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.

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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Mg 2+ 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.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca concentration ([Ca ] ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca response to Ach was mediated by InsP R3, TPC1-2, and store-operated Ca entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca buffer. Moreover, the pharmacological blockade of the Ca response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca signal via M5-mAchRs.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the target cells, which activates the Ca 2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca 2+ ] i and NO production. The current study assessed whether and how glutamate drives Ca 2+dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca 2+ ] i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca 2+ oscillations were triggered by rhythmic endogenous Ca 2+ mobilization and maintained over time by extracellular Ca 2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca 2+ release was mediated by InsP 3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) J Cell Physiol. 2019;234:3538-3554. wileyonlinelibrary.com/journal/jcp 3538 | gated two-pore channel 1. Constitutive store-operated Ca 2+ entry mediated Ca 2+ entry during ongoing Ca 2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca 2+ signals. Of note, glutamate induced Ca 2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca 2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain. K E Y W O R D S Ca 2+ oscillations, endothelial cells, glutamate, neurovascular coupling (NVC), nitric oxide

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Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca 2+-releasing messenger that increases the intracellular Ca 2+ concentration by mobilizing the lysosomal Ca 2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca 2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca 2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca 2+ store and TPC1 in circulating ECFCs by harnessing Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca 2+ sequestration by acidic organelles, caused endogenous Ca 2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP 3)sensitive endoplasmic reticulum (ER) Ca 2+ pool. Likewise, the amount of ER releasable Ca 2+ was reduced by disrupting lysosomal Ca 2+ content. Liposomal delivery of NAADP induced a transient Ca 2+ signal that was abolished by disrupting the lysosomal Ca 2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca 2+ release also required ER-embedded InsP 3 receptors. Finally, NAADP-induced lysosomal Ca 2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca 2+ oscillations and proliferation, while it did not contribute to adenosine-5′trisphosphate-induced Ca 2+ signaling. These findings demonstrated that NAADPinduced TPC1-mediated Ca 2+ release can selectively be recruited to induce the Ca 2+ response to specific cues in circulating ECFCs.

It has long been known that the conditionally essential polyunsaturated arachidonic acid (AA) regulates cerebral blood flow (CBF) through its metabolites prostaglandin E2 and epoxyeicosatrienoic acid, which act on vascular smooth muscle cells and pericytes to vasorelax cerebral microvessels. However, AA may also elicit endothelial nitric oxide (NO) release through an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we adopted Ca2+ and NO imaging, combined with immunoblotting, to assess whether AA induces intracellular Ca2+ signals and NO release in the human brain microvascular endothelial cell line hCMEC/D3. AA caused a dose-dependent increase in [Ca2+]i that was mimicked by the not-metabolizable analogue, eicosatetraynoic acid. The Ca2+ response to AA was patterned by endoplasmic reticulum Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptors, lysosomal Ca2+ mobilization through two-pore channels 1 and 2 (TPC1-2), and extracellular Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4). In addition, AA-evoked Ca2+ signals resulted in robust NO release, but this signal was considerably delayed as compared to the accompanying Ca2+ wave and was essentially mediated by TPC1-2 and TRPV4. Overall, these data provide the first evidence that AA elicits Ca2+-dependent NO release from a human cerebrovascular endothelial cell line, but they seemingly rule out the possibility that this NO signal could acutely modulate neurovascular coupling.

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H 1 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. K E Y W O R D SCa 2+ oscillations, cerebral blood flow, H1 receptor, histamine, nitric oxide