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.

An increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) controls virtually all endothelial cell functions and is, therefore, crucial to maintain cardiovascular homeostasis. An aberrant elevation in endothelial can indeed lead to severe cardiovascular disorders. Likewise, moderate amounts of reactive oxygen species (ROS) induce intracellular Ca²⁺ signals to regulate vascular functions, while excessive ROS production may exploit dysregulated Ca²⁺ dynamics to induce endothelial injury. Herein, we survey how ROS induce endothelial Ca²⁺ signals to regulate vascular functions and, vice versa, how aberrant ROS generation may exploit the Ca²⁺ handling machinery to promote endothelial dysfunction. ROS elicit endothelial Ca²⁺ signals by regulating inositol-1,4,5-trisphosphate receptors, sarco-endoplasmic reticulum Ca²⁺-ATPase 2B, two-pore channels, store-operated Ca²⁺ entry (SOCE), and multiple isoforms of transient receptor potential (TRP) channels. ROS-induced endothelial Ca²⁺ signals regulate endothelial permeability, angiogenesis, and generation of vasorelaxing mediators and can be exploited to induce therapeutic angiogenesis, rescue neurovascular coupling, and induce cancer regression. However, an increase in endothelial [Ca²⁺]_i induced by aberrant ROS formation may result in endothelial dysfunction, inflammatory diseases, metabolic disorders, and pulmonary artery hypertension. This information could pave the way to design alternative treatments to interfere with the life-threatening interconnection between endothelial ROS and Ca²⁺ signaling under multiple pathological conditions.

Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca²⁺ signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca²⁺ concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca²⁺-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.

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.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca²⁺ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABA_A receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABA_B receptors is still unknown. Preliminary evidence showed that GABA_A receptor stimulation can induce an increase in endothelial Ca²⁺ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca²⁺]_i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca²⁺ release from neutral and acidic Ca²⁺ stores, respectively, and sustained by store-operated Ca²⁺ entry. GABA_A and GABA_B receptors were both required to trigger the endothelial Ca²⁺ response. Unexpectedly, we found that the GABA_A receptors signal in a flux-independent manner via the metabotropic GABA_B receptors. Likewise, the full Ca²⁺ response to GABA_B receptors requires functional GABA_A receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.