Cracking the Endothelial Calcium (Ca2+) Code: A Matter of Timing and Spacing

Abstract

A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the main mechanism whereby vascular endothelial cells integrate the information conveyed by local and circulating cues. Herein, we describe the dynamics and spatial distribution of endothelial Ca²⁺ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca²⁺ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca²⁺ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca²⁺ signals. We further highlighted how distinct Ca²⁺ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.

Keywords: Ca2+ oscillations; Ca2+ pulsars; Ca2+ signaling; Ca2+ sparklets; Ca2+ wavelets; Ca2+ waves; angiogenesis; endothelial cells; myo-endothelial gap junctions; vasorelaxation.

Figure 1

Local Ca²⁺ signals in endothelial cells. Schematic representation of the wall of a microvessel presenting a myo−endothelial projection (MEP) that passes through the internal elastic lamina and establishes physical contact with the overlying vascular smooth muscle cells (VSMCs) in systemic resistance arterioles or pericytes at the arteriole-to-capillary transition zone. Local Ca²⁺ signals in endothelial cells (ECs) are produced by the opening of single InsP₃R or a cluster of InsP₃Rs in the ER, which, respectively, generate Ca²⁺ blips and Ca²⁺ puffs. The summation of spatially coupled Ca²⁺ puffs via the mechanism of Ca²⁺-induced Ca²⁺ release leads to global Ca²⁺ waves (not shown). Repetitive openings of smaller clusters of InsP₃Rs in ER cisternae protruding within MEP originate the Ca²⁺ pulsars. The basal production of InsP₃ is driven by tonic activation of PLCβ upon the activation of G_qPCRs located in the PM, such as muscarinic M3 receptors. ER Ca²⁺ refilling during repetitive Ca²⁺ puffs and Ca²⁺ pulsars is likely to be sustained by store-operated Ca²⁺ entry (SOCE), which is mediated by the interaction of the ER-resident Ca²⁺ sensor, STIM1, and one or more Ca²⁺-permeable channels on the PM, such as Orai1, TRPC1 and TRPC3. Local Ca²⁺ signals can be produced also by the activation of Ca²⁺−permeable channels on the PM, known as Ca²⁺ sparklets, including TRPV4, TRPV3 and TRPA1. Ca²⁺ sparklets are mainly coupled with SK_Ca/IK_Ca channels, thereby promoting endothelium-dependent hyperpolarization (EDH), which spreads to overlying VSMCs or pericytes to deactivate voltage-gated L-type Ca²⁺ channels and reduce contractility. The Ca²⁺-sensitive eNOS can also be present in systemic resistance arteries and is widely expressed in brain microcirculation. The Ca²⁺ source responsible for eNOS activation could be provided by the global increase in [Ca²⁺]_i, although the contribution of Ca²⁺ pulsars has also been postulated. In large conduit arteries and in brain microvascular endothelial cells, eNOS is mainly recruited by SOCE (not shown). Local and global InsP₃Rs-mediated Ca²⁺ signals could propagate toward adjacent endothelial cells via homo-cellular gap junctions (GJ), thereby generating intercellular Ca₂₊ waves.

Figure 2

Endothelial Ca²⁺ signaling controls CBF. Synaptic activity can stimulate endothelial G_qPCRs that induce PIP₂ hydrolysis, thereby promoting InsP₃-induced ER Ca²⁺ release and TRPV4-mediated Ca²⁺ entry at capillary level. Here, synaptic activity also results in the local accumulation of extracellular K⁺, which results in K_ir2.1-mediated endothelial hyperpolarization and amplifies extracellular Ca²⁺ entry. The overall increase in endothelial [Ca²⁺]_i stimulates eNOS to produce NO and induce vasorelaxation at the arteriole-to-capillary transition zone. Furthermore, neuronal activity can result in the local accumulation of extracellular ROS that gate brain capillary endothelial TRPA1-channels. TRPA1-mediated Ca²⁺ entry stimulates ATP release via Panx1 channels, thereby triggering an intercellular Ca²⁺ wave that is sustained by the activation of P2X receptors on adjacent endothelial cells. When the intercellular Ca²⁺ wave reaches the arteriole-to-capillary transition zone, it activates SK_Ca/IK_Ca channels and promotes endothelial-hyperpolarization. The negative shift in the V_M (−ΔVM; also known as endothelium-dependent hyperpolarization or EDH) spreads through hetero-cellular gap junctions to overlying pericytes, deactivates voltage-gated L-type Ca²⁺ channels and promotes the local increase in CBF. In parenchymal arterioles, synaptically released glutamate can also activate endothelial NMDARs, which engage eNOS and SK_Ca/IK_Ca channels to induce vasorelaxation.

Figure 3

Distinct pro-angiogenic Ca²⁺ signature induced by VEGF in vascular endothelial cells. VEGF elicits an increase in endothelial [Ca²⁺]_i by binding to VEGFR-2, a TKR that recruits PLCγ to induce ER Ca²⁺ release through InsP₃Rs. InsP₃-evoked ER Ca²⁺ mobilization may be supported by lysosomal Ca²⁺ efflux through TPCs (not shown) and is maintained over time by SOCE activation on the PM (not shown). High doses of VEGF elicit a biphasic increase in [Ca²⁺]_i that consists of a rapid Ca²⁺ peak followed by a sustained plateau phase and stimulates endothelial cell migration by activating myosin light chain kinase (MLCK). Low doses of VEGF elicit intracellular Ca²⁺ oscillations that promote endothelial cell proliferation by recruiting calcineurin and inducing the nuclear translocation of NFAT.