A current view of G protein-coupled receptor - mediated signaling in pulmonary hypertension: finding opportunities for therapeutic intervention

Pathological vascular remodeling is observed in various cardiovascular diseases including pulmonary hypertension (PH), a disease of unknown etiology that has been characterized by pulmonary artery vasoconstriction, right ventricular hypertrophy, vascular inflammation, and abnormal angiogenesis in pulmonary circulation. G protein-coupled receptors (GPCRs) are the largest family in the genome and widely expressed in cardiovascular system. They regulate all aspects of PH pathophysiology and represent therapeutic targets. We overview GPCRs function in vasoconstriction, vasodilation, vascular inflammation-driven remodeling and describe signaling cross talk between GPCR, inflammatory cytokines, and growth factors. Overall, the goal of this review is to emphasize the importance of GPCRs as critical signal transducers and targets for drug development in PH.

Vasodilator GPCRs that increase cAMP may also activate cAMP-binding domain in exchange factor EPAC1, a GEF for the small molecular weight G-protein Rap1, a member of Ras superfamily. Rap1 activates ARAP3, a Rho GAP, which in turn, inhibits RhoA, leading to reduced MLC phosphorylation and vasodilation [24,25] . Vasodilation also occurs via endothelial cell (EC)-dependent production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS), which is activated by Akt or ERK1/2 by phosphorylation on Ser 1177 residue [26] . Highly permeable NO readily enters VSMC, stimulates soluble guanylate cyclase (sGC) and activates cGMP-PKG, antagonizing Ca 2+ action on phosphoSer 19 -MLC and promoting vasodilation. More specifically, NO-sGC-cGMP-PKG-axis inhibits Ca 2+ increase by stimulating TRPC6 phosphorylation at Thr 69 , decreasing ROCE and increasing vasodilation [27] . PKG phosphorylates and activates RGS2, and RGS4, that leads to the inhibition of G i /G q ,-rergulated PLC activity and termination of the vasoconstrictor Ca 2+ signal [23] . Both PKG and PKA phosphorylate and inhibit RhoA and increase the activity of myosin light chain phosphatase (MLCP), thereby decreasing MLC contraction [28,29] . MLCP is also activated by vasodilators by PKG-mediated phosphorylation of a MLCP inhibitory subunit [20] . In addition, PKG and PKA reduce the ability of RhoA to inhibit the delayed rectifier potassium channel (KDR), which attenuates extracellular Ca 2+ entry [30] . The enzyme PDE5A, a target of sildenafil therapy in PH, hydrolyzes cGMP to counter the effects of NO-cGMP-PKG signaling. However, other PDEs, including cAMP PDEs, play important roles [31] . Vasoconstrictors activate PDE5A to reduce cGMP in VSMC by RhoA/PKCmediated inhibition of protein phosphatase 1 (PP1), thereby increasing phosphorylation of PDE5A and activating it [32] . GPCRs, including those for adenosine, ATP, adiponectin, apelin, prostaglandin E2 (PGE2,), PGI 2 generally increase NO from EC, which diffuses to VSMC, or directly increase cAMP in VSMCs [33][34][35][36][37][38][39] .
As a final summation statement, all current PH therapies intersect GPCR actions by modulating critical signaling effects. Firstly they, ultimately inhibit intracellular Ca 2+ signaling and vasoconstriction. This includes the cGMP-PDE inhibitors, soluble guanylate cyclase (sGC) activators, PGI 2 analogs, Ca 2+ -channel blockers, and ET-1 receptor antagonists. Secondly, they exert anti-inflammatory effects on vascular cells, as all of these therapeutics are known to do [2,40,41] .

Increased activity of vasoconstrictor GPCRs
GPCR activity is frequently altered in diseases via internalization, phosphorylation, and expression levels. In lung, increased activity of TxA 2 and its G q -coupled GPCR (TP) occurs via palmitoylation of TP and increasing the proportion of the active receptor at the plasma membrane, consistent with pathophysiological action of TP in PH [53][54][55][56] . Similarly, increased expression of other GPCRs involved in PH pathogenesis has been noted for ET1 (ET A ) and serotonin receptors, 5-HT 1B R and 5-HT 2B R in COPD-PH patients [54,55,57,58] .

Decreased activity of vasodilator GPCRs
In PH, decreased serum concentrations of PGI 2 is accompanied by decrease in levels of the receptor IP, reducing the effectiveness of PGI 2 therapy [59] . Similarly, chronic stimulation of PGI 2 -IP axis, occurring with prostacyclin therapy in PH patients, is likely to even further down regulate the PGI 2 -IP axis via heterologous desensitization, compounding a pathogenic situation [60][61][62] . GPCRs such as IP, which increase cAMP-PKA, frequently exert antiinflammatory effects, inhibiting key pro-inflammatory/pro-proliferative transcription factors, including NF-κB [63,64] , Hippo pathway transcription factors Yaz- Taz (co-factors for the proproliferative transcription factor TEAD1) and, no doubt, many others [65] . Induction of antiinflammatory/anti-proliferative PPAR γ is also another mechanism, by which PGI 2 acts [66] . PPAR γ, along with sibling, transcription factors PPAR β / δ all are protective in PH and other cardiovascular diseases [34,[66][67][68][69][70][71] . The induction of PPARγ activity by PGI 2 was once thought to be a direct binding event to the PPARγ, but it now appears to occur by indirect mechanism. Activation of PKA or p38MAPK by PGI 2 -IP stimulates the cAMP response element-binding protein (CREB) by phosphorylation. Activated CREB turns on the transcriptional co-activator, peroxisome proliferator-activated receptor gamma co-activator 1α (PGC1α) gene, increases PGC1a activity and stimulates PPARγ, leading to protective anti-inflammatory effects [71] Molecular targets of PPARγ include inhibition of NF-κB and hypoxic activation of HIF-1α [72] . HIF-1α is clearly important in VSMC proliferation occurring in PH, as it helps the cell switch to a glycolytic/Warburg metabolic phenotype and has been connected to the increased expression of Ca 2+ entry channel, TRPC6, both aiding VSMC proliferation [73][74][75][76] . Targeted KO of HIF-1α inhibitor protein, prolyl-hydroxylase domain containing protein 2 (PHD2), reduced O 2 -driven proteolysis of HIF-1α, thereby increasing HIF-1α -dependent proliferation of VSMC [76] . There are 3 PHD (PHD1-3) enzymes, which in presence of O 2 hydroxylate proline residues, 402 and 564, ultimately resulting in the proteolysis of HIF-1α. A small molecule drug, R59949, a PDH inhibitor, has shown potential to combat PH in the hypoxic mouse model [76] .

Post-receptor mechanisms leading to increased vasoconstrictor GPCR response
In VSMC, Angiotensin II (Ang II) up regulates G i expression, thereby increasing the activation of PLCβ and mobilization of Ca 2+ , further enhancing vasoconstriction and proliferation by a post-receptor mechanism [77] . Of the PH pre-clinical therapeutics, RhoA-ROCK inhibitor, fasudil and statins both act at post GPCR level [78,79] . Statins, such as simvastatin, can work in combination with sildenafil, the cGMP-PDE inhibitor, likely an important feature of any new therapy. Although some studies reported no drug combination yet tested, the combination could be more effective for patients' survival than any monotherapy [2,80,81] . Statins may work in PH models by inhibition of isoprenoid intermediates, farnesyl pyrophosphate and geranyl-geranyl pyrophosphate, essential for the post-translational isoprenylation, membrane localization, and activation of Ras and Rho small GTP-binding protein families, respectively, thus inhibiting RhoA-ROCK [82] .

Post-receptor mechanisms leading to decreased vasodilator GPCR responses
Post-receptor mechanisms also operate to limit vasodilator response in PH, such as the several hits to the critical NO-cGMP-PKG vasodilation system. Firstly, inflammatory cytokines down regulate eNOS and upregulate reactive oxygen species (ROS), including superoxide [83][84][85] . Secondly, due to peroxynitrite formation, NO level is depleted [86] . Thirdly, vasodilator response can be limited due to increased PDE5 A expression [87,88] . Up regulation of both cAMP-PDEs, and cGMP-PDE is an important pathological event, which decreases effectiveness of vasodilator GPCRs and needs further investigation [89] . The PDEs are a complex family of enzymes with 21 genes, and 11 subfamilies, and some share little sequence identity [31] . Due to a combination of post-receptor mechanisms, increased expression of cAMP-and cGMP-PDEs, inhibition of eNOS activity, and decreased NO availability (as a result of ROS production), the effects of vasodilators in PH are diminished.

REMODELING
GPCRs induce cytokine/chemokine production from leukocytes, VSMC, ECs, fibroblasts, and cardiac myocytes and are pathogenic in PH. Up regulation of SDF-1 in activated T cell results to the expression and secretion of RANTES and Monocyte Chemo-attractant protein 1 (MCP-1). These chemokines promote proliferation of VSMC, matrix remodeling, and ROS production [90][91][92] . Additionally, GPCRs like serotonin receptor and purinergic P 2 Y 14 R, promote migration of bone marrow derived blood cells, essential to the development of PH [93,94] .

VASCULAR INFLAMMATION IN PH
The driving forces behind vascular inflammation in PH are unclear, but it is likely that sterile inflammation-damage molecular pattern (DAMP) systems play a role. Purinergic receptors are also critical in DAMP responses. ATP, ADP, or adenosine are released from extracellular stimuli-activated, hypoxic, or damaged cells and play prominent roles in inflammatory and secretory responses associated with tissue repair. Of the 19 purinergic receptors, 12 are GPCRs nucleotide P2YR 1, 2, 4, 6, 11-14 and adenosine A 1 , A 2A , A 2B A 3 , and the remaining 7 purinergic receptors P2X 1-7 , are ligand gated cation channels [95][96][97][98][99][100] . Macrophage activation in PH is potentiated by the P 2 Y 6 [101][102][103] . Some data suggest antagonizing the ATP-activated P 2 X 1 purinergic receptor could be beneficial in PH [104] . Both P 2 Y 1 and P 2 Y 12 purinergic receptors have been shown to be partially responsible for PA pressure increase due to hypoxia [105] . Hypoxia-induced ATP release from PA adventitial fibroblasts and vasa vasorum endothelial cells (VVEC) induces mitogenic and angiogenic responses in VVEC in autocrine/paracrine manner [95,96,106] [ Figure 2]. Released ATP and ADP are degraded rapidly to adenosine. Activation of the A 2A adenosine receptor has been reported to be protective against PH, but the activation of A 2B -AR results in pathogenic effects [107][108][109][110][111][112] .
The involvement of DAMPS-GPCRs in PH is understudied, and therapeutic possibilities remain to be explored.

PATHOGENIC CHEMOKINE GPCRS Small G-proteins in chemokine receptor-stimulated VSMC proliferation
In VSMC, MCP-1 acting via G i -coupled CCR2, stimulates G i -dependent proliferation, that also involves activation of the small G proteins [113] . One of the mechanisms includes p115RhoGEF-dependent activation of the Rac and Nuclear factor of activated T-cells (NFAT1)-dependent up-regulation of cyclin D1 expression in VSMC [113] .

Involvement of ROS in chemokine receptor-stimulated responses
ROS is a pathogenic factor in PH by mechanisms, which include reducing NO; promoting VSMC proliferation; initiating sterile inflammation-DAMP response; and promoting vasoconstriction via increased membrane depolarization [74,114] . G i -coupled GPCRs, such as MCP-1, SDF-1, thrombin, PAF, and purinergic receptors, stimulate ROS production [115][116][117] . ROS are produced as bactericidal compounds in large amounts in phagocytes (neutrophils, monocytes, macrophages) and, in a lesser amounts, in vascular cells. In phagocytes, chemokines, such as N-Formylmethionyl-leucyl-phenylalanine, PAF, complement C5a (C5a), LTB 4 , and MCP-1 are G i -coupled-GPCRs and activate Rac1-NAD(P)H oxidasesuperoxide system. NOX2 is a neutrophil NADPH oxidase responsible for producing increased amounts of superoxide. There are 7 NOX like oxidases, NOX1-5 DUOX1, 2 of which are expressed in vascular cells, and their activation involves Rac1 stimulation by the GEFs, such as engulfment and cell motility protein 1 (ELMO1) [115,117,118] . The superoxide generated by NOX enzymes in the extracellular space, is converted to H 2 O 2 , some of which enters the cell to stimulate proliferation. H 2 O 2 induces proliferation by changing the balance in protein kinase-protein phosphatase networks by inhibiting key protein phosphatases via the oxidation of labile sensitive cysteine in the active site [119] .

WITH GPCRS SIGNALING IN PH
PDGF-induced proliferation of VSMC is believed to be a major factor in PH. It is known to be dependent on Akt activation that can occur in co-operation with some GPCRs, termed trans-activation [140] . Ang II receptor works in concert with PDGF-receptor tyrosine kinase, promoting Akt-dependent VSMC proliferation [77,[141][142][143] . Thrombin-PAR trans-activates the TGF-β receptor to promote VSMC proteoglycan synthesis [144] . It is of some interest that PGI 2 has been described as unable to significantly inhibit PDGF-induced VSMC proliferation, suggesting that other PDGF-neutralizing strategies are needed in PH [145] . MCP-1 and IL-6 also work together to induce VSMC proliferation [146] . Activation of inflammatory TXA 2 -TP inhibits FGF-2-or VEGF-stimulated angiogenesis, which could relate to vascular pruning in cardiac and pulmonary vessels, and is an example of GPCRcytokine interaction [41,[147][148][149] . Protective interactions of GPCRs with cytokines and growth factors could include the ability of PGI 2 -IP to inhibit the IFNγ-induced inflammation, dependent upon induction of suppressor of cytokine signaling 3 (SOCS3) [150] . The GPCR GPR4 expressed on ECs, promotes angiogenesis in a Notch-dependent manner [151] . Vessel architecture is maintained by the ligand-receptor pair jagged expression on EC and Notch expression on VSMC, keeping VSMC in a differentiated non-proliferating state [152][153][154][155][156] .
Both HIF-1 a -induced VEGF for reparative angiogenesis and hypoxia-induced epithelial to mesenchymal transition require Ras family member, RhoE, which activation involves SDF-1 GPCR, CXCR4 signaling [157] . RhoE aids in HIF-1α maintenance and is induced by cAMP via G s -coupled GPCRs [158] . Cardiac angiogenesis is believed to be critically protective in heart disease and potentially links SDF-1, cAMP, RhoE, HIF-1α, and VEGF into signaling networks [159] .

INFLAMMATION AND GPCR ACTION
Platelets from patients with the sub-form of PAH, due to thromboembolic PAH, exhibit increased reactivity to thrombin, which stimulates the G q /G i -coupled protease activated receptor 1 (PAR1), promoting VSMC proliferation [185,186] . Thrombin receptors exist on EC and have been reported to inhibit angiogenesis.

RV REMODELING AND FAILURE
Cardiac myocytes (CMs) are terminally differentiated cells. The compensatory cardiac hypertrophy is entirely due to increased CM cell size, rather than proliferation. The adult heart is 56% CM, 27% fibroblasts, 10% VSMC, and 7% ECs, and these ratios change little between the four chambers [187] . During PH, the ratios of fibroblasts increases, and the ratio of ECs/CMs decreases [188] . The transition to heart failure has been linked to endothelial dysfunction due to insufficient reparative angiogenesis -a loss of capillaries supplying cardiac myocytes with O 2 , leading to capillary pruning, inflammation, and ROS production [147][148][149][188][189][190][191][192][193] .

Pathological role of GPCRs in cardiac myocyte with respect to RV failure
The hypertrophy response is engaged when increased Ca 2+ -and cAMP-dependent contractile signals lead to activation of NFAT, MEF2, and GATA 4 . These signals are driven by GPCR agonists, such as Ang II, thrombin, ET1, PGF2α , β-AR [194][195][196][197] . Typical gene expression changes include decreased expression of sarcoplasmic reticulum Ca 2+ re-uptake channel (SERCA2), increased expression of slow twitch contractile protein myosin heavy chain β9 (β-MHC, a.k.a. MyH7), and decreased expression of the fast twitch a-MHC/MyH6, amongst others [198,199] . The transcription factor, Egr-1 has been linked to the down regulation of cardiac SERCA2 in hypertrophy and was found to be overexpressed in PAs of PH patients [200][201][202] . GPCR-induced increase in intracellular Ca 2+ stimulates PKD activity, promoting nuclear export of histone deacetylase 5 (HDAC5), thereby activating MEF2 to initiate hypertrophic gene program [203,204] . GPR91, a receptor for succinate expressed in CMs, promotes cardiac hypertrophy by coupling to G i /G q -PI3K-Akt signaling [205,206] . Succinate may be accumulated during cardiac remodeling due to changes in metabolism, and when released from the cells, promotes positive feedback loop by activating GPR91 leading to hypertrophy, or as also reported, to CM apoptosis via caspase3 [188] .

ACTION OF GPCRS ON ENDOTHELIAL CELLS WITH RESPECT TO RV FAILURE
ED, occurring in failing RV, interconnects with fibrosis, as this appears to be a factor in the decreased capillary density-ED observed in hypertrophy and with the altered metabolism of CM, critical towards HF [217,218] . ED can result in potentially uncontrolled inflammation of local RV tissue and in turn can lead to EC apoptosis, down regulation of eNOS and PGIS. TGF-β, which is pathologic in PH, is induced by inflammation, promotes lung and heart fibrosis, but also promotes ED by inhibiting differentiation of endothelial progenitor cells (EPCs) into ECs to repopulate damaged endothelium, counteracting the effects of endothelium protective GPCR ligand, apelin [219,220] . Cardiovascular protective GPER is found in ECs, promotes angiogenesis, and could be significant in defending against endothelial dysfunction [207,221,222] .

VASCULAR FIBROBLASTS AND CARDIAC FIBROSIS
Cardiac fibrosis, seen in animal models of PH, involves expansion of fibroblast populations, their differentiation to myofibroblast, and the stiffening of the extracellular matrix by synthesis of collagens [198] . Fibroblasts also can derive from EMT via conversion of EC to fibroblasts [175] . GPCRs promoting cardiac fibrosis include G q -PLC-Ca 2+ -coupled 5-HT 2B , Ang II, and endothelin CPCRs. The thrombin receptor, PAR1 is the most highly expressed GPCR in cardiac fibroblasts, therefore is a potentially important profibrotic GPCR [223][224][225] . P 2 Y 6 -purinergic receptors are reported to enhance pressure overload-induced fibrosis by increasing TGF-β1 and CTGF release [226] . The p38 a MAPK, activated by Ang-II or non-GPCR stimuli, such as TGF-β1, or cyclic stretch, has been identified as a master switch, common to many different receptors stimulating fibrosis [198] . The ligand relaxin and its GPCR, RFXP1-4, are Gs-coupled and exert anti-hypertrophic and anti-fibrotic effects [227] . In cardiac fibroblasts, PGI 2 -IP-PKA axis activates CREB to inhibit Ang II-induced SMAD2 activation, attenuating proliferation [228] .

ROLE OF GPCRS IN MONOCYTE/MACROPHAGE WITH RESPECT TO RV FAILURE
Macrophage features in the inflammation associated with heart failure, with resident macrophages being described as protective, while recruited being pathogenic [191] . Increasing activity of the transcription factor KLF4 in resident macrophages to aid their survival or inhibiting MCP-1-CCR2 activity of recruited monocytes, has been suggested as a potential therapy [191] . Macrophage polarization in PH is thought to contribute to cardiac and pulmonary inflammation-induced damage and remodeling. M1 macrophage phenotype is considered pro-inflammatory (versus the M2 phenotype), is involved in resolving inflammation, but implicated in tissue fibrosis [229] . Some studies in PH suggest that M2 macrophages are more damaging than M1. Antagonizing the CX3CR1 chemokine receptor reduces pathogenic M2 in favor of less damaging M1 phenotype [90,230] . Most chemokine receptors activate Gα i1 /Gα i3 , which have been linked to promotion of polarization to M1 macrophage via increased LPS-TLR4-NF-κB, in contrast to CX3CR1 signaling [76] . An interesting development in macrophage polarization/anti-inflammatory responses are the 6 atypical chemokine receptors, ACKR1-6, which are "duds" unable to activate G-proteins, and exert anti-inflammatory effects [229] . In particular, the atypical chemokine receptor, CCRL2 (tentatively ACKR5) polarizes in favor of M2 phenotype [229] . Other GPCRs aiding polarizing to M2 phenotype, include lipoxinA4-activated FPR2, PGE 2 -receptors, and adenosine A 2A /A 2B -receptors [231][232][233][234] . GPCRs clearly critically control macrophage polarization and might well be employed to diminish macrophage-induced inflammation occurring in PH. The role of GPCRs in cardiac inflammation is clearly complex, and it should be mentioned that increasing recruitment of pro-angiogenic monocytes may be beneficial in ED, and is also under control of GPCRs [235][236][237][238] .

GPCRS, WHICH MIGHT BECOME CLINICAL TARGETS IN PH
GPCRs activating cAMP-PKA axis in ECs or VSMCs, such as PGI 2 and adenosine (A2 B AR), generally induce vasodilation, are often anti-inflammatory and protective in PH. Secondly, GPCRs, such as for apelin, PGI 2 , opioids, which increase NO release from EC to promote vasodilation, are also usually protective. Thus, any signals increasing cAMP, cGMP, NO and inhibiting Ca 2+ are usually protective [178,179] . By contrast, any GPCR signaling increasing Ca 2+ in VSMC, or decreasing NO, cAMP, cGMP, or increasing inflammation, are usually pathogenic in PH. One very potent anti-inflammatory agent is adenosine, which exerts powerful anti-inflammatory effects acting at A 2A AR, and clearly plays a protective role in PH [111,239] . New drugs (such as AEA061) are positive allosteric modulators of A 2 AAR, that activate receptors without binding to the normal agonist binding site, offer a therapeutic possibility of fewer side effects as they do not act at A 1 , A 2B or A 3 ARs [239] . Activation of A 2A AR without activating A1, A2B, and A3ARs has been an issue in developing anti-inflammatory therapies. Other potentially protective GPCRs include FPR2, an atypical chemokine receptor on macrophages, was reported to exert anti-inflammatory action [229,240] . Other protective receptors in PH include ET-1 receptor ET B [241] , angiotensin II type 2 receptor [242] , adiponectin-receptor [36,243] , mas1 (a receptor for angiotensin 1-7) [244] , and relaxin receptors [245,246] . ET B receptor is also protective in porto-pulmonary hypertension, a disease secondary to liver failure, but in which the same therapeutics, PGI 2 -cGMP-PDE-ET-1 receptor antagonist therapies are utilized [247,248] .

CONCLUSION
Research has highlighted many examples of pathological GPCR signaling, which can be targets for novel PH therapeutics. In PH pre-clinical studies many targets have been identified, but only few are druggable [ Tables 1 and 2]. GPCRs, by contrast, represent good targets for pharmacological strategies and in all likelihood present one of the best opportunities for therapeutic intervention in PH. The heart alone is estimated to express some 200 different GPCRs, suggesting significantly better therapeutics based on targeting GPCRs are possible. The challenge is to devise the best pharmacological cocktail for the PH patient. At the moment, while much has been published with respect to GPCR action in PH, much more clearly awaits discovery.   Cefminox, a dual agonist of prostacyclin receptor and peroxisome proliferator-activated receptor- Schematic presentation of the mechanisms by which G protein-coupled receptors (GPCRs) regulate vascular tone and vascular smooth muscle cells (VSMC) proliferation. Vasoconstrictors like Ang II, ET1, thrombin, activate Gα i , Gα q , or G 12 / 13 -coupled GPCRs, increase Ca 2+ via PLC β activity, and receptor operated calcium channels such as TRPC6. Increase in PLC β activity decreases PIP2 relieving tonic inhibition of TRPC6. Increase in Erk1/2 activity by G i /G q -coupled GPCRs activates TRPC6 by phosphorylation leading to increased Ca 2+ entry and calmodulin-dependent protein kinase (CAMK) activation. CAMK increases MLCK activity by phosphorylation, which in turn phosphorylates MLC phosphorylation causing vasoconstriction. GPCRs coupled to G 12 / 13 increase RhoA activity and the downstream kinase ROCK. ROCK increases MLC phosphorylation by inhibiting MLCP, or by direct phosphorylation. Vasodilators, such as PGI 2 acting via G s -coupled receptors activate PKA thereby inhibit Ca 2+ increase by PKA-mediated phosphorylation of PLC β and TRPC6. In ECs, G i , or G q -coupled GPCRs, increase, PI3K-Akt signaling and activate eNOS by phosphorylation at Ser 1177 . NO diffuses to nearby VSMC, activating soluble guanylate cyclase, increasing cGMP, activating PKG, and inhibiting TRPC6 by phosphorylation. PKG also activates the GAPs for G q , RGS2 and RGS4 to inhibit PLC β Schematic diagram illustrating a role of PI3K, Rho and ROCK pathways in hypoxia-induced ATP release and ATP-mediated angiogenic effects in vasa vasorum endothelial cells. Activation of PI3K/Rho/ROCK pathway in response to hypoxia results in regulated ATP release from VVEC. In turn, extracellular ATP triggers/initiates P2YR-dependent activation of PI3K/Rho/ROCK pathway leading to angiogenic responses in vasa vasorum endothelial cells. VVEC: vasa vasorum endothelial cells