor which dramatically impairs endothelium- dependent vasodilation and leads to hypertension and vascular damage. The impairment of the vasomotor response to extracellular autacoids, e.g., acetylcholine, mainly depends on the reduced Nitric Oxide (NO) bioavailability, which hampers vasorelaxation in large conduit arteries. In addition, obesity may affect Endothelium-Dependent Hyperpolarization (EDH), which drives vasorelaxation in small resistance arteries and arterioles. Of note, endothelial Ca2+ signals drive NO release and trigger EDH. A structured search of bibliographic databases was carried out to retrieve the most influential, recent articles on the impairment of vasorelaxation in animal models of obesity, including obese Zucker rats, and on the remodeling of the endothelial Ca2+ toolkit under conditions that mimic obesity. Furthermore, we searched for articles discussing how dietary manipulation could be exploited to rescue Ca2+-dependent vasodilation. We found evidence that the endothelial Ca2+ could be severely affected by obese vessels. This rearrangement could contribute to endothelial damage and is likely to be involved in the disruption of vasorelaxant mechanisms. However, several Ca2+-permeable channels, including Vanilloid Transient Receptor Potential (TRPV) 1, 3 and 4 could be stimulated by several food components to stimulate vasorelaxation in obese individuals. The endothelial Ca2+ toolkit could be targeted to reduce vascular damage and rescue endothelium- dependent vasodilation in obese vessels. This hypothesis remains, however, to be probed on truly obese endothelial cells.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Inside the USCAP Journals

Inside the USCAP Journals

TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia

Sepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+]i in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-κB pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-κB pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases.

Inside the USCAP Journals

Inside the USCAP Journals

Endothelial Transient Receptor Potential Channels and Vascular Remodeling: Extracellular Ca2 + Entry for Angiogenesis, Arteriogenesis and Vasculogenesis.

Vasculogenesis, angiogenesis and arteriogenesis represent three crucial mechanisms involved in the formation and maintenance of the vascular network in embryonal and post-natal life. It has long been known that endothelial Ca2+ signals are key players in vascular remodeling; indeed, multiple pro-angiogenic factors, including vascular endothelial growth factor, regulate endothelial cell fate through an increase in intracellular Ca2+ concentration. Transient Receptor Potential (TRP) channel consist in a superfamily of non-selective cation channels that are widely expressed within vascular endothelial cells. In addition, TRP channels are present in the two main endothelial progenitor cell (EPC) populations, i.e., myeloid angiogenic cells (MACs) and endothelial colony forming cells (ECFCs). TRP channels are polymodal channels that can assemble in homo- and heteromeric complexes and may be sensitive to both pro-angiogenic cues and subtle changes in local microenvironment. These features render TRP channels the most versatile Ca2+ entry pathway in vascular endothelial cells and in EPCs. Herein, we describe how endothelial TRP channels stimulate vascular remodeling by promoting angiogenesis, arteriogenesis and vasculogenesis through the integration of multiple environmental, e.g., extracellular growth factors and chemokines, and intracellular, e.g., reactive oxygen species, a decrease in Mg2+ levels, or hypercholesterolemia, stimuli. In addition, we illustrate how endothelial TRP channels induce neovascularization in response to synthetic agonists and small molecule drugs. We focus the attention on TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPV1, TRPV4, TRPM2, TRPM4, TRPM7, TRPA1, that were shown to be involved in angiogenesis, arteriogenesis and vasculogenesis. Finally, we discuss the role of endothelial TRP channels in aberrant tumor vascularization by focusing on TRPC1, TRPC3, TRPV2, TRPV4, TRPM8, and TRPA1. These observations suggest that endothelial TRP channels represent potential therapeutic targets in multiple disorders featured by abnormal vascularization, including cancer, ischemic disorders, retinal degeneration and neurodegeneration.

Calcium-Permeable Channels in Tumor Vascularization: Peculiar Sensors of Microenvironmental Chemical and Physical Cues.

Calcium (Ca2+)-permeable channels are key players in different processes leading to blood vessel formation via sprouting angiogenesis, including endothelial cell (EC) proliferation and migration, as well as in controlling vascular features which are typical of the tumor vasculature.In this review we present an up-to-date and critical view on the role of Ca2+-permeable channels in tumor vascularization, emphasizing on the dual communication between growth factors (mainly VEGF) and Ca2+ signals. Due to the complexity of the tumor microenvironment (TME) as a source of multiple stimuli acting on the endothelium, we aim to discuss the close interaction between chemical and physical challenges (hypoxia, oxidative stress, mechanical stress) and endothelial Ca2+-permeable channels, focusing on transient receptor potential (TRP), store-operated Ca2+ channels (SOCs), and mechanosensitive Piezo channels. This approach will depict their crucial contribution in regulating key properties of tumor blood vessels, such as recruitment of endothelial progenitors cells (EPCs) in the early steps of tumor vascularization, abnormal EC migration and proliferation, and increased vascular permeability. Graphical abstract depicting the functional role of Ca2+-permeable TRP, SOCs and Piezo channels in the biological processes regulating tumor angiogenesis in presence of both chemical (oxidative stress and oxygen levels) and mechanical stimuli (ECM stiffness). SOCs store-operated Ca2+ channels, TRPA transient receptor potential ankyrin, TRPV transient receptor potential vanilloid, TRPC transient receptor potential canonical, TRPM transient receptor potential melastatin, TRPM transient receptor potential vanilloid, O2 oxygen, ECM extracellular matrix.

Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta.

An increase in intracellular Ca2+ concentration ([Ca2+]i) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The “Ca2+ add-back” protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.

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

Increased Vascular Contractility in Hypertension Results From Impaired Endothelial Calcium Signaling.

Endothelial cells line all blood vessels and are critical regulators of vascular tone. In hypertension, disruption of endothelial function alters the release of endothelial-derived vasoactive factors and results in increased vascular tone. Although the release of endothelial-derived vasodilators occurs in a Ca2+-dependent manner, little is known on how Ca2+ signaling is altered in hypertension. A key element to endothelial control of vascular tone is Ca2+ signals at specialized regions (myoendothelial projections) that connect endothelial cells and smooth muscle cells. This work describes disruption in the operation of this key Ca2+ signaling pathway in hypertension. We show that vascular reactivity to phenylephrine is increased in hypertensive (spontaneously hypertensive rat) when compared with normotensive (Wistar Kyoto) rats. Basal endothelial Ca2+ activity limits vascular contraction, but that Ca2+-dependent control is impaired in hypertension. When changes in endothelial Ca2+ levels are buffered, vascular contraction to phenylephrine increased, resulting in similar responses in normotension and hypertension. Local endothelial IP3(inositol trisphosphate)-mediated Ca2+ signals are smaller in amplitude, shorter in duration, occur less frequently, and arise from fewer sites in hypertension. Spatial control of endothelial Ca2+ signaling is also disrupted in hypertension: local Ca2+ signals occur further from myoendothelial projections in hypertension. The results demonstrate that the organization of local Ca2+ signaling circuits occurring at myoendothelial projections is disrupted in hypertension, giving rise to increased contractile responses.

Group 1 metabotropic glutamate receptors trigger glutamate-induced intracellular Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-D-aspartate receptors to mediate extracellular Ca2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca2+ response to glutamate was initiated by endogenous Ca2+ release from the endoplasmic reticulum and endolysosomal Ca2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca2+]i. These data demonstrate for the first time that glutamate may induce metabotropic Ca2+ signals in human brain microvascular endothelial cells. The Ca2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

Endothelial Ca2+ Signaling, Angiogenesis and Vasculogenesis: Just What It Takes to Make a Blood Vessel

It has long been known that endothelial Ca2+ signals drive angiogenesis by recruiting multiple Ca2+-sensitive decoders in response to pro-angiogenic cues, such as vascular endothelial growth factor, basic fibroblast growth factor, stromal derived factor-1α and angiopoietins. Recently, it was shown that intracellular Ca2+ signaling also drives vasculogenesis by stimulation proliferation, tube formation and neovessel formation in endothelial progenitor cells. Herein, we survey how growth factors, chemokines and angiogenic modulators use endothelial Ca2+ signaling to regulate angiogenesis and vasculogenesis. The endothelial Ca2+ response to pro-angiogenic cues may adopt different waveforms, ranging from Ca2+ transients or biphasic Ca2+ signals to repetitive Ca2+ oscillations, and is mainly driven by endogenous Ca2+ release through inositol-1,4,5-trisphosphate receptors and by store-operated Ca2+ entry through Orai1 channels. Lysosomal Ca2+ release through nicotinic acid adenine dinucleotide phosphate-gated two-pore channels is, however, emerging as a crucial pro-angiogenic pathway, which sustains intracellular Ca2+ mobilization. Understanding how endothelial Ca2+ signaling regulates angiogenesis and vasculogenesis could shed light on alternative strategies to induce therapeutic angiogenesis or interfere with the aberrant vascularization featuring cancer and intraocular disorders.

H2 O2 -induced microvessel barrier dysfunction: the interplay between reactive oxygen species, nitric oxide, and peroxynitrite.

Elevated H2O2 is implicated in many cardiovascular diseases. We previously demonstrated that H2O2‐induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO‐mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO‐derived peroxynitrite (ONOO−) in H2O2‐induced vascular barrier dysfunction by elucidating the interrelationships between H2O2‐induced NO, superoxide, ONOO−, and changes in endothelial [Ca2+]i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+]i, NO, and O2 − were assessed with fluorescence imaging. Perfusion of vessels with H2O2 (10 µmol/L) induced marked productions of NO and O2 −, resulting in extensive protein tyrosine nitration, a biomarker of ONOO−. The formation of ONOO− was abolished by inhibition of NOS with NG‐Methyl‐L‐arginine. Blocking NO production or scavenging ONOO− by uric acid prevented H2O2‐induced increases in endothelial [Ca2+]i and Lp. Additionally, the application of exogenous ONOO− to microvessels induced delayed and progressive increases in endothelial [Ca2+]i and microvessel Lp, a pattern similar to that observed in H2O2‐perfused vessels. Importantly, ONOO− caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO‐derived ONOO− is essential for H2O2‐induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self‐promoted amplifications of NO‐dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species‐mediated vascular dysfunction in cardiovascular diseases.

Human myometrial artery function and endothelial cell calcium signalling are reduced by obesity: Can this contribute to poor labour outcomes?

Determining how obesity affects function in human myometrial arteries, to help understand why childbirth has poor outcomes in obese women. Myometrial arteries were studied from 84 biopsies. Contraction (vasopressin and U-46619) and relaxation (carbachol, bradykinin, SNAP) was assessed using wire myography. eNOS activity was assessed using L-NAME. Cholesterol was reduced using methyl-β-cyclodextrin to determine whether it altered responses. Differences in endothelial cell intracellular Ca2+ signalling were assessed using confocal microscopy. The effects of BMI on relaxation were agonist specific and very marked; all vessels, irrespective of BMI, relaxed to bradykinin but 0% of vessels (0/13) from obese women relaxed to carbachol, compared to 59% (10/17) from normal weight women. Cholesterol-lowering drugs did not restore carbachol responses (n=6). All vessels, irrespective of BMI, relaxed when NO was directly released by SNAP (n=19). Inhibition of eNOS with L-NAME had a significant effect in normal but not overweight/obese vessels. Compared to bradykinin, a lower proportion of endothelial cells responded to carbachol and the amplitude of the calcium response was significantly less, in all vessels. Furthermore, a significantly lower proportion of endothelial cells responded to carbachol in the overweight/obese group compared to control. In contrast to relaxation, the effect of contractile agonists was unchanged with increasing BMI. The ability of human myometrial arteries to relax is significantly impaired with obesity, and our data suggest this is due to a deficit in endothelial calcium signalling. This inability to recover following compression during contractions, might contribute to poor labours in obese women.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow.

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.

Endothelial Calmodulin and CaMKII Play a Role in Leukocyte Transmigration

Inflammation results in the dilation of blood vessels and recruitment of circulating leukocytes into damaged tissues for repair. A transient increase in endothelial cytosolic free calcium (↑[Ca2+]i) is required for leukocyte transendothelial migration (TEM). We have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the calmodulin‐binding isoleucine‐glutamine (IQ) motif domain is critical for its function during TEM. Calmodulin (CaM) is an intracellular calcium‐binding protein, however, its role in TEM is undescribed. Therefore, we hypothesized that it is involved in the endothelial calcium signaling cascade essential for supporting leukocyte transmigration.In endothelial cells IQGAP1 knockdown reduced TEM substantially. Re‐expressing IQGAP1 truncation mutants containing both the actin‐binding and IQ‐motif domains rescued TEM but truncation mutants lacking the IQ motif domain but did not support TEM.Human endothelial cells treated with the calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Only 13% of leukocytes transmigrated through endothelial monolayers pretreated with 100 mM TFP compared to 84% TEM in control, untreated monolayers (p<0.05). We also looked at the role of calmodulin kinase II (CaMKII). Treating endothelial monolayers with 10 mM KN‐93, a CaMKII inhibitor, substantially reduced leukocyte TEM. Here 22% of leukocytes transmigrated which was significantly reduced compared to control TEM rates (p<0.05). Calmodulin pulldowns also showed that without the IQ‐motif domain IQGAP1 cannot substantially interact with calmodulin further suggesting a link between endothelial calcium, IQGAP1 and CaM.Current experiments focus on inhibiting CaM and CaMKII in endothelial cells using genetically encoded selective inhibitory peptides and inducible endothelial‐specific CaMKII‐deficient mice. Identifying the mechanism by which endothelial Ca2+ influx mediates TEM will provide novel insight into the signaling and also provide potential targets for anti‐inflammatory therapy.Support or Funding InformationR01 HL046849, R01 HL064774, F30 HL134202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alteration of Calcium and Electrical Dynamics in Cerebrovascular Endothelium During Development of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia characterized by a decline in cognitive function among the elderly. Currently, ~5.7 million Americans are living with AD and this number will increase to ~14 million by 2050. Age‐related AD alters blood flow to the brain and is associated with cerebral hypoperfusion, due in part, to impaired vascular endothelial function. However, it is unknown whether AD pathology impacts the biophysical functions of key cerebral vascular G‐protein‐coupled receptors (GPCRs) and K+ channels (KCa2.3 or SKCa, KCa3.1 or IKCa) that govern blood flow via an electrical vasodilatory pathway known as endothelium‐derived hyperpolarization (EDH). Thus, we tested the hypothesis that cerebrovascular endothelial purinergic receptors and Ca2+‐activated K+ channels become functionally dysregulated during progression of AD. We used the triple mutation mouse model of AD (3xTgAD) to examine endothelium isolated from posterior cerebral arteries in young control (YC; 1–2 mo), amyloid‐β plaques (Aβ; 6–8 mo), and plaques + neurofibrillary tangles (Aβ+Tau; ≥ 12 mo); n ≥ 3 male & n ≥ 3 female mice/group. Intracellular calcium concentration ([Ca2+]i) and membrane potential (Vm) were simultaneously measured using Fura‐2 photometry and sharp electrodes (pH 7.4, 37°C). EDH was demonstrated by an increase in [Ca2+]i (ΔF340/F380 ≥ 0.3) concomitant with ΔVm ≥ −10 mV in response to the P2Y agonist ATP (100 μM, 3 min). Membrane hyperpolarization to ATP during Aβ+Tau vs. YC decreased (ΔVm, mV; YC: −20±4, Aβ: −17±2, Aβ+Tau: −13±3) while [Ca2+]i responses were maintained. In contrast, hyperpolarization increased during Aβ and Aβ+Tau (ΔVm, mV; YC: −29±3, Aβ: −36±2, Aβ+Tau: −38±3) during direct activation of SKCa/IKCa alone with SKA‐31 (10 μM, 5 min). Finally, Δ[Ca2+]i and ΔVm responses to hydrogen peroxide (H2O2; 200 μM, 20 min) in young control animals were greater by > 40% as compared to Aβ & Aβ+Tau. These data suggest coupling of endothelial SKCa/IKCa function with Ca2+ signaling is reduced and adaptation to oxidative stress occurs during the development of AD. Altogether, endothelial K+ channel function may be directly calibrated for optimal cerebral blood flow to maintain a healthy brain with aging while helping to prevent neurodegenerative disease.Support or Funding InformationThis research is supported by Loma Linda University School of Medicine new faculty start‐up funds and National Institutes of Health grants R00AG047198 & R56AG062169 (EJB).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.