Endothelial remodelling and intracellular calcium machinery

Francesco Mocciaa, Franco Tanzia and Luca Munaronb

aLaboratory of Physiology, Department of Biology and Biotechnology “Lazzaro Spallanzani”, via Forlanini 6, 27100 Pavia, Italy; bDepartment of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123.

Corresponding author:
Dr. Francesco Moccia
Laboratory of Physiology, 
Department of Biology and Biotechnology “Lazzaro Spallanzani”
University of Pavia, 
Via Forlanini 6, 27100, Pavia, Italy.
Tel: 0039 382 987169
Fax: 0039 382 987527 
E-mail: francesco.moccia@unipv.it

Running title: Ca2+ signalling in damaged endothelium

Abstract

Rather being an inert barrier between vessel lumen and surrounding tissues, vascular endothelium plays a key role in the maintenance of cardiovascular homeostasis. The de-endothelialization of blood vessels is regarded as the early event that results in the onset of severe vascular disorders, including atherosclerosis, acute myocardial infarction, brain stroke, and aortic aneurysm. Restoration of the endothelial lining may be accomplished by the activation of neighbouring endothelial cells (ECs) freed by contact inhibition and by circulating endothelial progenitor cells (EPCs). Intracellular Ca2+ signalling is essential to promote wound healing: however, the molecular underpinnings of the Ca2+ response to injury are yet to be fully elucidated. Similarly, the components of the Ca2+ toolkit that drive EPC incorporation into denuded vessels are far from being fully elucidated. The present review will survey the current knowledge on the role of Ca2+ signalling in endothelial repair and in EPC activation. We propose that endothelial regeneration might be boosted by intraluminal release of specific Ca2+ channel agonists or by gene transfer strategies aiming to enhance the expression of the most suitable Ca2+ channels at the wound site. In this view, connexin (Cx) channels/hemichannels and store-operated Ca2+ entry (SOCE) stand amid the most proper routes to therapeutically induce the regrowth of denuded vessels. Cx stimulation might trigger the proliferative and migratory behaviour of ECs facing the lesion site, whereas activation of SOCE is likely to favour EPC homing to the wounded vessel.

Introduction

The endothelium consists in a continuous monolayer of endothelial cells (ECs) lining the inside of all blood (arteries, capillaries and veins) and lymphatic vessels of vertebrates, thereby providing a selective permeable barrier between flowing blood and surrounding tissues. Rather than being a metabolically inert tissue, the vascular endothelium represents a highly dynamic and disseminated endocrine organ that maintains cardiovascular homeostasis by releasing a plethora of vasoactive substances, such as adenosine, gasotransmitters, prostanoids, endothelins, reactive oxygen species (ROS), C-natriuretic peptide, angiotensin II (Ang II), and thromboxan A2, in response to both chemical (autacoids, circulating hormones, growth factors and cytokines) and physical (changes in pulsatile pressure, shear stress, and pH) stimuli (1-5). By properly integrating the incoming inputs from the surrounding environment, a healthy endothelium serves as a signaling platform that governs platelet activation, leukocyte adhesion to the vascular wall, vascular tone, blood-tissue exchange of solutes and fluids, and angiogenesis (1, 3, 6). Moreover, cardiac microvascular ECs exert a paracrine function by modulating cardiac metabolism, growth, contractility, and rhythm by liberating diffusible messengers, such as nitric oxide (NO), endothelin-1, Ang II and prostacyclin (PGI2) (7). As a consequence, significant wounding of the endotheliual monolayer may tip the balance towards a pro-inflammatory, pro-coagulatory, hypertensive, and proliferative phenotype (1, 2). This occurs as vessel injury exposes the thrombogenic subendothelial matrix, thereby inducing the activation of the coagulation cascade (8-10). In addition, the endothelial monolayer prevents vascular smooth muscle cell (VSMC) proliferation and migration by releasing NO and prostacyclins (2). Consequently, endothelial denudation stimulates VSMCs to switch from a contractile to a synthetic, proliferative and migratory phenotype. This is the key event in the so-called process of “restenosis, which narrows vascular lumen and reduces blood supply to heart, brain and limb arteries (11-13). Endothelial denudation has, therefore, been regarded as a triggering (co)-factor in several cardiovascular diseases as severe as hypertension, atherosclerosis, myocardial infarction, brain stroke and vascular forms of renal failure (8, 14).
The integrity of endothelial monolayer may be compromised by either disturbed blood flow or pathological conditions (6, 10-12). In this view, atherosclerotic lesions are more frequent at sites of laminar flow separation, where high shear stress may promote endothelial cell detachment into circulating blood (6, 13). Similarly, the prolonged and/or repeated exposure of the endothelial monolayer to well known cardiovascular risk factors (i.e. hypertension, hyperlipidemia, hypercholesterolemia, smoking, and ageing) has a dramatic impact on vascular endothelium (1, 8, 15). It causes ECs to lose integrity, progress to senescence, undergo apoptosis and ultimately slough off the vessel wall into the bloodstream (8). As a consequence, the number of circulating ECs is increased in a wide variety of disease conditions, including inflammatory, immune, infectious, and cardiovascular pathologies (16, 17). Additionally, endothelial integrity may be disturbed by surgical procedures aiming at restoring blood flow through clogged feeding arteries, such as percutaneous transluminal angioplasty (PTCA), intracoronary stents and coronary artery bypass surgery (13, 18). These manoeuvres may destroy the intimal layer and, depending on lesion severity, lead to a renarrowing of the arterial lumen by triggering which is termed “in-stent restenosis” (ISR). The incidence of ISR may range from 20% within 6 months after bypass surgery up to 40% after PTCA (18). Drug-eluting stents (DES) may be placed during or immediately after angioplasty to reduce the rate of ISR (13). Polymer-based sirolimus (Cypher [Cordis, Miami Lakes, FL]) and paclitaxel (Taxus [Boston Scientific, Natick, MA]) DES significantly improved the clinical outcome of surgical interventions and maintained arterial patency better than bare-metal stents (BMC); however, both early and late (1 month-1 year post implantation) side effects, including thrombosis and severe allergic reactions, have been reported to limit the therapeutic efficacy of these medical devices (19, 20). Indeed, although designed to inhibit VSMC remodelling, paclitaxel may arrest EC motility, while both drugs interfere with endothelial proliferation (13, 18-20). The subsequent delay in endothelial regeneration favours platelet activation and increases the risk of arterial renarrowing (19-21). Notably, the work carried out by Prof. Finn’s Group has demonstrated that >50% of stent struts in humans are not covered by ECs up to 24 months after DES placement (22). Lastly, regenerated endothelium is altered both morphologically and functionally, as compared to the healthy monolayer, in patients undergoing stent deployment, a feature which contributes to the development of the so-called in-stent neoatherosclerosis (20). Intriguingly, the delay in endothelial growth is much more evident with DES rather than with BMS, although a number of pre-clinical studies have suggested that second-generation zotarolimus-eluting stents are less detrimental to regenerated ECs as compared to first-generation devices (20). It is, therefore, not surprising that novel approaches are crucial for clinicians, particularly interventional cardiologists, to achieve the early restoration of a fully competent endothelium in order to gain more effective long-term results after vascular regenerative surgery (13, 18, 19, 23). This therapeutic goal is further supported by the pioneering observation that the accelerated restitution of endothelial integrity and function dampens neointimal proliferation (23-26). Furthermore, for patients requiring bypass surgery, saphenous vein, internal mammary artery, internal thoracic, or radial artery are the grafts of choic;, however, 5-30% of patients have no suitable veins/arteries available due to previous use or diseased vein wall. The design of alternative strategies to boost vascular healing will benefit from the better understanding of the signal transduction pathways responsible for endothelial regeneration (5, 20, 23, 27, 28).
Endothelial regeneration requires the activation of mature ECs proximal and distal to the wound and released from contact inhibition. Healing ECs move into the denuded area and re-cover the innermost layer of the arterial wall, thus retarding or even suppressing neointima hyperplasia in the injured vessel (8, 9, 15, 17, 29). Moreover, endothelial progenitor cells (EPCs) may be mobilized from bone marrow and home to the lesioned artery, thereby contributing to restore endothelial integrity (17). EPCs are mobilized into circulation and recruited towards the lesioned vessel by a concentration gradient of cytokines (stromal derived factor-1, or SDF-1) and growth factors (mainly vascular endothelial growth factor, VEGF) released into the bloodstream by the target organ. Herein, they attach to residual basement membrane proteins and differentiate into mature and healthy ECs (17, 30, 31). It has now been clearly established that both EC and EPC activation finely tuned by an increase in intracellular Ca2+ concentration ([Ca2+]i) (4, 5, 27, 28, 32, 33). Intracellular Ca2+ signalling is central to wound healing; however, the mechanistic information on injury-induced intracellular Ca2+ waves in vascular endothelium is still lacking. Similarly, the molecular machinery that guides EPC engraftment into lesioned vessels is far from being fully elucidated. This review aims at summarizing current knowledge on the role served by Ca2+ signalling in promoting endothelial regeneration and in driving the EPC-dependent repair of blood vessels. The molecular machinery responsible for EC/EPC activation is likely to highlight alternative targets to design therapeutic interventions aiming at restoring endothelial function and treating cardiovascular pathologies.

The Ca2+ signalling machinery in vascular endothelial cells
Intracellular Ca2+ signals are a highly conserved mode for the control of EC survival, proliferation, motility, and differentiation (4, 32, 34-37). It is, therefore, not surprising that the control of cardiovascular homeostasis by vascular ECs is tightly driven by the spatio-temporal profile of the Ca2+ responses to extracellular agonists, such as growth factors, hormones, transmitters, and to physical stimuli, such as pulsatile blood flow, shear stress, swelling, and changes in environmental temperature (4, 5, 38). A detailed description of the Ca2+ machinery exploited by ECs to properly react to a given extracellular input is far beyond the goal of the present article. We refer the reader to a number of recent reviews from our research group which extensively address this issue and will describe the main features of Ca2+ signalling in vascular endothelium that are indispensable to understand the molecular underpinnings of the Ca2+ response to mechanical damage (4, 5, 36, 37). 

Spatio-temporal profile of Ca2+ signalling in vascular endothelial cells
Despite the great variability in human EC phenotypes, which is mainly due to the micro-environmental factors, the basic mechanisms that regulate the [Ca2+]i are the substantially conserved throughout the entire vascular tree. ECs respond to physical and chemical stimuli acting from the extracellular compartment by increasing the [Ca2+]i in a reversible mode, usually recovering the resting conditions upon agonist removal or even in its presence, through the active extrusion of the ion mediated by the Na+/Ca2+ exchanger (NCX) and by calcium pumps, such as the SarcoEndoplasmic Reticulum Ca2+-ATPase (SERCA) and the Plasma Membrane Ca2+-ATPase (5, 39, 40). The spatio-temporal profile of the Ca2+ signal encodes the information controlling the transcriptional response in cells undergoing an oscillatory change in [Ca2+]i. Rapid and infrequent Ca2+ oscillations may activate two different transcription factors, such as NFAT and NF-B, respectively. In addition, while oscillations in cytosolic Ca2+ induce NFAT nuclear import, an increase in nuclear Ca2+ concentration is required to regulate other transcriptional factors, such as CREB and DREAM (32, 34, 41).  In particular, a critical role is played by the specific intracellular distribution of highly localized calcium microdomains that engage differential patterns of Ca2+-sensitive decoders and finely tune specific cellular events (5, 32). For instance, InsP3Rs-mediated Ca2+ release stimulate nuclear accumulation of NFAT, whereas the same process is inhibited by RyRs activation (42) (see below for the definition of both InsP3Rs and RyRs). 

Ca2+ release from intracellular stores: inositol-1,4,5-trisphosphate receptors, ryanodine receptors and NAADP-activated channels
An increase in [Ca2+]i is shaped by the gating of Ca2+-permeable ion channels expressed in the plasma membrane and in the membranes of endoplasmic reticulum (ER), the main intracellular Ca2+ store. This is a passive event that follows the physiological electrochemical gradient for Ca2+. Calcium release from ER gives raise to a fast calcium spike of short duration (minutes), typically due to the increase in cytosolic levels of inositol-1,4,5-trisphosphate (InsP3), a diffusible and ubiquitous intracellular second messenger (5). InsP3 is the product of phosholipase C (PLC) recruitment upon activation of tyrosine kinase and G-protein coupled receptors (TKRs and GPCRs, respectively) involved in the response to proangiogenic and vasoactive agonists. More specifically, the -isoform is engaged by GPCRs, while the -isoenzyme is enlisted by TKRs (5, 34). An alternative route is the so-called Ca2+-induced Ca2+ release (CICR) in which the ER-associated calcium sensitive-ryanodine receptors (RyRs) are secondarily activated by a previous Ca2+ influx through the plasma membrane or Ca2+ mobilization from InsP3Rs (5, 34). Finally, it has recently been found that vascular ECs express two-pore channel nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, which release Ca2+ stored within lysosomal acidic stores, but not from the ER (5). NAADP-induced Ca2+ discharge might, in turn, be amplified by CICR-mediated recruitment of RyRs, albeit such a mechanism is yet to be unveiled in ECs (5). 

Ca2+ inflow across the plasma membrane: Transient Receptor Potential channels and Store-Operated Ca2+ Entry
Calcium entry from the extracellular environment usually triggers long lasting cytosolic Ca2+ signals (up to tens of minutes) of smaller amplitude compared to the Ca2+ release-related spikes. The concerted interaction between InsP3-dependent Ca2+ mobilization and Ca2+ inflow results in a either a biphasic Ca2+ signal, consisting of a rapid Ca2+ elevation followed by a plateau of intermediate magnitude, or repetitive oscillations in [Ca2+]i (5, 32). The best characterized endothelial plasma membrane calcium channels are voltage-independent, even if voltage-dependent calcium fluxes have been described in primary ECs (5). Vascular endothelium express a large number of transient receptor potential (TRP) channels, which serve as cation channels and, thereby, allow both Ca2+ influx into the cells and membrane depolarization (38, 43). Six subfamilies of TRP channels have hitherto been identified in mammals, based on the homology of their amino-acid sequence: TRPC (canonical; TRPC1-TRPC7), TRPV (vanilloid; TRPV1-TRPV6), TRPM (melastatin; TRPM1-TRPM8), TRPP (polycystin; TRPP1-TRPP3), TRPML (mucolipin; TRPML1-TRPML3), and TRPA (ankyrin; TRPA1) families (44, 45). Each TRP channel subunit consists of 6 transmembrane (TM) domains with a pore between the 5th and 6th TM domain and intracellularly located NH2 and COOH tails. The only notable exception is provided by TRPP1 which is made of 11 TM domains, a rather long and complex 3000 aminoacid extracellular segment, and a 6 TM TRP-like channel motif at the COOH-terminal. The latter may interact with the COOH-terminal of TRPP2 through a coiled-coil domain, thus forming a hetero-complex where TRPP1 functions as a Ca2+-permeable channel (44-46). Functional TRP channels, thought to be composed as either homo- or heterotetramers of four subunits, serve as non-selective cation channels, with a Ca2+ : Na+ permeability ratio (PCa/PNa) ranging from <1 for TRPM1 to >100 for TRPV5 and TRPV6 (44, 46). Ca2+ entry through these pathways is gated by a conformational change in the channel protein in response to membrane receptor activation, changes in temperature, depletion of intracellular Ca2+ stores, and direct binding of ligands, such as exogenous synthetic compounds, natural products, or second messengers, such as diacylglycerol (DAG) and arachidonic acid (AA), liberated upon binding of membrane receptors to their cognate ligands (vasoactive agonists and peptidic growth factors such as ATP, bradykinin, VEGF, bFGF and others) (5, 38, 43). For instance, members of either TRPC or vanilloid TRP (TRPV) sub-families are sensitive to second messengers synthesized by the phosphoinositide pathway, such as InsP3, DAG, and AA and its derivatives (5, 36). In this view, TRPC3 and/or TRPC6 are directly stimulated by DAG to induce angiogenesis in human microvascular endothelial cells (47, 48) and in human umbilical vein endothelial cells (HUVECs) challenged with VEGF (49). Moreover, TRPC3-, TRPC4- and TRPC5-dependent Ca2+ oscillations underlie HUVEC tubulogenesis when plated in Matrigel plugs and control cell proliferation in vitro (50). Similarly, TRPV4 mediates the pro-angiogenic action exerted by AA on breast tumour endothelial cells (B-TECs) (51, 52) and is essential to drive the shear stress-induced growth of collateral vessels upon arterial occlusion in a number of vascular districts (53, 54). Conversely, TRPM7, which is gated upon a drop in intracellular Mg2+ levels, inhibits cell growth by arresting the cell cycle in G0/G1 and G2/M, so that its genetic suppression leads to an increase in EC proliferation (5, 36, 55). Other Ca2+ channels, called Orai1, physically interact with STIM1, a Ca2+-sensor localized in the ER membranes (56). When Ca2+ is released from ER, STIM1 is activated leading in turn to the recruitment of Orai1 and the consequent Ca2+ entry from the extracellular medium. This event, called store-operated calcium entry (SOCE), is particularly important to sustain endothelial proliferation, migration, gene expression, and NO synthesis (57-60). Briefly, STIM1, in turn, is a single-pass transmembrane protein endowed with two Ca2+-sensitive EF-hand motifs within the luminal NH2-tail: following InsP3-dependent Ca2+ release, Ca2+ dissociates from the canonical EF-hand domain (cEF), thereby stimulating STIM1 to oligomerize and translocate towards ER-plasma membrane junctions, termed puncta. Herein, STIM1 tethers and recruits Orai1 to induced SOCE (5, 36, 61). It should be pointed out that TRPC channels may also be gated by the emptying of the intracellular Ca2+ reservoir. In particular, TRPC1, TRPC3, and TRPC4 are linked to ER Ca2+ loading, although via a larger number of signal transduction pathways than Orai1. For instance, STIM1 itself may bind to TRPC1, TRPC4, and TRPC5 and determine their function as store-operated channels in a heterologous expression system (62, 63) as well as in naïve cells (50, 64). SOCE mediated by a STIM1-activated TRPC1/TRPC4 complex has been shown to control endothelial permeability in mice pulmonary artery ECs (65). Alternatively, TRPC1 may be recruited by the physical and functional interaction with either InsP3R2 or InsP3R3 and the following Ca2+ entry lead to the nuclear translocation of both NFAT and NF-kB (66-69). Finally, gasotransmitters, such as NO and hydrogen sulphide (H2S) are well known modulators of Ca2+ signalling in vascular endothelium, as recently reviewed in (3) and in (70). More specifically, nitrosylation of native TRPC5 upon purinergic stimulation evokes Ca2+ entry in cultured ECs, whereas TRPC1, TRPC4, TRPV1, TRPV3 and TRPV4 proteins have been shown to mediate NO-induced Ca2+ signals in heterologous expression systems (71). Either of these channels might underpin the spatially restricted pro-angiogenic Ca2+ microdomains observed within the submembranal space of bovine aortic ECs (72). Moreover, NO may inhibit endothelial SOCE and enhance the ER Ca2+ load upon SERCA stimulation (60). On the other hand, H2S may evoke Ca2+ entry in both B-TECs (73) and rat aortic endothelium (74), albeit the underlying signalling pathways are yet to be fully elucidated. It thus appears that a large repertoire of Ca2+-permeable channels is potentially available to ECs both on the plasma membrane and on the membrane enveloping the ER. It is conceivable that different routes may be selected by extracellular stimuli to enable one single ion, i.e. calcium, to regulate the distinct processes whereby ECs regenerate damaged vessels, namely proliferation, migration, and differentiation.

Mechanical scraping elicits an increase in intracellular Ca2+ concentration in neighbouring cells which is essential for tissue regeneration
Mechanical wounding has long been known to promote tissue healing by inducing an elevation in [Ca2+]i in nearby survivors (75) (76-79). Intracellular Ca2+ signals may be reliably elicited by scraping confluent monolayers with either sterile micropipette tips or a fresh 18-20 gauge needle, and have been observed in several cell types, including vascular ECs (75, 77, 80-86), rat alveolar type II cells (76), human urothelial cells (87), human and mouse corneal epithelial cells (78, 88), polarized hepatic cells (79), and rat aortic VSMCs (89). Before describing the current knowledge about the molecular underpinnings and functional role of injury-induced Ca2+ signals in vascular endothelium, it is worth of providing a brief description of the Ca2+ response to mechanical denudation in other cell models. 

Mechanisms of wound-induced intracellular Ca2+ signals in non-endothelial tissues
In non-endothelial preparations, wound-induce Ca2+ increase is not restricted to the lesion edge, but crosses cell boundaries and spreads out through multiple adjacent cells as a Ca2+ wave (76-79). The first row of cells nearby to the injury site experience either a single Ca2+ transient (76, 78, 88-90) or a biphasic signal (87), consisting in an initial Ca2+ peak followed by a plateau which rapidly decays to the baseline. Typically, the magnitude and/or the duration of the Ca2+ response drop off at increasing distance from the wound until elevation in [Ca2+]i can no longer be observed (76-79, 88, 89). The correlation between the extent of the local damage and the distance travelled by the Ca2+ wave has never been clearly investigated, although this type of analysis did not yield statistically relevant results in rat VSMCs (89), i.e. the propagation of the Ca2+ signal did not increase with the wound size. The dynamics of the Ca2+ signals elicited by mechanical wounding exhibit a tissue-dependent variability. For instance, the removal of a large path of cells (100 m wide and 4-6 mm long) triggers a Ca2+ signal that spreads more than 300 m away in the periphery in cultures of rat alveolar type II cells (76), while it only encompasses the first 10 rows of cells in the mouse mammary epithelial cell line 31EG4 (77). Similarly, the speed of Ca2+ wave propagation is not uniform across the different cell types analyzed, the average value ranging from around 10 m/sec in polarized hepatic cells (79) and rat alveolar type II cells (76) up to around 50 m/sec in human urothelial cells (87). The Ca2+ wave may decelerate on its way towards the outermost limit of the damaged culture, as described in rat alveolar type II cells (76) and human corneal epithelial cells (78). Table 1 summarizes the mean values of both the propagation rate and the length of the path traversed by the Ca2+ waves in injured monolayers. The molecular toolkit responsible for the onset and the propagation of the Ca2+ response to mechanical scraping is yet to be clearly decoded. It is, however, feasible to conclude that the Ca2+ signal arising at the wound edge may be mechanistically decoupled from the Ca2+ elevation detected in cells that are further back. Accordingly, the Ca2+ signal is generally absent or dramatically reduced in the cells directly facing the lesion area when the wound is carried out in the absence of extracellular Ca2+ (78, 87, 88). These findings hint at the activation of a stretch-sensitive Ca2+ channel as a consequence of the mechanical stress imposed to their plasma membrane. The molecular make-up of this pathway has not been thoroughly investigated; however, it has now widely accepted that mechanosensitive channels are likely to belong to the superfamily of TRP channels. In particular, an increasing number of TRP isoforms have recently been implicated in the sensing of mechanical stresses, such as osmotic pressure, membrane stretch and shear stress, in a variety of cell types (91, 92). More specifically, TRPC1, TRPC3, TRPC6, TRPM4, TRPM7, TRPN1, TRPA1, TRPY1, TRPP1, TRPP2, and notably, TRPV1, TPRV2 as well as TRPV4 may participate to cellular mechanosensing and mechanotransduction (91-93). Apart from vascular endothelium (see below), there is no hint at their involvement in the Ca2+ signal occurring at the wound edge in any of the preparations described above. A careful molecular and genetic analysis is, therefore, required to validate TRP channel contribution to injury-evoked Ca2+ influx into the cells directly facing the lesion area in non-endothelial tissues. As to the propagation of the Ca2+ wave outward across neighbouring cells, two main mechanisms have been proposed. According to the first, the Ca2+ signal is communicated to adjacent cells upon the release into the extracellular medium of one or more soluble factors, which in turn mobilize the InsP3-dependent intracellular Ca2+ reservoir (76, 78). This chain of events has been described in rat alveolar type II cells (76), rat VSMCs (89), human urothelial cells (87), and mouse (88) and human (78) corneal epithelial cells, where ATP released from injured cells stimulates purinergic P2Y2 receptors to trigger and propagate the Ca2+ elevation from the 2nd row of cells towards the periphery of the lesioned monolayer (88, 94, 95). This model does not imply the involvement of gap junctions-mediated intercellular communication, whose pharmacological blockade does not affect tissue excitation (78, 87). The intracellular Ca2+ discharge may be accompanied by an influx of Ca2+ that regulates the magnitude, speed and extent of the Ca2+ wave  (76). The membrane pathway responsible for this signal has never been addressed; it is, however, conceivable to attribute it to either a mechano-sensitive TRP channel or a Ca2+ channel activated upon depletion of the Ca2+ stores. Conversely, the propagation of injury-induced Ca2+ waves in hepatocytes requires both the cell-to-cell transmission of intracellular second messengers (i.e. InsP3 or Ca2+ itself) trough gap junctions and the release of trinucleotide messengers, such as ATP, in the extracellular space (79).

The role of wound-induced intracellular Ca2+ signals in non-endothelial tissues
The intracellular Ca2+ sweeps elicited by mechanical scraping accomplish the double functions of activating surviving cells at the wound edge and of transmitting local-damage signals to more distal sites. The propagation of the Ca2+ signal, in turn, enables the lesioned tissue to orchestrate the complex sequence of events, i.e. cell proliferation, spreading, and migration, that is mandatory to pursue the healing process (96). As such, the Ca2+-dependent signal transduction pathways recruited by the spreading wave are part of the intricate network of directional cues that drive tissue repair and regeneration and include: injury stimulation itself, chemotaxis, contact inhibition release, wound void, population pressure, stress/tensile forces, and electrical fields (97, 98). Accordingly, in human epithelial corneal cells, the genetic ablation of P2Y2 receptors prevented Src and ERK phosphorylation, thereby causing a dramatic decrease in migration rate and wound closure in a confluent monolayer of human corneal epithelial cells exposed to mechanical denudation (94). In addition, the same stimulus promoted cell adhesion and cytoskeletal reorganization at the trailing edge of motile cells by causing paxillin phosphorylation at Y31 and Y118 (99). At the same way, injury-induced Ca2+ waves stimulated VSMC replication and advancement into the wound space by stimulating Ca2+/Calmodulin (CaM) to phosphorylate ERK1/2 (89). Finally, in human urothelial cells, the sustained influx of Ca2+ observed in the cells immediately adjacent to the wound edge has been associated to their higher migration rate as compared to the cells further back in the monolayer, that undergo a brief InsP3-dependent Ca2+ release (87). This study was corroborated by the observation that, in mouse corneal epithelial cells, ERK1/2 phosphorylation is more sustained in proximity to the lesion site rather than in more peripheral cells (88). These latter findings suggested that the coordinated response to physical injury manifested by different areas of a wounded cell monolayer (i.e. migration at the damage site vs. proliferation in the innermost sheet) might specifically be dictated by either the signature of the Ca2+ signal, defined both by its amplitude and spatio-temporal profile, or the underlying machinery (i.e. stretch-sensitive Ca2+ channels in the cells proximal to the lesion vs. intracellular Ca2+ release channels at more distal sites) or both. In addition, based on the well known amplitude- and frequency-modulation of Ca2+ signals, the possibility that distinct Ca2+ sensors are selectively recruited by the decelerating Ca2+ waves (i.e. speed-sensitive decoders of the Ca2+ signal) emanated from the injury site towards the periphery of the tissue should not be ruled out. It is, however, evident that, whereas the components of the Ca2+ signalling toolkit involved in cell proliferation, spreading, and migration are under intense scrutiny (4, 32, 36, 100-104), the molecular underpinnings of the Ca2+ response to mechanical scraping still await to be fully elucidated. They represent a prospectively promising field of research for cell biologists, pharmacologists, and clinicians aiming at pursuing tissue repair and regeneration. Interestingly, a recent study demonstrated that PMCA4 reversibly relocated from the apical and lateral plasma membranes to the basal membranes of rabbit corneal epithelial cells adjacent to the wound edge during the healing process (105). Such a redistribution of one of the most important transporters extruding Ca2+ out of the cytosol might contribute to the delayed recovery of the Ca2+ increase induced by Ca2+ influx at the injury site (76-78, 87, 88).

Vascular endothelium as a suitable model to study the mechanisms of tissue regeneration and repair
Vascular endothelium has long provided the most suitable preparation to investigate the role of intracellular Ca2+ as mediator of tissue repair and regeneration. Classically, normal tissue repair progresses through coagulation, inflammation, proliferation and remodelling phases without complication resulting in wound closure (97). The formation of the haemostatic plug depends on the interaction between activated platelets and the components of the extracellular matrix, such as fibronectin and vitronectin, that are exposed to blood flow upon de-endothelialization. The formation of the fibrin clot is essential to cease hemorrhagic events and to pave the way for the subsequent migration of neighbouring ECs into the lesioned area. The subsequent infiltration of inflammatory cells, which are recruited to the injured vessels by the up-regulation of endothelial adhesion receptors, such as E-selectin, P-selectin, VCAM-1 and ICAM-1, is essential to clear wound debris, remove infectious agents, and stimulate local EC proliferation and tubulogenesis. Accordingly, both neutrophils and macrophages may release a number of pro-angiogenic growth factors and cytokines, such as VEGF, interleukin-8, transforming growth factor- (TGF-), TGF-, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF). During the proliferative phase, endothelial regeneration occurs to cover the wound surface with concomitant growth of granulation tissue to fill the wound space. Granulation tissue formation involves proliferation of fibroblasts, deposition of collagens and other extracellular matrices, and development of new blood vessels. Once the new tissue within the wound is formed, the remodelling phase begins to restore vessel structural integrity and functional competence (97). The intricate network of signalling pathways controlling the 4 phases of wound healing involve an increase in [Ca2+]i, which drives platelet aggregation (106), leukocyte activation and accumulation (107), EC proliferation and motility (5, 36), and VSMC remodelling (108). Relevant to the subject of our review, is the acute Ca2+ response evoked by mechanical scratching to ECs adjacent to the lesion area which triggers the proliferative and migratory phase of the process.

Intracellular Ca2+ signals in injured endothelial confluent monolayers: role and molecular mechanisms
The first observation of injury-evoked endothelial Ca2+ signals has been provided by Drumheller and Hubbell in 1991; in this seminal paper they described how single-cell wounds elicit a local elevation in [Ca2+]i in a confluent monolayer of human venous ECs (75). These authors hypothesized for the first time the involvement of intracellular Ca2+ signals in endothelial regeneration due to the well known role served by such an ion in cellular proliferation and motility (109, 110). Subsequently, it was shown that a relatively larger de-endothelialisation induces a propagating Ca2+ wave in calf pulmonary artery ECs (CPAE) (77), thereby activating the Ca2+-mediated events necessary to regenerate the lesioned monolayer (83-85). Surprisingly, this topic remained unexplored until recently, when three separate groups described the Ca2+ response to mechanical damage in rat aortic ECs (RAECs) (6, 80, 81), human umbilical vein ECs (HUVECs) (86), and mouse brain microvessel endothelial cells (bEnd3) (82). The first investigations on the spatio-temporal pattern of the elevation in [Ca2+]i evoked by wounding in surviving cells were rather descriptive and failed in analyzing the molecular machinery underlying the Ca2+ response; this feature severely dampened the quest for novel strategies to recapitulate endothelial structure and function. In the pioneering study that Drumheller and Hubbell conducted on confluent monolayers of human venous ECs, the mechanical disruption of a single cell induced surrounding cells to undergo an elevation in [Ca2+]i, that radially spread as a Ca2+ wave until 6-7 rows away from the lesion site (75). As a consequence, the loss of a single cell caused the Ca2+ activation of around 200 neighbouring ECs. The Ca2+ wave propagated by the denuded area was not supported by a regenerative mechanism, as implied by the decrease in both the time-to-peak (TTP) and maximum height of the Ca2+ signal arising at more distal sites (75). Accordingly, the latency of the Ca2+ response increased with increasing distances from the injury point. The intracellular Ca2+ peak returned to the baseline in about 1-2 minutes and was significantly dampened by removal of extracellular Ca2+ or by the addition of Ni2+, an unspecific inhibitor of Ca2+ inflow, or heptanol, which interferes with gap junctions (75). Intriguingly, in the view of the findings subsequently obtained in rat aortic endothelium, heptanol did not only affect the propagation of the Ca2+ excitation away from the lysed cells: it significantly attenuated injury-induced Ca2+ entry in the 1st row cells (75). However, the molecular identity of the Ca2+-conducting pathway recruited by the injury and sensitive to heptanol was still unknown at this stage. Subsequent studies on CPAE focussed on the Ca2+ signal induced by larger endothelial denudation, which was obtained out by scratching a 100-200 m wide and 1 cm long swath from a confluent monolayer with a hypodermic 18 Gauge needle (77). The authors nicely demonstrated that the cells placed beyond the 1st row survived the injury and were viable since they were not able to ingest membrane-impermeable dyes, such as trypan blue, propidium iodide and rhodamine dextran (77). Conversely, the cells immediately adjacent to the lesion were irreversibly damaged and were discarded from following analysis. The resulting Ca2+ wave spread out with a constant rate of 28 m/sec and travelled from the 2nd up to the 12th row of cells away from the wound (77). Similar to human venous ECs (75), both the TTP and the magnitude of the Ca2+ mobilization were significantly dampened at increasing distance from the wound edge (77). The Ca2+ signal adopted biphasic kinetics, whereas the initial Ca2+ peak rapidly decayed to a plateau level within 1 min and then recovered to pre-wounding levels within further 8  min. The Ca2+ response described in injured CPAE confluent monolayers was shaped by the concerted interaction between extracellular Ca2+ influx and Ca2+ release from intracellular InsP3Rs (77). Indeed, the Ca2+ wave still occurred in the absence of extracellular Ca2+ entry, but its magnitude, duration, range, and speed were significantly abated. The propagation of the Ca2+ elevation throughout the monolayer was mediated by an unidentified soluble factor released from damaged cells, while it did not impinge on gap junctional communication (77). The signalling transmitter responsible for Ca2+ wave propagation in CPAE might be provided by ATP, as described in HUVECs (86) and rat aortic endothelium (80, 111). Importantly, injury-induced Ca2+ entry promoted the resealing of the endothelial surface by stimulating surviving ECs to move into the denuded area and proliferate. In mode detail, the spreading Ca2+ wave enrolled CaM to stimulate the rapid (within minutes) expression of the two immediate early genes, c-fos and c-jun, which reached their peak at 2 hours post-injury (84). Subsequently, c-fos triggered the transcriptional programme responsible for the synthesis of the molecular machinery supporting EC movement and wound closure. Notably, c-fos is preferentially expressed at the leading edge of migrating cells in lesioned endothelial monolayers (112). Moreover, flickering high-Ca2+ microdomains have recently been detected at the front of migrating fibroblast, where they steer directional cell movement (113). It was observed that an average rate of 24 m/h was sufficient to recover the scraped area in about 3 hours (84, 85). At the same time, the Ca2+ sweep evoked by the mechanical trauma orchestrated the intricate sequence of changes in cell in cell shape, actin polymerization, and cell-substrate adhesion generating the net traction force that drives EC advancement (85). In particular, the strength of the adhesive contacts to the underlying substrate was tightly regulated by v3 integrin (or vitronectin receptor) clustering along the leading edge of moving cells (85). v3 integrins, in turn, enable ECs to interact with the surrounding environment by mediating the molecular link between the extracellular matrix and intracellular actin cytoskeleton and signalling proteins (114). In addition to promoting EC movement, the spreading Ca2+ wave triggered the second physiological event which is essential to regenerate larger endothelial wounds, i.e. CPAE replication (83). This process was first detectable at 18 hours post-injury and involves only the cells undergoing the elevation in [Ca2+]i, i.e. those that are located within the first 10-15 rows from the denuded zone (83). Although not experimentally tested, it is conceivable that secondary cascade of gene expression elicited by c-fos dictates the proliferative response to mechanical scraping as well. The pioneering findings on the Ca2+ response to injury in confluent CPAE monolayers were somehow conflicting with those described on HUVEC cultures. In this preparation, the injury-evoked Ca2+ wave spread for a longer distance (up to the 25th cell row after the lesion edge) and at a significantly lower average speed (about) 2 m/sec (86). Moreover, the propagation of the Ca2+ signal along the injured HUVEC culture required the interaction between paracrine signalling via extracellularly released messengers and gap-junction mediated intracellular communication. More specifically, the transmitter liberated by ruptured cells was identified as ATP (86). This finding concurs with the notion that ATP concentration may elevate up to 50-100 M nearby the plasma membrane of ECs adjacent to the wound area (115). Two pieces of observations supported the mechanistic conclusions on the spreading of the Ca2+ wave. First, the amplitude of the Ca2+ signal at the wound margin and the distance traversed by the Ca2+ elevation were dampened by suramine, a non specific blocker of purinergic P2Y receptors (86). Second, the pharmacological blockade of the gap junctions with oleamide significantly hindered both the magnitude and the speed of the Ca2+ sweep (86). Zhao and coworkers’ work on HUVECs was the first one to shed light on the molecular undepinnings of injury-evoked Ca2+ signals. They showed that the Ca2+ wave was generated by the interplay between Ca2+ entry and InsP3-dependent Ca2+ release. Unlike CPAE, Ca2+ inflow was essential to initiate the propagating Ca2+ sweep, albeit the molecular nature of the underlying channel was not identified (86). Indeed, Ca2+ inflow was required along with intracellular Ca2+ discharge to initiate endothelial regrowth in around 24 hours. Unfortunately, this study did not try to discriminate between the Ca2+-permeable pathways gating Ca2+ entry at the lesion edge (where it might be mediated by a mechano-sensitive channel) and in the cells further back in the monolayer (where it might be attributed to receptor-operated channels, such as ionotropic P2X receptors, store-operated channels or DAG-sensitive channels). Interestingly, the Ca2+ signal still arose in the presence of suramin, i.e. in the absence of purinergic membrane receptor stimulation. This feature strongly concurs with the hypothesis that Ca2+ entry at the wound margin is decoupled from this signal transduction pathway and might well be supported by a mechano-sensitive pathway. Finally, a recent study was carried out on cultured mouse brain microvessel bEnd3 cells to unveil the molecular make-up of injury-activated Ca2+-permeable pathway(s). This investigation provided the first evidence that two members of the TRP super-family, i.e. TRPC1 and TRPP1, mediated a rapid influx of Ca2+ in ECs exposed to a traumatic injury (82). This finding gains further relevance when considering that TRPC1 and TRPP2 may assemble as a novel heteromeric channel with a 2 : 2 subunit stoichiometry and an alternating subunit arrangement (116, 117). Future studies will have to assess whether TRPC1 and TRPP2 provide the pathway for Ca2+ entry to occur also in ECs belonging to different vascular beds, such as CPAE and HUVECs. In this view, the involvement of TRPM7, the stretch-sensitive channel mediating the Ca2+ flickers that guide fibroblasts towards a chemoattractant source (113), in the Ca2+ response to endothelial injury should also be evaluated. 
Only little mechanistic information is, therefore, available regarding the signalling machinery underlying the Ca2+ response to mechanical scraping in cultured endothelial monolayers: 1) TRPC1 and TRPP2, which serve as the mechanosensitive channel activated by injury at the lesion site and, perhaps, in more remote areas of the tissue; 2) P2Y receptors, which prime the Ca2+ wave propagating from the 2nd row of cells towards the innermost periphery of the lesioned monolayer; 3) InsP3Rs, which mobilize the Ca2+ reservoir to permit the intercellular transmission of the Ca2+ sweep to the outermost tissue; 3) gap junctions, which may sustain the cell-to-cell communication of the Ca2+ signal. Our knowledge of the Ca2+ toolkit machinery that might be harnessed to regenerate the endothelial function lacks several key components. Which are the P2Y receptor subtypes binding to injury-derived ATP? Are ryanodine receptors involved in the transmission of InsP3-activated Ca2+ mobilization? Is SOCE, that is regarded among the most important pathways for Ca2+ influx into vascular endothelium, involved in the Ca2+ response to wounding? Is the acute increase in [Ca2+]i responsible for the up-regulation of the adhesion receptors that drive leukocyte homing to the injured vessels? In this view, it has long been known that the gene expression of E-selectin ICAM-1 and VCAM-1 is controlled by the Ca2+-dependent transcription factor, NF-kB (118). Are the information provided by ECs isolated from a specific vascular district useful to accomplish the regeneration of a different vascular area? This feature should be taken in account when considering that CPAE typically require paracrine cell-to-cell communication, while HUVECs also impinge on the direct transfer of the Ca2+ wave between two adjacent cells. The diversity in endothelial signalling along the vascular tree is, however, not surprising, as it has long been known that ECs display a remarkable heterogeneity in structure and function not only between different organs, but even between different branches of the vascular network within the same organ (119, 120). It is now established that in vitro and in vivo ECs present with important phenotypic differences when analyzing their blend of ion channels and transporters. This feature renders it necessary to compare the results obtained from cultured cell monolayers with those provided by in situ preparations (33, 121), such as rat aortic rings, which integrate the advantages of both in vitro and in vivo preparations (122).

The importance of Ca2+ influx in endothelial regeneration
Unveiling the molecular nature of the membrane pathway gating Ca2+ influx into injured endothelium is an imperative challenge to utilize Ca2+ signals for therapeutic purposes. Endothelial healing is significantly delayed when extracellular Ca2+ is prevented by either ion removal from the perfusate or by unspecific blockers of Ca2+ inflow (83, 85, 86). Accordingly, Ca2+ inflow has long been known to control cell proliferation and motility, which are two essential mechanisms for tissue regeneration (4, 5, 27, 36, 110). As earlier outlined, Ca2+ entry generates local Ca2+ microdomains which may selectively engage the Ca2+-sensitive decoders mediating the cellular response to Ca2+ signals (123). For instance, an increase in submembranal Ca2+ levels may lead to NO production by recruiting the endothelial NO synthase (eNOS) (124), thereby stimulating the angiogenic process (3). Moreover, microdomains of elevated Ca2+ nearby the inner mouth of plasmalemmal channels control the cell division cycle and stimulate DNA synthesis by promoting G0 to G1 and G1 to S phase transitions (125). Accordingly, Ca2+ influx may specifically bind to and activate CaM, which in turn modulates cell cycle machinery by controlling cyclins and cyclin-dependent kinases (cdk) (102, 125). In addition, a local Ca2+ elevation may promote centrosome duplication and separation in mitotic cells (102). Finally, high localized Ca2+ fluctuations control lamellipodia retraction and focal adhesion by enlisting the Ca2+-sensitive effector, m-calpain, that orchestrates the remodelling of actin cytoskeletal during EC migration (126, 127). Attention should be paid to the functional differences observed in regenerating endothelium between cells located at the wounding edge and those located at distance from it. Indeed, whereas the former both proliferate and advance into the lesioned area, the latter are not induced to migrate (29, 128, 129). It is conceivable that distinct Ca2+-conducting pathways are gated at different locations such that those opening in proximity of the wound are physically linked to the Ca2+-dependent machinery involved in cell motility (130). This hypothesis is physiologically corroborated by the evidence that activated cells are freed from contact inhibition and may divide and move over the exposed membrane basement. It turns out that elucidating the route responsible for Ca2+ entry into injured ECs might pave the way to device alternative treatments to favour endothelial repair.

Ca2+ signalling in “in situ” damaged endothelium: the unveiled role of connexin hemichannels
With the aim to understand at molecular level the nature of injury-evoked Ca2+ response in damaged endothelium, our group has recently benefited from a physiologically relevant preparation, such as excised rat aorta (27, 80, 81). Measuring [Ca2+]i in in situ endothelium is crucial to decipher the molecular underpinnings of Ca2+ fluctuations in nearby the wound edge, as enzymatic and culturing protocols alter the endothelial expression of cell surface receptors and ion channels (5, 27, 33, 121). A remarkable example is provided by the absence of inward rectifying K+ channels, which set the resting membrane potential (Vm) at around -60 mV in rat aortic endothelium (131), in cultured rat cardiac microvascular ECs (CMECs). Accordingly, CMECs exhibit a more positive Vm of around -30 mV (132). Moreover, in vitro ECs are featured by a decrease in the expression of the surface M3 receptor (133), as well as of type 3 RyRs (134). Finally, studies on intact tissue have found that ectonucleotidase activity is higher as respect to in vitro cell monolayers. The Ca2+ response to 20 M ATP lacks an evident plateau, but quickly decays to the baseline (111). Conversely, even lower concentrations (i.e. 5 M) of the nucleotide induce a biphasic Ca2+ signal in CPAE monolayers (60). This difference is attributable to the high ectonucleotidase activity degrading ATP to ADP, AMP and adenosine on the luminal side of the arterial vessel, thereby dampening the elevation in [Ca2+]i (111).
The procedure that we adopted for mechanical scraping, i.e. scraping with a micropipette tip one-to-two rows of cells (80, 81), mimics the focal regions of spontaneous injury and EC detachment from the vascular wall that have been previously described in the endothelium of rat aorta (135, 136). These denuded areas are rapidly recovered by neighbouring ECs, which spread into the lesioned area and undergo mitosis. Accordingly, the daily rate of EC replication is increased by 50% at these sites (136). Mechanical wounding of an excised rat aortic ring caused surviving cells to experience an immediate elevation in [Ca2+]i, which mainly reflected Ca2+ release from intracellular stores and was followed by a long-lasting decay phase supported by extracellular Ca2+ influx (80). This pattern was detected in a high percentage (about 75%) of cells facing the wound. The remaining 25% of cells manifested asynchronous oscillations in [Ca2+]i  that superimposed the recovery to the baseline and augmented the heterogeneity in the Ca2+ response to injury (81). Interestingly, whereas the monophasic signal was always propagated to neighbouring cells and did not travelled farther than the 3rd to 4th row of cells, [Ca2+]i oscillations were restricted to the lesion site. The hallmark of the Ca2+ response to mechanical scraping in rat aortic endothelium was the sustained decay to the resting Ca2+ levels which could last 1 hour in our longest recordings (80, 81). Therefore, injury-induced Ca2+ movements display different spatio-temporal features between in vitro and in situ endothelial monolayers. First, repetitive Ca2+ spikes have never been illustrated in cultured ECs exposed to a significant wounding. Second, mechanical scraping triggers a brief (1-2 min) increase in [Ca2+]i which travel a long way (up row 12) from the lesion site in in vitro confluent monolayers (75, 77, 86), while the Ca2+ signal is significantly longer and confined to the wound edge in in situ tissues. As a consequence, the Ca2+ signal elicited by injury in blood vessels in vivo might be more spatially restricted (i.e. in close proximity of the lesion) and persistent than we previously speculated. The spatial limitation of the Ca2+ wave might prevent more distant cells from being stimulated and utilizing undemanded metabolic energy; conversely, they could later provide the cells at the lesion margin with either ATP or other pro-survival molecules, such as glutathione, via gap junctional communication (137). Moreover, the long-lasting Ca2+ entry concurs with the central role fulfilled by extracellular Ca2+ in promoting endothelial repair. The initial increase in [Ca2+]i mainly depends on the activation of P2Y receptors, which leads to PLC recruitment. A careful pharmacological analysis unveiled that ATP liberated at the wound edge could stimulate rat aortic ECs (RAECs) by binding to P2Y1, P2Y2 and P2Y12 receptors, which are widely expressed in vascular endothelium and trigger InsP3-induced Ca2+ release (138). We hypothesize that the spatial limitation of the Ca2+ signal at the lesion site is due to the high ectonucleotidase activity found in rat aortic rings (80, 111). Likewise, when the hydrolysis of extracellular ATP is pharmacologically abrogated by ectonucleotidase inhibitors, the conduction of flow-induced vasodilation is dramatically attenuated in rat small mesenteric arteries (139). functional RyRs are not present in RAECs (unpublished data), so that the InsP3Rs-dependent Ca2+ mobilization cannot be amplified by CICR.  Despite the fact that mechanical damage causes the initial depletion of the InsP3-sensitive Ca2+ pool, the subsequent Ca2+ inflow is not contributed by store-operated channels. Accordingly, 30 min pretreatment with 20 M 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide (BTP-2/YM-58483), which is regarded among the most specific SOCE inhibitors (61, 140), prevented ATP-elicited Ca2+ inflow, while it did not affect the Ca2+ response to injury. This finding demonstrated that SOCE was not involved in the onset of the Ca2+ signal (80). Two further pieces of evidence suggested that the decay phase was underlain by a non-conventional membrane pathway. Both SK&F, a popular inhibitor of TRP channels (45), and Gd3+, which selectively targets mechano-sensitive channels, did not dampen Ca2+ entry into ECs adjacent to the lesion site. Surprisingly, injury-induced Ca2+ inflow  in rat aortic endothelium was suppressed by gap junction blockers, such as the aliphatic alcohols, octanol and heptanol, the fatty acids, oleamide and palmitoleic acid, and the trivalent cation, La3+ (80). These compounds inhibited both the prolonged decay-phase and the overlapping Ca2+ transients (80, 81): these latter findings hint at Ca2+ entry as the trigger of the repetitive Ca2+ spikes. Gap junction blockers do not only abrogate intercellular communication by reducing dye and current transfer across endothelial monolayers (141-143); these drugs have extensively been employed to interfere with uncoupled connexons (or connexin hemichannels) in vascular endothelium. Therefore, our pharmacological results indicated that Ca2+ entry into damaged endothelium was mediated by a connexin hemichannel (CxHc). This discovery adds to the growing repertoire of mechanistic differences observed between cultured ECs and those maintained in their physiological extracellular environment in the presence of mechanical wounding; indeed, as described above, the involvement of gap junctional channels has previously been described in terms of intercellular communication in in vitro monolayers, whereas they serve as Ca2+ influx routes in in situ preparations.

Connexin hemichannels: molecular structure and involvement in wound-induced Ca2+ entry
Gap junctions are specialized intercellular connections between the cytoplasm of two adjacent cells that allow the direct transfer of small molecules (up to 1 kDa; ATP, Ca2+, InsP3, glutamate) (144). A number of comprehensive review articles have recently detailed both the structure and functional role served by Cx within the cardiovascular system (145-148). Gap junction channels are formed by the end-to-end docking of two hemichannels (termed connexon), with each hemichannel constructed by a ring of six connexon monomers that contain four transmembrane domains and two extracellular peptide loops (143, 149). Recent evidence indicates that connexons may also remain as uncoupled hemichannels in the plasma membrane, thereby serving as an ion channel which presents a relatively high (up to 350 pS) conductance and mediate the movement of transmitters, second messengers and signal metabolites (150, 151). Thus far, 21 different Cx genes have been detected in humans and 20 in mice (144). Vascular endothelium mainly express Cx37, 40 and 43, depending on the vessel type and its location along the vascular tree (145). However, a variety of cultured human ECs and inferior vena cava ECs may also be endowed with Cx32 (152). When considering the role played by Cxs in endothelial repair, one should bear in mind that Cxs 37 and 40 are present in the lumen of rat aorta, while Cx43 is more abundant at vessel bifurcations that experience higher haemodynamic stress (153). Hemichannels exhibit a constitutive activity consisting of transient openings, which might be difficult to record during patch-clamp measurements (150). The open probability of CxHc in ECs, as well as other cell types, augments during metabolic inhibition (154), upon mechanical stimulation (155, 156) and after InsP3-dependent Ca2+ release (157). Consistently, Cx43-containing gap junctions underlie the propagation of pro-inflammatory Ca2+ sweeps in mouse lung capillaries (158) and support ATP-induced Ca2+ spikes in ovine uterine artery ECs (159). Moreover, Cx43Hcs gates Ca2+ influx and activates Ca2+-sensitive downstream decoders in mouse embryonic stem cells (160), Cx43-EGFP HeLa transfectants (90), neonatal cardiac myocytes and cardiac atrial cells (161), Cx26- and Cx43-expressing HeLa cells (161), and MDCK cells (162). Finally, both Cx37Hcs and Cx43Hcs underlie ATP-elicited Ca2+ influx in RBE4 (rat brain endothelial) cells and bovine brain capillary ECs (BBCECs) (163).
Classical gap junction blockers may interfere with both spontaneous and induced hemichannel openings and, as extensively illustrated above, suppress Ca2+ influx into lesioned endothelium (27, 80, 81, 141, 142, 150). The triggering role served by CxHc-mediated Ca2+ influx in the Ca2+ response to injury might depend by their sensitivity to physical stimulation. CxHcs are prompted to open in response to mechanical forces exerted on the plasma membrane by either the gentle contact with a glass micropipette (156) or laminar shear stress (155). Endothelial scraping causes a strong tensile deformation in the plasma membrane of cells facing the lesion site; this mechanism might gate the unapposed connexons sitting at the wound edge. However, mechanical singe-cell stimulation with a glass pipette did not trigger any detectable increase in [Ca2+]i in rat aortic endothelium (unpublished data). These results pointed towards an alternative scheme to understand CxHc signalling in rat aortic endothelium. We have hypothesized that de-endothelialization uncouples CxHcs located on surviving cells when they are grossly separated from their injured neighbours. These connexons adopt a close configuration, which renders them impermeable to both Fura-2 and ethidium bromide, which otherwise flow through fully open channels (80). Nevertheless, CxHc closure is not complete, such that a sustained Ca2+ influx may occur at the lesion site. This behaviour prevents the harmful consequences of persistent CxHcs opening (such as collapse of metabolic and ionic gradients across the plasma membrane). However, it endows the surviving cells with an alternative pathway which may recruit the Ca2+-sensitive effectors involved in endothelial healing. Accordingly, metabolic inhibition causes rat neonatal cardiomyocytes to undergo cell injury and death by inducing a massive Ca2+ entry through fully open Cx43Hc (164). This hypothesis has been corroborated by recent single-channel recordings of Cx43Hcs expressed in HeLa cells, which unveiled a sub-conductance state, termed the residual state (165). The latter represents a narrowing of the channel pore, which presents an inwardly-rectifying IV-relationship and is permeable by electrolytes, but not charged dyes. Ca2+ entry through unapposed connexons would be consistent with the spatial restriction of the Ca2+ wave to the lesion site, as ECs at more remote sites are not abruptly freed from their adjoining cells. Recent studies put forward pannexins, which are structurally unrelated to Cxs, as a molecular alternative to form membrane hemichannels. Pannexins comprise only three isoforms, called Px1 to Px3 (149), and may assembly into heteromeric conduits in the non-junctional area of cell membrane (149, 166). In astrocytes, for instance, Px1 mediates ATP efflux in the extracellular space, thereby stimulating P2Y receptors to increase [Ca2+]i in nearby cells (167). However, Px expression has been detected only in microvascular ECs, but not in macrovessels, such as aorta (168). Moreover, Px1 hemichannels are insensitive to La3+ and heptanol, which inhibit CxHcs and prevent injury-elicited Ca2+ influx in RAECs (80, 81, 169).

The role of connexin hemichannels in wound-induced intracellular Ca2+ oscillations in vascular endothelium
As mentioned above, CxHc-mediated Ca2+ influx ignites the oscillations in [Ca2+]i that overlap the prolonged decay phase in a fraction of cells nearby the lesion site in injured endothelium. The following observations permit to appreciate the physiological potential of these repetitive Ca2+ signals. First, as mentioned before, they do not arise in cultured endothelial monolayers. Second, intracellular Ca2+ oscillations carry the information required to initiate the genetic programme driving endothelial proliferation, migration and assembly into capillary-like tubulary structures (5, 170-172). Unlike the biphasic signal, injury-induced repetitive Ca2+ transients did not propagate in a 1 : 1 to ratio to neighbouring cells and randomly arose on both sides of the wound. Notably, the oscillatory response to injury impinged on the finely tuned interplay between NCX-mediated Ca2+ entry (3 Na+ out : 1 Ca2+ in) and the subsequent InsP3-dependent Ca2+ release (81). CxHcs are permeable to any ion and solute with a molecular weight lower that 1000 kDa and, therefore, gate Na+ influx into the cells. It is conceivable that, in oscillating cells, CxHcs are selectively linked to NCX, thereby generating a submembranal Na+ microdomain that turns the transporter into the backward mode. The following Ca2+ influx mayl, in turn, activate the Ca2+-sensitive PLC to synthesize InsP3, thereby driving the cell in and out of oscillations (81). The perculiar organization of the transporters (NCX), enzymes (PLC) and ion channels (InsP3Rs) required to initiate each oscillatory cycle might explain low fraction of spiking RAECs. PLC activation is not induced by either P2Y receptors or tyrosine kinase receptors. Therefore, we suggested that InsP3 is produced by PLC, which might directly be stimulated by the sustained Ca2+ inflow occurring in damaged endothelium (81, 173). The interaction between Cx43Hcs-dependent Ca2+ inflow and  Ca2+ release through InsP3Rs may occur in RBE4 cells, BBCECs, and MDCK cells (162, 163). The biphasic Ca2+ signal and the periodic Ca2+ fluctuations induced by injury at the wound edge may accomplish endothelial repair by activating distinct Ca2+-sensitive mediators. For instance, the sustained Ca2+ influx occurring during the decay phase is, perhaps, the most suitable signal to recruit eNOS in the vascular wall (3, 5, 32). Our preliminary observations show that mechanical scraping triggers an increase in NO levels in surviving RAECs; this signal is abrogated by the same gap junction blockers impairing the concomitant Ca2+ response. Endothelial-derived NO governs several processes which could protect the arterial vessel from EC disruption. For instance, NO exerts an anti-coagulation and anti-inflammatory action and might promote the subsequent processes of endothelial proliferation and migration (3, 32, 174). A delayed NO synthesis at the wound edge has been previously hypothesized, due to the increase in eNOS activity arising in regenerating endothelium at 48 hours from the scraping, but not experimentally probed due to the intrinsic difficulty of real-time NO measurements both in situ and in vivo (129). At the same time, intracellular Ca2+ oscillations could ignite a cascade of gene expression that outlasts the duration of the spiking signal and exerts a long-term impact on the healing process (123, 170).

Further evidences about the role of connexin hemichannels in endothelial regeneration
 The observation that CxHcs gate Ca2+ entry into ECs at the lesion site permits to read under a different light the well known contribution provided by Cxs to endothelial regrowth. The expression profile of Cx subtypes changes during the regeneration of injured endothelium (145, 175, 176). The expression of Cxs 37, 40 and 43 in rat carotid artery ECs is reduced by ballooning injury as compared to intact vessels. At 28 days after the lesion, Cx40 levels recover to the baseline, while Cxs 37 and 43 are over-expressed (177). These findings suggested that Cx could participate to blood vessel re-endothelization. Accordingly, wound repair is delayed when the dominant negative Cx inhibitors are expressed in in vitro bEnd.3 cells (178). This result is mimicked by the pharmacological blockade of gap junctional communication with 18-a-glycyrrhetinic (178). Again, bEnd.3 cell migration was featured by an increased expression of Cx43, but not Cx37 (178). The same feature has been reported in adrenal cortex-derived bovine microvascular ECs (179). The tight relationship between Cxs and endothelial regrowth is further underscored by the consequences of Cx43 ablation on ISR in vivo in hypercholeresterolemic mice subjected to carotid angioplasty (180). A recent investigation confirmed these data at single-cell by showing that siRNA-mediated Cx43 silencing affects human aortic EC proliferation, viability, and angiogenesis (181). In addition to regulating the acute response to mechanical damage, Cxs might contribute to maintain endothelial structure in presence of cardiovascular risk factors, such as hypercholesterolemia, obesity, hypertension and diabetes (145, 175, 182). Accordingly, a series of studies carried out on transgenic animal models lacking endothelial Cx isoforms gained the evidence that: 1) the surface of atherosclerotic lesions is larger in apolipoprotein-E knockout (KO) mice devoid of Cx40 display (145) and 2) the onset of atherosclerotic lesions in the thoracic-abdominal aorta and in the aortic sinus is accelerated by deleting Cx37 in apolipoprotein-E KO mice (183). Moreover, the profile of Cx expression is altered at branching sites, where atherosclerotic plaques normally develop in the absence of exogenous cardiovascular risk factors. Accordingly, Cx43 is highly localized at sites exposed to turbulent blood flow, such as ostia of bifurcating vessels and flow dividers, and vessel coartaction significantly enhances its expression after 8 days (153). Further experiments are necessary to unveil the impact of distinct Cx isoforms on endothelial regrowth. In this light, a major hurdle to our investigation is that we could not assess the molecular composition of the hemichannels mediating Ca2+ influx into injured rat aortic endothelium. Albeit the pharmacological profile of the Ca2+ response may be attributed to a gap junction/connexin pathway, it is worth of recalling that the classic gap junction blockers we employed may dissolve in the membrane lipid bilayer and alter general ionic permeability (184-186). Nevertheless, our preliminary experiments gained the evidence that, at least octanol, does not affect either InsP3-dependent Ca2+ release or Ca2+ buffering from the cytoplasm. Moreover, both heptanol and octanol have already been shown to act as selective gap junction blockers in rat aortic rings (89, 187). The following scenarios may be, therefore, envisaged to relate CxHc-dependent Ca2+ inflow with endothelial regeneration: 1) the Ca2+ permeability and the amplitude of Ca2+ entry into damaged endothelium are dictated by the Cx subtypes expressed by the tissue; 2) different Cx isoforms are selectively linked to distinct Ca2+-sensitive decoders, such as eNOS, CaM and adenylate cyclase (188); 3) endothelial Cxs assembly into hetero-oligomers, thus increasing their variability in Ca2+ permeability and the blend of nearby Ca2+-dependent mediators activated by Ca2+ influx.

Endothelial progenitor cells as an alernative tool to enhance endothelial regrowth
Endothelial progenitor cells (EPCs) are recruited from bone marrow upon vascular injury in order to repair damaged endothelium by physically lodging within the monolayer. In addition, they may stimulate local ECs to initiate the complex and multi-stepped process of angiogenesis via the paracrine release of a number of growth factors, including VEGF (17, 28, 61, 189, 190). EPCs are selectively directed to the target organ by various signalling molecules, such as VEGF and stromal derived factor-1 (SDF-1), that reach bone marrow after their release into peripheral circulation by injured tissues. Herein, VEGF stimulates stromal cells to increase eNOS expression and, consequently, NO synthesis, thereby leading to the secretion of several proteases, including matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), CD26, cathepsin G, and neutrophil elastase (191-193). These enzymes, in turn, cleave the adhesive bonds on the stromal cells that interact with integrins on EPCs and retain the latter in the bone marrow niche. Furthermore, MMP-9 converts membrane-bound Kit ligand (mKitL) to soluble Kit ligand (sKitL) favouring EPC release into circulation. EPCs then migrate by following the angiogenic gradient via chemokine receptors, such as CXCR-4 (which recognizes and binds to SDF-1) and VEGFR-2 (which mediates the pro-angiogenic effect of VEGF) (28, 191-193). Once homed to sites of arterial injury, bone marrow-derived EPCs adhere to the denuded extracellular matrix and replace the lost and damaged cells by differentiating into mature ECs (8, 28, 194, 195). Notably, EPC engraftment within the wounded area promotes vascular healing and inhibits neointimal proliferation and restenosis associated with percutaneous coronary intervention (23, 196, 197). Accordingly, a number of clinical trials (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth, HEALING; and Tri-stent Adjudication Study, TRIAS) have been carried out to demonstrate that EPCs-capturing stents accelerate the re-endothelialization of the damaged arterial segment and reduce the risk of neointimal hyperplasia and VSMC proliferation (195, 198). These procedures overcome the shortcomings intrinsic to the injection of autologous EPCs into peripheral circulation, i.e. 1) the amount of blood volume (12 L) required to isolate a therapeutically sufficient amount of EPCs (0.52.0 · 104 human EPCs/g body weight) (199); 2) the low percentage (10%) of EPCs retention and survival within the injury site (61, 200, 201); and 3) the differentiation fate of transplanted EPCs, which might acquire a phenotype other than the endothelial lineage (61, 201, 202). A host of hematopoietic and vascular EC subsets express the panel of surface antigens that have been attributed to EPCs (190, 203); moreover, a unique pattern of gene expression cannot be selectively associated to a given EPC subtype. A number of recent reviews have addressed the phenotypic and functional characterization of the three main populations of putative circulating EPCs, i.e. colony forming units-ECs (CFU-ECs), circulating angiogenic cells (CACs), and endothelial colony forming cells (ECFCs) (61, 190, 201, 203). Whereas CFU-ECs and CACs are rather pro-angiogenic cells and display a hematopoietic phenotype, ECFCs are considered truly endothelial. Indeed, they present all the typical surface antigens of the endothelial lineage (CD105, CD31, CD144, von Willebrand factor, VEGFR-2) but not the CD45 and CD14 antigens, thus ruling out the possibility that ECFCs emerge from the hematopoietic lineage. In addition, they display a great proliferative potential and form tubular lumenized structures both in vitro and in vivo. In particular, ECFCs stably integrate within the vasculature of the host animal, thereby permitting blood flow, when suspended in a collagen scaffold and transplanted into immunodeficient mice (61, 190, 201, 203). Therefore, ECFCs seem to represent the most suitable EPC subtype to repair lost ECs and that the regeneration rate of damaged arterial vessels might be significantly augmented by presenting a larger number of this cell population to EPC-capturing stents. Unfortunately, their frequency in peripheral blood is extremely rate (0.05-0.2 cells/ml). Therefore, the genetic manipulation of the signal transduction pathways controlling their proliferation and homing to the injured site might aid in improving their regenerating potential (61, 199, 201, 204). 

Intracellular Ca2+ signals drive EPC proliferation and recruitment from bone marrow
A growing body of evidence hinted at the Ca2+ machinery as an alternative molecular target to enhance the therapeutic outcome of ECFCs in cell-based therapy. A recent work investigated the role of the insulin-like growth factor 2 (IGF2)/IGF2 receptor (IGF2R) system in driving ECFC homing to the target organ. Local hypoxia may induce human ECFCs to secrete IGF2, which in turn autocrinally acts on its IGF2R to drive cell chemotaxis towards a VEGF, SDF-1, and IGF gradient and to increase cell adhesion to a fibronectin matrix (205). These in vitro experiments were corroborated by in vivo observations, according to which the IGF2/IGF2R system stimulates exogenously administered ECFCs to lodge into the ischemic area and give raise to postnatal vasculogenesis (205). Remarkably, both U73122, a widely employed PLC blocker, and BAPTA, an intracellular Ca2+ buffer, dampened IGF2-induced EPC chemotaxis and adhesion, a result which highlighted the pivotal role served by Ca2+ signalling in cell homing in vivo (205). Consistently, silencing the 2 isoform of PLC with small interference RNA (siRNA) reversed EPC adhesion to extracellular matrix, whereas pharmacological blockade of alternative signalling pathways, such as PI3K and mitogen-activated protein kinase kinase (MEK), did not exert any significant effect (205). These results clearly suggest a relevant role for calcium ions in promoting the formation of new blood vessels by ECFCs, but did not address the primary source of the underlying Ca2+ signal. Indeed, whereas InsP3 mobilizes Ca2+ from intracellular pools, the subsequent drop in ER Ca2+ levels leads to SOCE activation in mature ECs [5, 166-169]. In the aforementioned study, therefore, Ca2+ release as much as Ca2+ entry could be involved in ECFC migration and adhesion. As aforementioned, SOCE has been referred to as the predominant Ca2+ influx pathway in mature endothelium (5, 33, 56), where it regulates proliferation (57, 58), NO synthesis (124), gene transcription [169, 170], migration (206), and tubulogenesis (57). The central role served by SOCE in differentiated endothelium recently prompted us and other groups to investigate such pathway in human ECFCs isolated from peripheral blood. We have shown that both cell types are endowed with a SOCE pathway that is activated by emptying of InsP3-sensitive Ca2+ stores and is blocked by the selective inhibitors, YM-58483/BTP-2 and low micromolar doses of La3+ and Gd3+ (27, 61, 140, 201). Gene silencing experiments demonstrated that SOCE is abolished in ECFCs deficient for each one of the following proteins, STIM1, Orai1, and TRPC1 [168, 172]. In the absence of further data, this finding may be explained by two alternative mechanisms. First, the formation of a dynamic TRPC1-STIM1-Orai1 ternary complex, which is essential for SOCE activation, has been demonstrated in a number of cell types, including hematopoietic cells (64). In this context, Orai1 might mediate the communication between STIM1 and TRPC1 and render TRPC1 sensitive to store depletion (207). Alternatively, TRPC1 and Orai1 might constitute distinct store-operated Ca2+ channels, each featured by distinct biophysical properties (208): both are gated by STIM1 in response to store depletion and contribute to SOCE in the same cell. The critical step in the activation of TRPC1 is its insertion into the plasma membrane, which is governed by Orai1-mediated local Ca2+ entry (209). The physiological stimulus that causes the drop in ER content responsible for SOCE activation in human ECFCs is provided by VEGF (57, 210), which causes STIM1 re-arrangement into near plasma membrane puncta, thereby enabling it to bind to and gate both TRPC1 and Orai1 (57, 140). The interplay between InsP3-dependent Ca2+ release and SOCE leads to the onset of asynchronous Ca2+ waves that drive NF-kB translocation into the nucleus, where it triggers the transcriptional programme controlling ECFC proliferation and assembly into a bidimensional tubular network (210, 211). It should, however, be pointed out that ECFCs lack a number of DAG-sensitive Ca2+ permeable pathways that mediate proangiogenic Ca2+ entry in mature endothelium (5, 36), such as TRPC3 (47, 50) and TRPC6 [181, 182]. It is, therefore, likely that ECFCs are instructed to express such Ca2+ channels once incorporated within the target vasculature under the guidance of signals emitted by the local environment (119). This suggests that ECFCs present only the conductances that are vital to accomplish their regenerating purposes. In this light, it is worth of noting that they also express functional TRPV4, which operates as integrator of both mechanical (shear stress, cyclic strain, cell swelling) and chemical (arachidonic acid, 5’,6’-epoxyeicosatrienoic acid, phorbol esters) stimuli to sustain the sprouting of collateral vessels that occur in response to arterial occlusion (5). TRPV4-mediated Ca2+ influx regulates ECFC proliferation and tubulogenesis, albeit the Ca2+-sensitive decoders that translate this signal into a biological response are yet to be fully elucidated (Dragoni, Rosti, and Moccia, unpublished observations). It has been suggested that, similar to mature endothelium (54, 212, 213), TRPV4 impacts on ECFC behaviour by engaging one or more of the following Ca2+-sensitive transcription factors: NFAT, cytoplasmic, calcineurin-dependent 1 (NFATc1), Kv channel interacting protein 3, calsenilin (KCNIP3/CSEN/DREAM), and myocyte enhancer factor 2C (MEF2C). Future work will have to assess whether transducing ECFCs with any of these Ca2+ entry-related proteins, i.e. Stim1, Orai1, TRPC1, or TRPV4, will: 1) lead to an improvement of cell motility in vitro; 2) improve the percentage of cells incorporating within the ischemic tissue in vivo; 3) enhance the extent of blood perfusion and organ/tissue savage in vivo (28, 61).

The Ca2+ toolkit as a novel signalling pathway to promote endothelial remodelling and vascular healing
The regeneration of injured arterial vessels is accomplished by the sole activation of the ECs neighbouring the lesion site and/or by the accompanying mobilization of EPCs from bone marrow niches, depending on the extent of injury and on the endogenous capacity for repair, which may be impaired in presence of cardiovascular risk factors (8, 190, 194, 195). Importantly, the frequency and the proangiogenic properties of bone marrow-derived EPCs may be affected in subjects burdened by risk factors and atherosclerotic disease (202). The central role played by Ca2+ signalling in the activation of both cell types, i.e. mature ECs and circulating EPCs, renders the endothelial Ca2+ machinery a promising target to devise a more appropriate clinical treatment of patients undergoing vascular surgery. This proposal is corroborated by the experimental evidence that in vitro regeneration may be boosted by properly intervening on intracellular Ca2+ dynamics. Accordingly, the addition of platelet lysate, which contains a host of growth factors (bFGF, VEGF, platelet-derived growth factor, and transforming growth factor ) stimulates wound scratch closure in HUVECs and human dermal fibroblasts by inducing repetitive Ca2+ spikes, that in turn result in ERK1/2 activation [186, 187]. In addition, a faster wound healing rate has been measured in HUVECs transfected with PLC (173), i.e. the PLC isoform that might sustain [Ca2+]i oscillations in injured endothelium (81). Finally, the administration of EGF to mouse corneal epithelial cells sustains the repair process by igniting a Ca2+ wave that propagates well beyond the injury site and excites nearby cells (94), as depicted above (78). These latter findings are particularly intriguing when considering that: 1) therapeutic application of VEGF stimulates endothelial growth in denuded vessels and inhibits intimal proliferation in vivo (204, 214, 215); and 2) the proangiogenic effect of growth factors, such as VEGF, bFGF, and epidermal growth factor (EGF) is mediated by an increase in intracellular Ca2+ concentration in both mature endothelium (4, 5, 36, 72, 171, 216-219) and bone marrow-derived EPCs (57, 210). 

The pharmacological stimulation of connexin channels/hemichannels at the wound site 
In the first instance, the time course of healing might be accelerated by utilising stents that release drugs able to rapidly recruit the proangiogenic Ca2+ machinery in ECs immediately adjacent to the wound. As described above, a major limitation to this approach is currently represented by our scarce knowledge of the ion channels patterning injury-induced Ca2+ signals. In addition, most of the data available have been obtained in in vitro rather than in in situ or in vivo preparations, a feature which renders them less amenable of immediate clinical translation (see below). It should, however, be pointed out that all the studies hitherto carried out hint at the pivotal role served by gap junction channels/hemichannels in driving either the onset or the spreading of the Ca2+ wave in injured endothelium. In this view, Cx43, eNOS and von Willebrand factor are down-regulated in ECs growing onto different stent materials, such as those utilized to coat DES, and this feature is associated to an impairment in cell growth (220). The slower rate of endothelial proliferation in injured arteries may depend on the lower extent of Ca2+ entry through CxHcs at the lesion site (80, 81) and/or an impaired propagation of the Ca2+ signal throughout the lesioned endothelium (77, 86). This hypothesis is supported by the relation between the outcome of balloon injury and the distinct profiles of Cx expression, which led to conclude that Cxs could be targeted to adverse in stent restenosis in vivo (145, 175). A number of drugs have been synthesized to enhance gap junctional conductance: these compounds might be utilized to enhance Ca2+ signalling at the wound edge. The so-called hexapeptide, designated as AAP (antiarrhythmic peptide) and first discovered in bovine atria, improves intercellular coupling due to its ability increase gap junctional conductivity. The identification of its amino acid sequence (H2N-Gly-Pro-4Hyp-Gly-Ala-Gly-COOH) paved the way for the development of synthetic analogues which were obtained by exchanging single amino acids and generating propionyl derivatives, such as the lead compound AAP10 (H2N-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH2). A combination of two-dimensional nuclear magnetic resonance spectroscopy and mutational analysis disclosed a horseshoe-like bended conformation, which is due to the presence of proline and hydroxyproline and is essential for the normal functionality of this compound (221-223). Along with the semicyclic structure, the electron density in the tyrosine-benzene ring, van der Waals bonds and intramolecolar H-bonds were important for the antiarrhythmic activity (221, 222). AAP10 improves gap junctional conductance by binding to a membrane GPCR, thereby initiating a signalling intracellular cascade culminating in Cx43 phosphorylation by PKCα. It is worth of noting that AAP10 is more efficient when the target cells which are partially uncoupled, such as the ECs adjacent to the wound edge (221-223). AAP10 presents a short half-life in human plasma, therefore, it has been chemically modified into more resistant compounds, such as ZP123 or rotigaptide (H2N-Gly-D-Ala-Gly-D-4Hyp-D-Pro-D-Tyr-Ac). Rotigaptide is a rotation-inversion of AAP10 that incorporates unnatural D-amino acids to provide an increase in proteolitic stability and a reduced clearance in rat and human plasma. These features prompted the design of Phase I and II clinical trials that were carried out on patients suffering from atrial fibrillation, unstable angina or myocardial infarction (141, 221). Preliminary demonstrated the safety and tolerability of this treatment; however, AAP10 has been substituted by the first non-peptide analogue (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride (GAP-134) (221, 222). GAP-134 has been proposed as a novel and orally-active antiarrhythmic drug for the treatment of postoperative atrial fibrillation and has been advanced into clinical trials on November 2008 (http://clinicaltrials.gov/ct2/show/NCT00783341). An alternative pharmacophore of peptidomimetics have been developed by taking advantage from the core sequence “RXP” (arginine, any amino acid, proline) that binds to the COOH-terminal domain of Cx43 (Cx43CT). This strategy led to the synthesis of an antiarrhytmic drug containing a particular 34-amino acids peptide within this RXP series (termed RXP-E) and preventing the block of action potential propagation. This drug was indeed able to enhance the stability of the open state of cardiac gap junctions (224, 225). RXP-E  has a high molecular weight and is poorly permeable across the plasma membrane; subsequent studies focussed on the development of a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY, that bind to Cx43CT and prevent Cx43Hc closure (226). These novel compounds have a lower molecular weight as compared to previous Cx43CT-interacting drugs and CyRP-71 has been exploited as a template to produce ZP2519, i.e. the first RXP-derived peptidomimetic with preserved activity as gap junction opener (227). Therefore, pharmacologists and interventional cardiologists may therapeutically interfere with the propagation of Ca2+ signals in damaged endothelium. It is worth of noting that AA10 evokes [Ca2+]i oscillations in cultured rat ventricular myocytes (228). Gap junction channels/hemichannels might be stimulated to enhance Ca2+ activity in regenerating endothelium by luminal release of their specific agonist(s) through DES.

The therapeutic increase in the expression of connexin channels/hemichannels at the wound site 
An alternative strategy might pursue the increase in Cx levels at the injury site. Selective gene delivery might lead to the sustained expression of gap junctional proteins for therapeutic purposes in situ. For instance, local injection of adenoviruses encoding for Cx40 and Cx43 may maintain conduction velocity and prevent persistent atrial fibrillation (AF) in a porcine model of AF (229-231). Future studies will have to ascertain whether this strategy augments Cx expression by damaged endothelium in vivo, thereby enhancing the extent of propagation of the Ca2+ waves and accelerating vascular remodelling. 
In addition, the transcription of endothelial Cx-encoding genes might be pharmacologically promoted. For instance, statins, which are widely used as HMG-CoA-reductase inhibitors and include lovastatin, fluvastatin, simvastatin, and pravastatin, up-regulate Cx43 levels in HUVECs and restore Cx37 expression in hyperlipidemic mice (145, 175, 176, 223). This effect is independent on their ability to prevent cholesterol production by the liver by decreasing L-mevalonate availability. As pointed out in (223), it should be experimentally probed whether the increase in Cx levels at the wound edge would lead to their correct insertion within the plasma membrane. A plethora of hormones and cytokines may prime ECs to synthesize  Cx proteins. In more detail, endothelin-1 enhances the transcription of the gene encoding for Cx43 by activating the Gq/11-coupled ETA-receptors to generate DAG, which stimulates PKC to activate the MAPK (ERK1/2) pathway (223). Likewise, VEGF and bFGF may augment Cx43 expression, albeit the underlying mechanism is still unclear (232, 233). This finding gains particular relevance as VEGF and bFGF might further promote endothelial regrowth by activating ECs at the lesion edge and by herein attracting the rare circulating ECFCs. In other words, this treatment might engage both the main repair mechanisms of damaged endothelium, i.e. stimulation of local ECs and recruitment of bone marrow-derived EPCs. 

Growth factor delivery to promote EPC engraftment into injured vessels: is there a role for store-operated Ca2+ entry? 
VEGF is more attractive than bFGF to adverse vessel wall injury as the former selectively stimulates ECs and endothelial committed progenitors, while the latter might also induce VSMC remodelling (234). Consistently, therapeutic delivery of VEGF has long been known to accelerate re-endothelialization and dampen intimal thickening (234, 235). Unfortunately, the systemic administration of the high doses of recombinant VEGF required to achieve the biologically active concentration at the lesioned site causes severe thwarted effects, such as hypotension and oedema (236). As widely established in the literature (236), the ideal regimen for therapeutic restoration of endothelial integrity would be a single bolus providing a transient but sustained (2-3 weeks) focal elevation of the growth factor at injured vessels. A limited rise in systemic VEGF levels would be expected to reduce the potential for distant, unwanted side-effects. An elevation in the local levels of the growth factor may, therefore, be obtained by either indirect or ex vivo gene transfer into the arterial wall (214, 234). The first approach implies the genetic manipulation of ex vivo expanded EPCs, which are selectively targeted to the injured vessel, to overexpress the angiogenic growth factors before being injected into circulation (61, 204, 214). Accordingly, the transplantation of heterologous EPCs transduced with adenovirus encoding for VEGF was shown to rescue impaired neovascularisation in an animal model of limb ischaemia and amplify re-endothelialization, thereby inhibiting neointimal hyperplasia (237). Notably, the number of EPCs required to restore blood perfusion in these in vivo experiments was 30 times less than that required by employing unmodified EPCs (237). Future work should be devoted to assess whether the regenerative outcome of these treatments is somehow associated to an increase in Cx expression in injured endothelium and to the Ca2+-dependent activation of cells adjacent to the lesion site. It is noteworthy that regenerative therapy conducted with cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified to overexpress VEGF and platelet derived growth factor (PDGF) led to an increase in Cx43 expression in rat infarcted hearts (238). The second option utilizes the intra-arterial gene transfer of the VEGF165 (the most mitogenic among the four known VEGF isoforms) gene to pursue endothelial remodelling. Indeed, the local deliver of a plasmid encoding human VEGF165 may accelerate the development of an intact luminal endothelial monolayer and inhibit neointimal formation after vessel wall injury (214, 215, 239, 240). The feasibility of this approach to treat cardiovascular diseases has been shown by the Kuopio Angiogenesis Trial (KAT): VEGF gene transfer during angioplasty and stenting restored endothelial function and prevented restenosis in human subjects (241). A number of recent reviews has provided for an exhaustive description of the most popular strategies currently adopted for direct transfer of VEGF165 encoding genes or ex vivo transfection of stem cells (61, 242).
Needless to say, genetic strategies are by no means the only approach to cause a sustained elevation in VEGF levels in proximity of injured endothelium. Vascular grafts might be properly engineerized to let them release growth factors in such manner as to retain their pro-angiogenic properties in shear stress environments over a time lapse that can cover the denuded area. In particular, VEGF-eluting stents (VES) have been proposed as a promising strategy to boost vascular healing (21), albeit an earlier study failed to demonstrate their beneficial effect on either endothelialization or intimal hyperplasia (243). Nevertheless, they did reduce the stent thrombosis rate, which may make VES less thrombogenic and amenable for further investigation (243). The reason for the conflicting results achieved upon pharmacological or genetic delivery of VEGF remains to be elucidated, although more preclinical studies with VES are required. Nevertheless, differences in the local VEGF concentration and in the route of growth factor administration might easily be envisaged. The regenerative outcome of a strategy based on an increase in gap junctional communication associated to EPC recruitment at the lesion site by a focal increase in VEGF levels might be limited by the time required for Cx channels/hemichannels to be expressed on the plasma membrane of injured endothelium. Indeed, it is the acute Ca2+ response to mechanical wounding that activates the repair process, as widely discussed above. A more suitable approach to induce endothelial remodelling might require: 1) an increase in the open probability of Cx channels/hemichannels by delivering selective modulators of the isoform(s) expressed at the lesion site through the proper stent combined to 2) the injection of autologous ECFCs overexpressing SOCE to increase their sensitivity to chemoattractants (i.e. VEGF and SDF-1) released by ruptured cells. As recently suggested (61), we hypothesize that engineering the cells with Stim1 and/or Orai1, and/or TRPC1 before injection will enhance the resealing of the denuded artery. The nature and the exact stoichiometry ratio of the proteins to express will, however, depend on the full elucidation of the molecular make-up of SOCE in bone marrow-derived human ECFCs. Indeed, we will have to understand whether store-dependent Ca2+ inflow is mediated by two distinct Ca2+-permeable routes, namely Orai1 and TRPC1, each gated by Stim1, or by a ternary complex formed by each of the aforementioned proteins.

Potential shortcomings of endothelial regeneration as therapeutic strategy
The Ca2+ signalling toolkit appears as a novel therapeutic molecular target for vascular reconstructive strategies (28, 61). Our current information hints at gap junction channels/hemichannels as the most feasible signalling pathway to interfere with to enhance the extent of endothelial activation and consequent remodelling. In this context, it will be necessary to ascertain whether these two distinct events (i.e. engagement of Cx-based channels at the wound site and reendothelialization of damaged vessels) are mechanistically related. The cross-species differences existing between humans and the animal models harnessed to investigate endothelial remodelling might complicate the clinical translation of gap junction channels/hemichannels as therapeutic target. These results will have to be confirmed in in situ endothelium of human vessels, when available, or on freshly dissociated human ECs. A number of drugs, which are gaining a therapeutic interest in anti-arrhythmic therapies due to their ability to increase the open conductance of Cx43 channels, might be harnessed to treat damaged endothelium. It should, however, be pointed out that Cx43, which is the most abundant Cx isoform in myocardium, is far less present in the lumen of vascular tree. Indeed, Cx37 and Cx40 are normally co-expressed in ECs, whereas Cx43 is restricted at the branch points of elastic (aorta) and muscular (coronary) arteries (145, 175). Albeit a significant mechanical trauma may activate Cx43 transcription by surviving cells, the acute response is dominated by Cx37 and Cx43. This feature suggests that the latter isoforms might be the ideal target of an alternative therapy aiming at regenerating damaged endothelium. For instance, novel drugs could be designed to selectively bind to and enhance the conductance of Cx37- and/or Cx40-based channels. As recently outlined in (221), peptide-based drugs constitute the perfect template to start from to synthesize a specific Cx-directed compound. When keeping this in mind, caution should be taken when promoting gap-junctional communication in damaged vessels. Despite the evidence provided by our research Group about the requirement for Cx hemichannels in triggering the Ca2+ response to injury, a number of studies have shown that connexons provide the pathway for the entry of cell death or the loss of cell survival signals (244). At the same time, gap junctions could enable the long distance transfer of apoptotic signals to cells that are located far away from the stimulation point. As a consequence, the extent of tissue damage might be increased, rather than diminished, by therapeutic strategies aimed at stimulating Cx-based channels. However, gap-junctions may also enable the transfer of rescue signals, such as the energy molecules ATP and glucose or the free radical scavenger glutathione and ascorbic acid, from remote, healthy cells to ECs near the lesion edge, i.e. the so-called “Good Samaritan Effect” (221, 244).
The genetic manipulation of bone marrow-derived ECFCs presents a number of potentially relevant pitfalls, too (61). Indeed, it has recently been shown that  Orai1-mediated Ca2+ inflow promotes physiological postnatal cardiac growth upon the selective engagement of both Ca2+/CaM-dependent protein kinase II (CaMKII) and calcineurin A (245) and interferes with myocardial automaticity (246). These features make it indispensable to assess the long-term safety of treatments aiming at inoculating ECFCs engineerized to over-express Orai1 into patients undergoing PTCA. The repopulation of the injured coronary monolayer might, indeed, be associated to the subsequent perturbation of the normal heart rhythm or to cardiac hypertrophy.
Finally, an additional limitation to therapies aiming at adversing vessel injury concerns the functionality of regenerated endothelium. As recently reviewed by Prof. Vanhoutte (1, 15), the latter often fails in recapitulating the vasorelaxing features of the intact monolayer. For instance, NO-dependent vasodilation may be impaired after endothelial regrowth due to a decrease in NO synthesis and bioavailability (1, 15). Moreover, regenerated ECs display irregular sizes and polygonal shapes and are not aligned in the direction of blood flow (234). In this view, it is worth of noting that somatic gene transfer of VEGF165 after balloon injury restored endothelium-dependent vasomotor responses to acetylcholine stimulation due to an increase in NO production (240, 247, 248). These findings were corroborated by preclinical studies showing that VEGF165 gene delivery in patients with severe vascular disease significantly ameliorate the endothelial reactivity of lower extremity vessels (249).

Conclusions
Mechanical disruption of endothelial integrity upon medical interventions may lead to late ISR and limit the beneficial outcome of vascular regenerative surgery. Unveiling the signal transduction pathway that stimulates ECs at the lesion site to replicate and re-cover the lesioned area might open a new avenue in design of drug- and gene-eluting stents and improve the long-term treatment of cardiovascular disorders. The increase in [Ca2+]i observed within surviving cells might be central in recruiting the Ca2+-sensitive mediators of endothelial proliferation and motility and is spread to adjacent cells by both intracellular communication and paracrine signalling. These data have previously been interpreted in the light of the well known process of intercellular gap-junctional communication, which would serve to coordinate DNA synthesis and motility during repair of the endothelial sheet. The endothelium of excised rat aorta represents an ideal model to investigate the molecular underpinnings of the endothelial response to damage, since the cells reside in their native environment and are not exposed to enzymatic digestion. Recent work performed on such preparation suggested a role for Cx-based channels in mediating a route for Ca2+ entry in injured endothelium. Altogether, the key role served by gap junction channels/hemichannels in the Ca2+ response to injury shed new light on the notion that the rate of wound repair is reduced in ECs transfected with dominant negative Cx inhibitors. Moreover, these results agree with those provided by transgenic mice, where selective ablation of endothelial Cxs accelerates the onset of atherosclerotic lesions, which are associated to endothelial dysfunction. In addition to stimulating ECs that are located nearby to the wound edge, intracellular Ca2+ signals are involved in EPC recruitment from bone marrow. This event is essential to facilitate the regeneration process when the extent of vessel damage is far too large to be repaired by surviving cells. These evidences hint at the endothelial Ca2+ toolkit as a promising, yet hitherto underestimated, target to rapidly restore vessel integrity after PTCA. Future experiments are necessarily required: 1) to assess which is the most suitable combination of ion channels and cell types (such as, various Cx isoforms in the damaged vessel and Stim1/Orai1/TRPC1 in circulating EPCs) to exploit for therapeutic purposes and 2) to device the most proper means to interfere with channel activity and/or expression (for instance, local delivery of genes encoding for a given Cx isoform or VEGF and accompanying administration of ECFC transduced to overexpress SOCE).

FIGURE LEGENDS

Figure 1. Chemical structure of AAP (A), AAP10 (B), ZP-123 (C), and GAP-134 (D). These drugs serve as enhancers of gap junctional conductance and might, therefore, be exploited to promote the Ca2+-dependent regeneration of damaged endothelium. Modified from (221).

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