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

Abstract

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.

Keywords: TRPC channels.; basic fibroblast growth factor; endothelial cells; endothelial colony forming cells; inositol-1,4,5-trisphosphate; nicotinic acid adenine dinucleotide phosphate; store-operated Ca2+ entry; stromal derived factor-1α; vascular endothelial growth factor.

Figure 1

The pro-angiogenic Ca²⁺ toolkit in vascular endothelial cells. Pro-angiogenic cues, such as growth factors and chemokines, bind to specific receptor tyrosine kinases (RTK) and G_q/11-protein Coupled Receptors (G_q/11PCR) thereby activating multiple phospholipase C (PLC) isoforms, which in turn cleave phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol-1,4,5-trisphosphate (InsP₃) and diacylglycerol (DAG). InsP₃ triggers Ca²⁺ release from the endoplasmic reticulum (ER) through InsP₃ receptors (InsP₃R), while DAG stimulates extracellular Ca²⁺ entry through TRPC3 and TRPC6. However, the major Ca²⁺-entry pathway in vascular endothelial cells is provided by store-operated Ca²⁺ entry (SOCE), which is mainly mediated by the physical interaction between STIM1 and Orai1. In addition, SOCE may be sustained by the interplay among STIM1, Transient Receptor Potential (TRP) Canonical 1 (TRPC1) and TRPC4, with [35] or without the involvement of Orai1. Endogenous Ca²⁺ release may also be sustained by ryanodine receptors (RyR, not shown) and by endolysosomal two-pore channel 1-2 (TPC1-2), which are gated by nicotinic acid adenine dinucleotide phosphate (NAADP). Multiple Ca²⁺-transporting systems maintain resting Ca²⁺ concentration and clear cytosolic Ca²⁺ after the pro-angiogenic signal. These include Sarco-Endoplasmic Reticulum Ca²⁺-ATPase 2a (SERCA2a), Plasma Membrane Ca²⁺-ATPase 1 (PMCA1) and PMCA4, and Na⁺/Ca²⁺ exchanger (NCX). Please, see the text for a more detailed description of how the endothelial Ca²⁺ toolkit is recruited by pro-angiogenic cues.

Figure 2

Ca²⁺-dependent activation of the extracellular signal-regulated kinase (ERK) pathway and of the Nuclear Factor of Activated T-cells (NFAT). VEGF binding to VEGFR2 triggers an increase in intracellular Ca²⁺ concentration that may stimulate the ERK1/2 phosphorylation cascade or NFAT nuclear translocation. VEGF-induced endothelial Ca²⁺ signals may be mediated by multiple Ca²⁺-entry/release pathways, depending on species and vascular bed. The Ca²⁺ response to VEGF may recruit the Ca²⁺-dependent PKCβ2 (cPKCβ2), which engages the downstream RAF1–MEK–ERK1/2 cascade to induce gene expression. An increase in [Ca²⁺]_i is required to promote cPKCβ2 translocation to the plasma membrane, where it is activated by DAG. Moreover, VEGF-induced endothelial Ca²⁺ signals may be sensed by calmodulin (CaM), which in turn activates calcineurin to dephosphorylate NFAT, thereby inducing its nuclear translocation. See Figure 1, Section 4.1 (ERK) and Section 4.3 (calcineurin and NFAT) for further details.

Figure 3

The Ca²⁺-dependent activation of Ca²⁺/Calmodulin (CaM)-dependent protein kinase 2 (CaMKII). Endothelial Ca²⁺ oscillations recruit CaMKII, which, in turns, stimulate angiogenesis by phosphorylating multiple targets, as widely illustrated in Section 4.4. VEGF-induced endothelial Ca²⁺ oscillations are sensed by CaM, which in turn stimulates CaMKII to phosphorylate multiple targets, including FAK to promote endothelial cell migration and Akt, JNK and Src to induce gene expression. The Ca²⁺ entry/release pathways that are recruited by VEGF to engage endothelial CaMKII are yet to be fully elucidated.

Figure 4

The Ca²⁺-dependent activation of the endothelial nitric oxide (NO) synthase (eNOS). VEGF binding to VEGFR2 causes an increase in intracellular Ca²⁺ concentration that displaces caveolin 1 (CaV1) from eNOS, thereby removing the tonic inhibition and inducing NO release. VEGF may impinge on several Ca²⁺ entry/release pathways to engage eNOS, including InsP₃R, TPC2 and Orai1. See Figure 1 and Section 4.6. for further details.

Figure 5

The Ca²⁺ toolkit in endothelial colony forming cells (ECFC). Growth factors, such as VEGF, and chemokines, such as stromal derived factor-1α (SDF-1α), bind to specific RTK and G_q/11PCR, thereby activating multiple PLC isoforms, which in turn cleave PIP₂ into InsP₃ and DAG. InsP₃ triggers ER-dependent Ca²⁺ release through InsP₃R, while DAG gates TRPC3 exclusively in umbilical cord blood-derived ECFCs. SOCE is the major Ca²⁺ entry pathway also in ECFC, in which it is mediated by the dynamic interplay among STIM1, Orai1 and TRPC1. Endogenous Ca²⁺ release is further supported by NAADP, which evokes EL Ca²⁺ release through TPC1. SERCA and PMCA contribute to maintain resting Ca²⁺ levels and clear cytosolic Ca²⁺ after a pro-angiogenic Ca²⁺ signal. Pro-angiogenic Ca²⁺ signals may also be delivered by TRP Vanilloid 1 (TRPV1) and TRPV4. See Section 5.2. (SDF-1α) and Section 5.3. (VEGF and NAADP) for further details.