Next Article in Journal
Preventive Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury after Modelling Stroke in a Rat
Next Article in Special Issue
New Insights of the Zn(II)-Induced P2 × 4R Positive Allosteric Modulation: Role of Head Receptor Domain SS2/SS3, E160 and D170
Previous Article in Journal
DNA Methylome Distinguishes Head and Neck Cancer from Potentially Malignant Oral Lesions and Healthy Oral Mucosa
Previous Article in Special Issue
P2 Receptors Influence hMSCs Differentiation towards Endothelial Cell and Smooth Muscle Cell Lineages
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases

1
The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA
2
Vascular Biology Center, Augusta University, Augusta, GA 30912, USA
3
The Department of Pediatrics, Division of Critical Care Medicine, University of Colorado Denver, Aurora, CO 80045, USA
4
Center for Blood Disorders, Augusta University, Augusta, GA 30912, USA
5
The Department of BioMedical Engineering, University of Wisconsin, Madison, WI 53706, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(18), 6855; https://doi.org/10.3390/ijms21186855
Submission received: 25 August 2020 / Revised: 11 September 2020 / Accepted: 15 September 2020 / Published: 18 September 2020
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)

Abstract

:
Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

1. Extracellular Nucleotides and Purinergic Receptors

Extracellular nucleotides (ATP, ADP, UTP) are emerging as important autocrine/paracrine regulators of the vascular, immune, and hematopoietic systems [1,2,3,4,5,6,7,8,9,10]. Under pathological conditions, such as inflammation, hypoxia, and vascular injury, high amounts of ATP can be released locally and promote vascular responses through the activation of purinergic receptors [8,9,11,12,13,14,15]. There are 19 different cell surface purinergic receptors [16], divided into P1 types (binding adenosine) and P2 types (binding ATP, ADP, UTP, β-NAD) [3,17,18]. P2 types are further divided into ligand-gated ion channels (P2X) and membrane-bound G-protein-coupled receptors (P2Y). In endothelial cells (ECs), extracellular nucleotides act through P2Y (metabotropic) receptors to regulate endothelial barrier function in both physiological and pathological conditions [1,2,3,4,5,6,19,20]. So far, eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been identified. Interestingly, all P2Y receptors have been shown to belong to the gamma-subgroups of class A GPCRs. P2Y1, 2, 6, and 11 are coupled to Gq. P2Y11 is also coupled to Gs, which increases the activity of adenylate-cyclase and thus increases the level of cAMP [21]. P2Y12, 13, and 14 are coupled to Gi, which inhibits adenylate cyclase and activates additional effector systems, including phosphatidyinositol-3 kinase (P3IK). P2Y4 is coupled to Gq and Gi [3,10,17,22,23]. We have recently demonstrated the expression of all P2Y receptors in lung microvascular EC (LMVEC), with P2Y6 being the most abundant and P2Y4 being the least abundant [24]. However, within the P2Y12,13,14 group, only P2Y13 has been observed in vasa vasorum EC (VVEC) [25], indicating that P2Y receptor expression may differ across EC subtypes.
The wide distribution of purinergic receptors in the vascular system, together with the expression of multiple receptor subtypes in endothelial cells, point to their physiological and pathophysiological importance. It is also becoming evident that alterations in the purinergic signaling may result in the development of a variety of neurogenerative, immune, and vascular pathologies [9,10,18]. Some additional reports indicate that extracellular ATP has been implicated in the hyperplasia and hypertrophy of arterial walls in spontaneously hypertensive rats [3], in the regulation of vascular barrier function, and the control of proliferation and migration of vascular smooth muscle cells and hematopoietic stem cells [26,27,28,29,30,31]. Purinergic antithrombotic drugs have been shown to reduce the risk of recurrent strokes and heart attacks [9]. While numerous studies demonstrated that P2 receptors are involved in the progression of lung diseases, they primarily focused on the role of ATP-induced P2X receptor activation [32,33]. The information on the role of P2Y receptors is more limited. Early studies demonstrated that ATP-induced activation of P2Y receptors is involved in acute lung inflammation and ventilator-induced lung injury [34]. More recently, we have shown that P2Y receptor agonists, ATPγS, and β-NAD enhance the lung endothelial barrier function in vitro and in vivo via activation of specific P2Y receptors [24,35,36,37].

2. P2Y Receptor Signaling and Endothelial Dysfunction

The vascular endothelium is a semi-selective diffusion barrier between the plasma and interstitial fluid and is critical for normal vessel wall homeostasis. In addition, due to its secretory and adhesive properties, the endothelium is recognized as an active part of the vasculature [38,39]. In terms of EC physiology, P2Y purinergic receptors are involved in the regulation of vascular tone and barrier function, activation of vascular inflammation and thrombosis, and regulation of vascular growth-all processes are tightly linked with vascular diseases. Importantly, P2Y-mediated signaling is integrated in a crosstalk with many more regulatory systems, including cytokines, growth factors, and toll-like receptor (TLR) input, and is counterbalanced by PGI2, extracellular adenosine and others. As an example of stepwise activation, P2Y2 activity is most effective at stimulating EC proliferation when working in conjunction with other growth factors or cytokines [40]. Other evidence supporting this observation comes from the cancer therapy field, showing that activation of EC P2Y receptors is protective against the DNA damaging activity of cancer chemotherapies toward the cardiovascular system [41]. There is growing evidence for a new role or P2Y subtype-specific signaling in ECs in various pathophysiological conditions.

2.1. P2Y Receptors and Vascular Tone Regulation

2.1.1. Hypertension

Vasodilation occurs via multiple mechanisms and is protective against high blood pressure, a major pathological factor in cardiovascular disease (CVD) [42]. Fluid shear stress, as found in systemic hypertension or in pulmonary arterial hypertension (PAH), exerts a negative feedback response to decrease pressure by causing local vasodilation. The mechanism involves the activation of the endothelial mechanosensitive cation channel PIEZO1, leading to ATP release and the stimulation of P2Y1,2,4,6/Gαq-PLC-Ca2+ pathways in the endothelium, in turn leading to eNOS-NO-mediated vasodilation [14]. Mice with endothelium-specific P2Y2 or Gαq/Gα11 deficiency lacked flow-induced vasodilation and developed hypertension accompanied by reduced eNOS activation [42]. Endothelium-specific PIEZO1 null mice do not generate high-flow-induced NO and vasodilation and, therefore, develop hypertension [14]. The small molecule activator of mechanosensitive PIEZO1, Yoda1 replicated to some degree, the effect of fluid shear stress on ECs and induced vasorelaxation in a PIEZO1-dependent manner. Activation of EC eNOS is considered paramount in EC-dependent vasodilation and is partially Ca2+-dependent [43]. Activation of TRPV4 channels creates localized Ca2+ increased-sparks in the vicinity of TRPV4-GPCR, leading to localized eNOS activation through the calmodulin-binding domain [43,44].
Mechanisms contributing to decreases in blood pressure are mediated by the activation of P2Y2. [45]. Intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells), as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), are all hypothesized to be part of the endothelium-dependent hyperpolarization (EDH) response. Studies in wild-type mice and mice lacking KCa3.1, KCa1.1, Cx37, or Cx40 demonstrated that loss of endothelial KCa3.1 channel and Cx40 decreased the vasodilator response to P2Y2 activation. These data indicate the presence of eNOS-NO-independent vasodilation response, in addition to eNOS-NO-dependent response [45]. In pressure-overload-induced CVD, such as that occurring in PAH and systemic hypertension, attenuation of P2Y1,2,4,6-mediated vasorelaxation has been observed, also indicating a possible connection to the increased level of the vasoconstrictor TXA2 [46].

2.1.2. Vascular Tone and Aging

Experimental evidence points to the involvement of P2YRs in aging-dependent hypertension [47]. Aging has been found to selectively diminish vasodilation that was mediated by P2Y2, but not muscarinic and nicotinic receptors [48]. Experimental aged rats showed impaired cardiac contractility and aortic blood flow, increased vascular inflammation, and persistently activated CaMKII [49]. Endothelial cells isolated from young and aged aorta exhibit differences in cell phenotype and physiology. A giant cell phenotype is typical in senescent cells, which are far more common in aged vessels and have a reduced capacity for proliferation or vasodilation in response to vascular stress. CaMKIIδ expression is significantly increased and activated in the endothelium of aged aorta [49]. As such, CaMKIIδ could serve as an essential marker of and maybe an appropriate candidate for investigating targeted therapy at the endothelial dysfunction that accompanies the aging process [49]. Reduction of NO-mediated vasodilation in aged blood vessels was due to decreased phosphorylation of eNOS on critical Ser1177 residue, and increased ceramide-activated phosphatase 2A might be responsible for decreased eNOS Ser1177 phosphorylation [50]. Another factor in the increased occurrence of hypertension with age is P2Y6 dimerization with the angiotensin II type 1 receptor (AT1R) [51]. With age, P2Y6 expression in VSMC increases, and heterodimers with AT1R are reported. In P2Y6 null mice, chronic over-administration of AngII resulted in less cardiac hypertrophy, vascular remodeling, and lower blood pressure [51].

2.1.3. Vascular Tone and Diabetes

Type 2 diabetes (T2D) is known to impair vascular perfusion through poorly defined mechanisms. It was found that diabetics have impaired vasodilator responses to purinergic, but not to muscarinic or nicotinic receptor-induced vasodilation, similar to the situation in aging [52]. In the streptozotocin-induced Type 1 diabetes (T1D) model, impaired P2Y1-mediated vasodilation has been attributed to decreased activation of eNOS-NO-PKG [53]. At the same time, P2Y1 receptor expression remains unchanged despite the frequent occurrence of receptor expression changes in diabetic disease states [54]. In general, alteration of purinergic receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes [55]. Additional data from the mice model demonstrated that diet-induced T2D resulted in vascular inflammation and insulin resistance, accompanied by decreased eNOS-NO activity [53,56]. Sepsis-promoted endothelial dysfunction, monitored by the ex vivo examination, was also associated with reduced activity of eNOS-NO generation [57]. Therefore, it is suggested that amplifying the eNOS-NO signaling axis might decrease the inflammation-induced vascular damage observed in several CVDs.

2.2. P2Y Receptors and Regulation of Oxidative Stress and Vascular Inflammation

2.2.1. Oxidative Stress

Oxidative stress is a feature of many CVDs and often closely correlates with inflammation, as both professional phagocytes bound to ECs and ECs activated by cytokines/TLRs produce ROS [58]. P2Y1 stimulation produces ROS via the NADPH oxidase catalytic component, NOX2, which produce superoxide [59,60]. Oxidative stress is reported in the ED observed in sepsis [57]. P2Y1,2,4,6 probably all activate ROS production in ECs, as all activate PKC, which seems to be at least one of the critical steps to activate the superoxide generating complex of NOX enzymes [59]. Indeed, angiotensin II and thrombin, which both have virtually identical signaling mechanisms to the P2Y1,2,4,6 group, also activate ROS production in ECs via activation of PKC [61]. One can then appreciate the clinical importance of studies proclaiming that ROS are intrinsic mediators of purinergic signaling [62]. Considering this, the role of ROS (including hydrogen peroxide) in the signaling of purinergic receptors should be examined, as was shown for P2Y1 [62].

2.2.2. Vascular Inflammation

Inflammation is a characteristic feature of CVD and is mediated by released cytokines, chemokines, eicosanoids, and ATP by vascular and blood cells. ECs represent a primary cellular target for many pro-inflammatory stimuli. Potent pro-inflammatory mediators, such as LPS and TNFα, significantly induce the P2Y6 receptor expression in EC in vitro and in vivo [63]. In vascular ischemic injury models, the ischemic conditions led to the activation of P2Y2 via released ATP [64]. P2Y2 was determined to have a pathogenic role by increasing local vascular inflammation and up-regulating TNFα, IL-1, and LT-α [64]. Additionally, these inflammatory conditions promoted VSMC proliferation, resulting in neo-intimal thickening, hyperplasia of VSMCs, and monocyte adhesion to EC. P2Y2 activity amplifies the release of IL-1 by a mechanism involving NFκB and Panx1 channels [65]. While overall P2Y2 effects are pro-inflammatory, it can initiate a negative feedback mechanism, upon which it down-regulates the ICAM-1 to limit inflammation in a miR-22 dependent manner [66]. P2Y4 also exhibits a pathogenic role in ischemic myocardial injury [67]. In the mouse ischemic heart model, loss of P2Y4 nucleotide receptor protected against myocardial infarction through endothelin-1 downregulation and MMP-1,-8,-9 expressions [67]. In ischemic injury, hypoxia due to poor perfusion is a significant factor of endothelial dysfunction. P2Y2 (and probably P2Y1,4,6) works together with the P1 receptor subtype A2B adenosine receptors to protect the ECs from hypoxia-induced apoptosis. This makes some sense, as A2B receptors are anti-inflammatory, and the reported effects involve the activation of the P2Y2-Gq-PI3K-Akt and the PI3K-Akt arms, well known for their cell survival action [68,69].
The P2Y11 receptor, activated by ATP, has anti-inflammatory actions, which could be implicated in EC protection in CVDs [70]. In ECs, antagonism of P2Y11 with a specific antagonist (NF157) increases the effectiveness of oxidized-LDL to promote inflammation of endothelial cells [71]. P2Y11 expression is up-regulated by oxidized-LDL activation of ECs, reminiscent of a classic negative feedback loop. NF157 decreases IL-6 and TNFα cytokine production and the activation of p38 MAPK in ECs and the consequent binding of monocytes to EC due to up-regulation of E-selectin and VCAM-1 [71]. Other reports show the anti-inflammatory effect of P2Y11 in EC, where the receptor can block the signaling activated by IL-1, TNFα, and other cytokines, particularly the activation of JNK [72]. Thus, agents activating P2Y11 could have protective therapeutic value to combat the atherosclerosis and inflammation that drives the disease.
The mechanism by which P2Y11 exerts anti-inflammatory action are not known, but P2Y11 is a dual-specificity GPCR, activating both Gq and Gs. The activation of Gs has been reported to be potentially more circumstance- or cell-type-dependent [73]. However, a significant clue is the activation of the endothelial Gs-AC-cAMP-PKA-EPAC axis, one of the most potent anti-inflammatory and barrier-protective cellular pathways. This pathway has been known for decades, but many of the key targets of (protein kinase A) PKA, those by which PKA exerts its anti-inflammatory effect, are still unclear. One target is the pro-inflammatory cytokine receptor for TNFα, which is inactivated by PKA-mediated phosphorylation leading to anti-inflammatory actions. One example is the PKA phosphorylation of TNFαR at Thr411 and, which blocks all downstream signaling [74], similar to the action of other cytokine receptors. However, this long-known phenomenon is incompletely characterized at the molecular level and deserves further investigation.
P2Y11 receptor has demonstrated anti-inflammatory effects on ECs in other settings, such as in tumors, where ECs often migrate from the tumor locus. In this setting, cytokine- growth factor-, and chemokine-driven migration can be inhibited by activation of P2Y11. Such effects require adenylate cyclase 10 isoforms of the enzyme [75,76]. In addition, activation of EPAC1, the cAMP-activated guanosine nucleotide exchange factor (GEF) for a small GTPase, Rap1, often opposes the pro-inflammatory action of RhoA-ROCK via Tiam-1 and Vav-mediated Rac activation that strengthens endothelial adherens junctions leading to the prevention of vascular leak and inflammation [77].

2.2.3. Atherosclerosis

In atherosclerosis, the P2Y2 receptor apparently has pro-inflammatory actions, as ApoE-/-mice with additional deletion of P2Y2 have lower inflammatory indices in an atherosclerotic lesion, which includes lower VCAM-1 and LT-α, a member of TNFα family known to be pathogenic in the disease [78]. As such, the investigations suggest blocking P2Y2 might be therapeutic for atherosclerosis by reducing the difficulty of treating inflammation. Oxidized-LDL promotes activation of ECs by releasing ATP, auto-activating the P2Y2 receptors, which serve to facilitate leukocyte binding to the ECs, increasing inflammation in the atherosclerotic lesion [79]. This oxidized-LDL-driven inflammation appeared to involve activation of the inflammasome, and mitochondrial ROS production to generate IL-1. Additional indications of TLR9 activity were evident, probably acting as a DAMP (damage-associated molecular pattern) response [80]. Other evidence supports the pro-inflammatory phenotype of P2Y12 in atherosclerosis, as P2Y2 null mice are protected against the disease [81].
Activation of endothelial P2Y2 also has been linked to the mechanism by which oxidized LDL promotes vascular inflammation in atherosclerosis. P2Y2 up-regulates cell adhesion molecules such as VCAM-1 and ICAM-1 to secure monocyte adherence and infiltration into the atherosclerotic lesion [82]. P2Y6 also plays a pro-inflammatory pathogenic role in the disease, with its expression increasing in atherosclerotic lesions [82]. P2Y6 null mice had decreased inflammation associated with lesions, and the macrophages prevent uptake of less cholesterol [82]. These results indicate that P2Y6 antagonists may be beneficial to protect against atherosclerosis.
Additional evidence suggests that GPR120, a GPCR for omega-3 fatty acid, seems to counteract oxidized-LDL/P2Y2-mediated inflammation in atherosclerosis, inhibiting the binding of monocytes to ECs [83]. GPR120 is downregulated by exposure to oxidized-LDL, suggesting a role for GPR120 in mediating oxidized-LDL insult. The activation of GPR120 inhibits ROS and cytokine generation induced by oxidized-LDL by elevating the KLF2 transcription factor’s expression level. KLF2 has been determined to exhibit vascular protective actions by inducing eNOS and thrombomodulin and inhibiting VCAM-1 and E-selectin expression [84]. In addition, a pro-thrombotic state is a part of endothelial dysfunction. Activation of P2Y2 leads to a pro-thrombotic state by up-regulating tissue factor (TF), the initiator of thrombosis [85,86].

2.2.4. Metabolic Syndrome

It is thought that in a hyper-adiposity state associated with metabolic syndrome, a high turnover of adipocytes is observed despite increased fat content. Death of adipocytes promotes sterile inflammation, and this is the connection to CVD. ATP, being released from EC and other cell type, is pro-inflammatory and instigates leukocyte infiltration via P2Y2 receptors. P2Y2-null mice show blunted responses on a high-fat diet, gain less weight, and have better insulin sensitivity and lower cholesterol levels [81]. Adipose tissue is highly vascularized, and some of the P2Y2 action probably spills over to effects on EC P2Y2.

2.3. P2Y Receptors and Regulation of Vascular Barrier Function

Increased vascular permeability is an important pathophysiological component of many CVD and lung diseases, including acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), which arise from a wide range of lung injuries such as toxins or inflammatory mediators, resulting in significant morbidity and frequently in death [87,88,89]. In addition, thrombotic events, like stroke, myocardial infarction, and pulmonary embolism, are associated with significant risk factors of lung injury [90,91,92,93]. A major cause of ALI is a dysfunction of the pulmonary vascular endothelial barrier resulting in pulmonary infiltrates, hypoxemia, and pulmonary edema [87]. Recent studies suggest a dual role of P2Y receptor signaling in vascular barrier regulation.

2.3.1. P2Y Receptors and Vascular Permeability

In addition to the regulation of vascular tone, P2Y1,2,4,6 receptors seem to potentiate vascular inflammation and increase vascular leak, decreasing the barrier function of the endothelium [94]. It has been shown that vascular leak is transient when only stimulated by P2Y1,2,4,6-receptor-mediated activation of Gαq-PLC, and much longer-lasting responses occur when ECs are co-stimulated with P2Y2 and LPS or other pro-inflammatory cytokines. Other GPCR ligands that mediate Gq-PLC activation, such as thrombin, histamine, PAF, and bradykinin, also transiently increase vascular permeability. P2Y1-mediated EC permeability involves the activation of CaMKIIδ6, the predominant CaM-activated protein kinase in ECs [95]. Similarly, Gαq-coupled thrombin receptor PAR1 activates CaMKIIδ6 to promote EC permeability. This pathway involves the activation of the RhoA-ROCK, but not ERK1/2; however, at higher concentrations, thrombin activates additional kinases [95]. These data imply that P2Y1-mediated activation of CaMKII is likely responsible for EC barrier dysfunction.

2.3.2. P2Y Receptors and Vascular Barrier Protection

Although the role adenosine, the ligand for P1 receptors, in the enhancement of endothelial barrier function has been established [96,97,98,99,100], less is known about the effects of ATP. Recent findings in lung microvascular EC presented new evidence on the role of P2Y receptors in vascular barrier protection. It was demonstrated that ATP and its stable analog, ATPγS, can significantly enhance the human lung microvascular EC (HLMVEC) barrier via P2Y4 and P2Y12 receptors coupled to Gq and Gi heterotrimeric proteins [24,101]. Quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated the expression of all three Gαi subtypes in HLMVEC with Gαi2 expressed at the highest and Gαi1 at the lowest (almost negligible) levels (Figure 1A). Specific depletion of Gαi2 significantly attenuated the ATPγS-induced increase in the trans-endothelial electrical resistance (TER), an inverse permeability index (Figure 1B) confirming the involvement of Gi2 in the ATPγS-induced barrier enhancement. In addition, it was shown that ATPγS attenuates the gram-negative bacterial toxin LPS-induced lung inflammation and vascular leak in vivo [35]. While the mechanisms involved in ATPγS-induced EC barrier preservation remain ill-defined, some data suggest that they likely include unconventional Gi-mediated PKA activation and specific association of Gi and PKA with AKAP2 (PKA anchoring protein 2) [24]. In this signaling cascade, downstream mechanisms include the activation of myosin light chain (MLC) phosphatase (MLCP), followed by dephosphorylation of MLC, leading to inhibition of contractile responses and enhancing of cell-cell junctions [24,101]. Importantly, depletion of Gαi2 reversed ATPγS-induced MLC dephosphorylation, suggesting the involvement of P2Y4/P2Y12/Gi2-mediated signaling in MLCP activity regulation [24].
Nicotinamide adenine dinucleotide (NAD) is a cofactor that is central to metabolism. In addition to its metabolic functions, NAD+ emerges as an adenine nucleotide that can be released from neurons and blood vessels spontaneously and by regulated mechanisms [102,103]. In recent years, NAD+ has also been recognized as an extracellular signaling molecule involved in cell-to-cell communication [102,104,105]. Recent in vitro studies demonstrated that extracellular β-nicotinamide adenine dinucleotide (β-NAD) protects the pulmonary endothelial cell barrier integrity from injury caused by thrombin and bacterial toxins, LPS, and pneumolysin (PLY) [36]. In addition, β-NAD induced rearrangement of the adherens junction protein VE-cadherin, supporting a tightening of the cell–cell contacts and strengthening of endothelial barrier function. Pharmacological and genetic inhibitory approaches in human pulmonary artery EC revealed the participation of P2Y1 and P2Y11 receptors in β-NAD-induced TER increases. The signaling mechanisms involve the activation of PKA, EPAC1 (the cAMP activated GEF for Rap1), small GTPase Rac1, and MLCP [17,36]. In addition, evidence suggests that P2Y11-mediated activation of EPAC1 opposes the pro-inflammatory action of RhoA-ROCK via Tiam-and Vav-mediated Rac activation that strengthens the endothelial adherens junction leading to the prevention of vascular leak and inflammation [77].
Consistent with the in vitro studies, β-NAD attenuated the LPS-induced inflammatory cells infiltration and decreased permeability in the lungs of LPS/β-NAD-treated mice compared to mice treated with LPS alone. Histological specimens from the lung showed increased, and prominent interstitial edema with infiltration of neutrophils in the lung parenchyma of mice treaded with LPS alone compared to the LPS/β-NAD-treated lungs [37]. Therefore, similar to ATP and ATPγS, P2Y agonist β-NAD is a potent positive regulator of the pulmonary endothelial barrier integrity both in vitro and in vivo (Figure 2).

2.4. P2Y Receptors and Regulation of Vessel Growth

Purinergic regulation of vascular growth and development is a rapidly developing concept in vascular biology, focusing on the role of specific P2YR subtypes. In addition to vasoprotective vasodilation, P2Y receptors are critical to angiogenesis, vasculogenesis, and endothelial differentiation. Several cell and animal studies have provided evidence on the importance of nucleotide signaling for vascular growth. A study on P2Y4 knockout mice demonstrated a role for this receptor in cardiac micro-vessel growth, migration, and PDGF-B secretion in response to UTP. This study also showed that P2Y4 is an essential regulator of endothelial-cardiomyocyte interactions in post-natal heart development [106]. Decreased expression of CD39/ENTPDase, the enzyme responsible for extracellular ATP degradation, was observed in plexiform lesions from PAH patients. It was hypothesized that increased extracellular ATP level in the pulmonary vascular endothelium, as well as within the angiomatoid proliferative lesions might promote excessive endothelial proliferation [107]. Importantly, functional crosstalk was observed between purinergic and growth factor receptors. For example, P2Y1 operates via transactivated vascular endothelial growth factor receptor (VEGFR2) in angiogenic signaling [108]. ATP, ADP, and their synthetic analogs (ATPγS, ADPβS, MeSATP, MeSADP) in combination with platelet extract growth factors synergistically increased the number of functional neovessels in matrigel plugs subcutaneously injected in mice [109].

2.4.1. Vasa Vasorum Neovascularization

Proliferative vascular remodeling has a central role in the pathology of many cardiovascular diseases [110,111,112,113,114,115,116]. Vasa vasorum (VV) is the microcirculatory network of the bronchial (systemic) circulation and, similar to its role in systemic vessels, contributes to vascular integrity through the supply of oxygen and nutrients to the outer part of the pulmonary artery (PA) wall. Studies on animal models of PH revealed marked adventitial thickening and expansion of the VV network, which are especially prominent components of the pulmonary vascular remodeling process [117,118]. An increasing body of experimental data has demonstrated that the expansion of the VV might also contribute to the progression of certain vascular diseases in systemic circulation including atherosclerosis, restenosis, vasculitis, and ascending aortic aneurism suggesting that neovascularization of the VV might be an important common feature of specific pulmonary and systemic vascular diseases [119,120,121,122,123,124,125]. Expansion of the bronchial vessels in the ischemic lung parenchyma and the PAs has also been demonstrated in patients with chronic thromboembolic disease, which suggests a unique proliferative and invasive capacity VV endothelial cell (VVEC) [126]. At present, the precise cellular mechanisms and endogenous molecular factors contributing to this process of neovascularization in the vessel wall are not entirely understood. Considering that within the PA adventitial compartment, ATP can be released as a result of the combined action of hypoxia, inflammation, mechanical forces, and sympathetic stimulation, elevated extracellular ATP levels may contribute to purinergic regulation of the pulmonary artery VV neovascularization [118].

2.4.2. Angiogenic Purinergic Signaling in VVEC

The findings from a neonatal bovine model of PH demonstrated that ATP is a potent angiogenic factor for PA VVEC [12,127]. Moreover, comparative studies on the pro-angiogenic effects of ATP in various EC subtypes revealed that extracellular ATP is a more potent mitogen for microvascular ECs (VVEC and lung microvascular EC) than for ECs of large vessels (main PA and aorta EC) [127]. It can be speculated that VVEC in the hypoxic PA adventitia has distinct phenotypic characteristics, with a particular reliance on extracellular nucleotides as pro-angiogenic stimuli. The exceptional sensitivity of microvascular ECs to stimulation with ATP and ADP is supported by other studies showing a mitogenic effect of P2Y agonists like ATP, UTP, and MeSATP on brain capillary and corneal endothelial cells [128,129]. Similarly, it was found that ATP and the other P2Y agonists exerted only weak increases in DNA synthesis in bovine aortic EC, supporting the idea that purinergic signaling has little impact on differentiated endothelial cell proliferation [130,131,132]. In addition, PA adventitial VVEC are a potent source of extracellular ATP, which is released via regulated exocytosis and acts as an autocrine/paracrine factor augmenting hypoxia-induced VVEC angiogenesis [12,25,127]. Pharmacological and genetic (siRNA) approaches revealed that the angiogenic effects of extracellular nucleotides in VVEC are mediated through dramatic and prolonged activation of P2Y1 and P2Y13 receptors and PI3K/mTOR and ERK1/2 pathways, as well as the elevation of cytoplasmic and nucleoplasmic Ca2+, [25,127,133,134,135,136,137,138]. Recent studies on VVEC also demonstrated a critical role of the P2Y-PI3K-Akt-mTOR axis in the activation of c-Jun, c-Myc, and Foxo3a transcription factors; showed a functional significance of these proteins in VVEC angiogenic responses; and identified target genes involved in tissue remodeling, cell cycle control, cell adhesion, and barrier function [69]. Therefore, data on VVEC and lung microvascular EC provide critical evidence on the regulatory role of extracellular nucleotides in angiogenesis [19,109,127,139]. Considering that the responses to extracellular ATP might be particularly important in the hypoxic and inflamed adventitial microenvironment, targeting P2Y receptors may have a potential translational significance in attenuating pathological vascular remodeling associated with many CVDs.

2.4.3. P2Y Receptor Subtypes and VV Neovascularization

As indicated above, pulmonary artery VV angiogenic expansion is a characteristic feature of hypoxia-induced pulmonary vascular remodeling in PH. The expression of P2Y13 has been previously shown in spleen, brain, and circulating CD34+ progenitor cells, but not in vascular endothelium [9,140]. The involvement of P2Y13 in VVEC proliferation is intriguing. Studies on VVEC isolated from PA adventitia demonstrated the expression of P2Y1 (known as an endothelial-specific purinergic receptor) in most VVEC populations isolated from the PA of control (VVEC-Co) and hypoxic (VVEC-Hx) animals, in contrast, the expression of P2Y13 (known as a progenitor cell receptor) was observed to a more considerable extent in VVEC-Hx (Figure 3A,B). Furthermore, the heterogeneous pattern of P2Y1 and P2Y13 expression together with co-expression of P2Y13 with progenitor cell markers CD31/PECAM-1, CD133, and CD34 observed in VVEC-Hx (Figure 3C) suggests that hypoxia-induced VV angiogenic expansion may involve the emergence of P2Y13 expressing highly proliferative progenitor-like cell populations residing in the VV [117]. The expression of P2Y13, well as P2Y1 and P2Y11, was also observed in the VV of the PA adventitia chronically hypoxic Sprague Dawley rats, supporting a potential involvement of these receptors in endothelial differentiation and angiogenesis (Figure 4). Evaluation of the P2Y13 involvement in endothelial angiogenic responses would validate these receptors as novel pharmacological targets for VV neovascularization and pathologic vascular remodeling in PH and other cardiovascular diseases.

2.5. P2Y Receptors and Infantile Hemangioma Development

Infantile hemangiomas are vascular tumors characterized by excessive proliferative/dysregulated endothelial proliferation and spontaneous involution. However, the mechanisms underlying their unique “lifecycle” remain largely unknown. Previous studies on hemangiomas have been restricted by elucidating canonical angiogenic pathways that include vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, IGF-I, Tie-1, Tie-2, bFGF, and angiopoietin [141]. Considering that P2Y-mediated signals are essential for the coordinated cell proliferation, migration, and differentiation in the developing organs and tissues [9,18,142], and the alteration of P2 receptor subtype expression can be implicated in pathologic cellular responses in the vasculature [9,18,143,144,145], it can be expected that hemangioma EC growth and/or regression may be regulated by extracellular nucleotides. Immunohistochemical analysis demonstrated the expression of P2Y13 in both proliferating and involuting infantile hemangiomas (Figure 5). Remarkably, expression of P2Y13 in proliferating hemangiomas was dramatically higher compared to those observed in involuting hemangiomas, suggesting that P2Y13 may be functionally involved in hemangioma expansion. Similar to P2Y13, P2Y1 expression was found to be higher in proliferating hemangiomas. It was also shown that involuting hemangiomas exhibit an overlapping pattern of the expression of endothelial marker CD31/PECAM and P2Y1, indicating that decreased hemangioma vascularity correlates with P2Y1 expression level. These data suggest that the expression of P2Y1 might be more attributable to a more differentiated endothelial phenotype, whereas the expression of P2Y13 is related to less-differentiated progenitor-like phenotype.

3. Conclusions and Perspectives

Endothelial purinergic signaling in CVD remains the area of intensive investigation. With the growing interest in pathological vascular remodeling, inflammation, angiogenesis, acute lung injury, stem cell differentiation, and vascular tumors, the regulatory circuits of extracellular nucleotides that operate via the activation of purinergic receptors may bring a new dimension to a better understanding of CVD mechanisms (Figure 6). Research on purinergic receptor signaling in inflammatory, neuronal, and cancer cells significantly advanced the understanding of the physiological and pathological purinergic signaling mechanisms in many diseases. In contrast, fewer studies have been focused on purinergic signaling in vascular bed-and organ-specific endothelium, though this research direction may uncover previously overlooked involvement of P2Y receptors in endothelial function. Studies on P2Y1, P2Y2, P2Y4, and P2Y6, as well as P2Y13 and P2Y13 receptor knock-out mice, revealed the biochemical and functional diversity of these receptors in various organs. In perspective, a combination of the genetic and advanced pharmacological approaches could recognize vascular bed-specific expression and function of P2Y receptor subtypes and distinguish their contribution to endothelial physiology in CVD. It should also be acknowledged that advances in medicinal chemistry and high-throughput screening of novel P2Y agonists and antagonists will identify the new therapeutic application for targeting P2Y receptors. An existing challenge now relates to identifying purinergic antagonists for therapeutic use, as the most purinoceptors have many polymorphic variations. There is also a need for developing orally bioavailable and stable in-vivo purinergic compounds. A better understanding of R2Y receptor covalent modifications, homo-and hetero-dimerization with purinergic and, possibly, other G-protein-coupled receptors will promise exciting and vital observations in the future. It would not be surprising if purinergic signaling mechanisms, like the one of the most conservative, fundamentally important, and ubiquitous, would be re-discovered as clinically relevant regulatory mechanisms in vascular endothelium.

Author Contributions

Conceptualization: E.G., D.S., and A.V.; Resources: E.G., K.R.S., and A.V.; Data curation: H.N., N.B., R.B., and A.K.-K.; Original draft preparation: E.G., D.S., A.V., U.S., J.K., and Y.S.G.; Review and editing: E.G., A.V., R.B., N.S.U., K.R.S., and V.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Institute of Health R01 HL086783 (E.G.), PPG-HL-14985 (K.R.S), the American Heart Association (AHA) Postdoctoral Fellowship 20POST35210753 (R.B.), the NHLBI Program Project Grant HL101902 (A.V.), and the AHA Grant AHA00161 (A.K.).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

ACAdenylate cyclase
ADPAdenosine triphosphate
ADPβSAdenosine-5′-0-(2-thiodiphosphate)
AKAP2PKA anchoring protein 2
AktSerine/threonine protein kinase
ALIacute lung injury
ApoEApolipoprotein A
ARDSAcute respiratory distress syndrome
ATPγSAdenosine 5′-(3-thiotriphosphate)
AT1R Angiotensin II type I receptor
CAMCell adhesion molecule
CaMKIICalmodulin kinase
cAMPCyclic adenosine monophosphate
CD39/ENTPDaseEctonucleoside triphosphate diphosphohydrolase
CVDsCardiovascular diseases
Cx 37, 40Connexin 37, 40
DAMPdamage-associated molecular pattern
ECEndothelial cells
ECMExtracellular Matrix
EDEndothelial dysfunction
EDHEndothelium-dependent hyperpolarization
eNOSEndothelial nitric oxide synthase
EPACExchange Protein Activated by cAMP
ERK1/2Extracellular signal regulated kinases 1/2
E-selectin CD62 antigen-like family member E
GEFGuanine nucleotide exchange factor
GPCRG-protein coupled receptor
GPR120G-protein coupled receptor 120
Gs, GsG protein subtypes
ICAM-1Intercellular Adhesion Molecule 1
IGF-IInsulin-like growth factor 1
IL-1, -6Interleukin-1, -6
JNKc-Jun N-terminal kinase
KCa3.1The calcium-activated potassium channel KCa3.1
KLFThe Krüppel-like family of transcription factors
LDLLow-density lipoproteins
LMVECLung microvascular endothelial cells
LPSLipopolysaccharide
MeSADP2-(Methylthio)adenosine 5’-diphosphate
MeSATP2-(Methylthio)adenosine 5′-triphosphate
MLCMyosin light chains
MLCPMLC phosphatase
MMPsMatrix metallopeptidases
mTORThe mammalian target of rapamycin
β-NADβ-Nicotinamide adenine dinucleotide
NFkBnuclear factor kappa-light-chain-enhancer of activated B cells
NONitric oxide
NOX2NADPH oxidase catalytic component
PAF Platelet-activating factor
PBBarometric pressure
PIEZOMechanically activated cation channels
PGI2Prostacyclin
PKAProtein kinase A
PKCProtein kinase C
PKGProtein kinase G
PLCPhospholipase C
PLYPneumolysin
PI3KPhosphatidyl inositol 3 kinase
PAHPulmonary arterial hypertension
p38 MAPKp38 Mitogen-activated protein kinases
Rac 1A small GTPase protein
Rap 1A small GTPases protein
ROSReactive oxygen species
ROCKRho protein kinase
TERTransendothelial electrical resistance
Tiam 1T-Lymphoma invasion and metastasis-inducing protein 1
TNFαRTumor necrosis factor alpha receptor
TXA2Thromboxane A2
TLRToll-like receptor
TRPV4Transient receptor potential cation channel subfamily V member 4
T1D, T2DType 1,2 diabetes
UTPUridine triphosphate
Vav Guanine nucleotide exchange factor
VCAM-1Vascular cell adhesion protein 1
VEGFR2Vascular endothelial growth factor receptor-2
VSMCVascular smooth muscle cell
VVECVasa vasorum endothelial cells

References

  1. Di Virgilio, F.; Solini, A. P2 receptors: New potential players in atherosclerosis. Br. J. Pharmacol. 2002, 135, 831–842. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Abbracchio, M.P.; Burnstock, G. Purinergic signalling: Pathophysiological roles. Jpn. J. Pharmacol. 1998, 78, 113–145. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Ralevic, V.; Burnstock, G. Receptors for purines and pyrimidines. Pharmacol. Rev. 1998, 50, 413–492. [Google Scholar] [PubMed]
  4. Burnstock, G. Purine and pyrimidine receptors. Cell Mol. Life Sci. 2007, 64, 1471–1483. [Google Scholar] [CrossRef]
  5. Motte, S.; Pirotton, S.; Boeynaems, J.M. Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells. Br. J. Pharmacol. 1993, 109, 967–971. [Google Scholar] [CrossRef] [Green Version]
  6. Wang, L.; Karlsson, L.; Moses, S.; Hultgardh-Nilsson, A.; Andersson, M.; Borna, C.; Gudbjartsson, T.; Jern, S.; Erlinge, D. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J. Cardiovasc. Pharmacol. 2002, 40, 841–853. [Google Scholar] [CrossRef]
  7. Burnstock, G.; Ulrich, H. Purinergic signaling in embryonic and stem cell development. Cell Mol. Life Sci. 2011, 68, 1369–1394. [Google Scholar] [CrossRef]
  8. Bours, M.J.; Swennen, E.L.; Di Virgilio, F.; Cronstein, B.N.; Dagnelie, P.C. Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol. Ther. 2006, 112, 358–404. [Google Scholar] [CrossRef]
  9. Burnstock, G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol. Rev. 2006, 58, 58–86. [Google Scholar] [CrossRef]
  10. Erlinge, D.; Burnstock, G. P2 receptors in cardiovascular regulation and disease. Purinergic Signal. 2008, 4, 1–20. [Google Scholar] [CrossRef] [Green Version]
  11. Gerasimovskaya, E.V.; Ahmad, S.; White, C.W.; Jones, P.L.; Carpenter, T.C.; Stenmark, K.R. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J. Biol. Chem. 2002, 277, 44638–44650. [Google Scholar] [CrossRef] [Green Version]
  12. Woodward, H.N.; Anwar, A.; Riddle, S.; Taraseviciene-Stewart, L.; Fragoso, M.; Stenmark, K.R.; Gerasimovskaya, E.V. PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 2009, 297, L954–L964. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Sitkovsky, M.; Lukashev, D. Regulation of immune cells by local-tissue oxygen tension: HIF1 alpha and adenosine receptors. Nat. Rev. Immunol. 2005, 5, 712–721. [Google Scholar] [CrossRef]
  14. Wang, S.; Chennupati, R.; Kaur, H.; Iring, A.; Wettschureck, N.; Offermanns, S. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J. Clin. Investig. 2016, 126, 4527–4536. [Google Scholar] [CrossRef]
  15. Corriden, R.; Insel, P.A. Basal release of ATP: An autocrine-paracrine mechanism for cell regulation. Sci. Signal. 2010, 3, re1. [Google Scholar] [CrossRef] [Green Version]
  16. Jacobson, K.A.; Delicado, E.G.; Gachet, C.; Kennedy, C.; von Kugelgen, I.; Li, B.; Miras-Portugal, M.T.; Novak, I.; Schoneberg, T.; Perez-Sen, R.; et al. Update of P2Y receptor pharmacology: IUPHAR Review 27. Br. J. Pharmacol. 2020, 177, 2413–2433. [Google Scholar] [CrossRef]
  17. Zemskov, E.; Lucas, R.; Verin, A.D.; Umapathy, N.S. P2Y receptors as regulators of lung endothelial barrier integrity. J. Cardiovasc. Dis. Res. 2011, 2, 14–22. [Google Scholar] [CrossRef] [Green Version]
  18. Burnstock, G.; Ralevic, V. Purinergic signaling and blood vessels in health and disease. Pharmacol. Rev. 2014, 66, 102–192. [Google Scholar] [CrossRef]
  19. Burnstock, G. Purinergic signaling and vascular cell proliferation and death. Arterioscler. Thromb. Vasc. Biol. 2002, 22, 364–373. [Google Scholar] [CrossRef]
  20. Gerasimovskaya, E.V.; Tucker, D.A.; Stenmark, K.R. Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation. J. Appl. Physiol. 2005, 98, 722–731. [Google Scholar] [CrossRef]
  21. Van der Weyden, L.; Conigrave, A.D.; Morris, M.B. Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor. Immunol. Cell Biol. 2000, 78, 369–374. [Google Scholar] [CrossRef] [PubMed]
  22. Burnstock, G. Purinergic receptors as future targets for treatment of functional GI disorders. Gut 2008, 57, 1193–1194. [Google Scholar] [CrossRef] [PubMed]
  23. Burnstock, G. Purinergic signalling and disorders of the central nervous system. Nat. Rev. Drug Discov. 2008, 7, 575–590. [Google Scholar] [CrossRef] [PubMed]
  24. Bátori, R.; Kumar, S.; Bordán, Z.; Cherian-Shaw, M.; Kovács-Kása, A.; MacDonald, J.A.; Fulton, D.J.R.; Erdődi, F.; Verin, A.D. Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening. J. Cell. Physiol. 2019, 234, 5863–5879. [Google Scholar] [CrossRef] [PubMed]
  25. Lyubchenko, T.; Woodward, H.; Veo, K.D.; Burns, N.; Nijmeh, H.; Liubchenko, G.A.; Stenmark, K.R.; Gerasimovskaya, E.V. P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells. Am. J. Physiol. Cell Physiol. 2011, 300, C266–C275. [Google Scholar] [CrossRef] [Green Version]
  26. Kaczmarek, E.; Erb, L.; Koziak, K.; Jarzyna, R.; Wink, M.R.; Guckelberger, O.; Blusztajn, J.K.; Trinkaus-Randall, V.; Weisman, G.A.; Robson, S.C. Modulation of endothelial cell migration by extracellular nucleotides: Involvement of focal adhesion kinase and phosphatidylinositol 3-kinase-mediated pathways. Thromb. Haemost. 2005, 93, 735–742. [Google Scholar]
  27. Jacobson, J.R.; Dudek, S.M.; Singleton, P.A.; Kolosova, I.A.; Verin, A.D.; Garcia, J.G. Endothelial cell barrier enhancement by ATP is mediated by the small GTPase Rac and cortactin. Am. J. Physiol. Lung Cell Mol. Physiol. 2006, 291, L289–L295. [Google Scholar] [CrossRef] [Green Version]
  28. Lemoli, R.M.; Ferrari, D.; Fogli, M.; Rossi, L.; Pizzirani, C.; Forchap, S.; Chiozzi, P.; Vaselli, D.; Bertolini, F.; Foutz, T.; et al. Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo. Blood 2004, 104, 1662–1670. [Google Scholar] [CrossRef] [Green Version]
  29. Rossi, L.; Manfredini, R.; Bertolini, F.; Ferrari, D.; Fogli, M.; Zini, R.; Salati, S.; Salvestrini, V.; Gulinelli, S.; Adinolfi, E.; et al. The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration. Blood 2007, 109, 533–542. [Google Scholar] [CrossRef] [Green Version]
  30. Satterwhite, C.M.; Farrelly, A.M.; Bradley, M.E. Chemotactic, mitogenic, and angiogenic actions of UTP on vascular endothelial cells. Am. J. Physiol. 1999, 276, H1091–H1097. [Google Scholar] [CrossRef]
  31. Noll, T.; Holschermann, H.; Koprek, K.; Gunduz, D.; Haberbosch, W.; Tillmanns, H.; Piper, H.M. ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i. Am. J. Physiol. 1999, 276, H1892–H1901. [Google Scholar] [CrossRef] [PubMed]
  32. Wirsching, E.; Fauler, M.; Fois, G.; Frick, M. P2 Purinergic Signaling in the Distal Lung in Health and Disease. Int. J. Mol. Sci. 2020, 21, 4973. [Google Scholar] [CrossRef] [PubMed]
  33. Burnstock, G.; Brouns, I.; Adriaensen, D.; Timmermans, J.P. Purinergic signaling in the airways. Pharmacol. Rev. 2012, 64, 834–868. [Google Scholar] [CrossRef] [Green Version]
  34. Matsuyama, H.; Amaya, F.; Hashimoto, S.; Ueno, H.; Beppu, S.; Mizuta, M.; Shime, N.; Ishizaka, A.; Hashimoto, S. Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: An experimental study. Respir. Res. 2008, 9, 79. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Kolosova, I.A.; Mirzapoiazova, T.; Moreno-Vinasco, L.; Sammani, S.; Garcia, J.G.; Verin, A.D. Protective effect of purinergic agonist ATPgammaS against acute lung injury. Am. J. Physiol. Lung Cell Mol. Physiol. 2008, 294, L319–L324. [Google Scholar] [CrossRef] [Green Version]
  36. Umapathy, N.S.; Zemskov, E.A.; Gonzales, J.; Gorshkov, B.A.; Sridhar, S.; Chakraborty, T.; Lucas, R.; Verin, A.D. Extracellular beta-nicotinamide adenine dinucleotide (beta-NAD) promotes the endothelial cell barrier integrity via PKA- and EPAC1/Rac1-dependent actin cytoskeleton rearrangement. J. Cell. Physiol. 2010, 223, 215–223. [Google Scholar] [CrossRef] [Green Version]
  37. Umapathy, N.S.; Gonzales, J.; Fulzele, S.; Kim, K.M.; Lucas, R.; Verin, A.D. beta-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury. Exp. Lung Res. 2012, 38, 223–232. [Google Scholar] [CrossRef] [Green Version]
  38. Aird, W.C. Phenotypic heterogeneity of the endothelium: II. Representative vascular beds. Circ. Res. 2007, 100, 174–190. [Google Scholar] [CrossRef] [Green Version]
  39. Aird, W.C. Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms. Circ. Res. 2007, 100, 158–173. [Google Scholar] [CrossRef]
  40. Li, W.H.; Qiu, Y.; Zhang, H.Q.; Tian, X.X.; Fang, W.G. P2Y2 Receptor and EGFR Cooperate to Promote Prostate Cancer Cell Invasion via ERK1/2 Pathway. PLoS ONE 2015, 10, e0133165. [Google Scholar] [CrossRef]
  41. Aho, J.; Helenius, M.; Vattulainen-Collanus, S.; Alastalo, T.P.; Koskenvuo, J. Extracellular ATP protects endothelial cells against DNA damage. Purinergic Signal. 2016, 12, 575–581. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Wang, S.; Iring, A.; Strilic, B.; Albarran Juarez, J.; Kaur, H.; Troidl, K.; Tonack, S.; Burbiel, J.C.; Muller, C.E.; Fleming, I.; et al. P2Y(2) and Gq/G(1)(1) control blood pressure by mediating endothelial mechanotransduction. J. Clin. Investig. 2015, 125, 3077–3086. [Google Scholar] [CrossRef] [Green Version]
  43. Marziano, C.; Hong, K.; Cope, E.L.; Kotlikoff, M.I.; Isakson, B.E.; Sonkusare, S.K. Nitric Oxide-Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries. J. Am. Heart Assoc. 2017, 6, 12. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Aoyagi, M.; Arvai, A.S.; Tainer, J.A.; Getzoff, E.D. Structural basis for endothelial nitric oxide synthase binding to calmodulin. EMBO J. 2003, 22, 766–775. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Dominguez Rieg, J.A.; Burt, J.M.; Ruth, P.; Rieg, T. P2Y(2) receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37. Acta Physiol. 2015, 213, 628–641. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Zhou, Z.; Lankhuizen, I.M.; van Beusekom, H.M.; Cheng, C.; Duncker, D.J.; Merkus, D. Uridine Adenosine Tetraphosphate-Induced Coronary Relaxation Is Blunted in Swine with Pressure Overload: A Role for Vasoconstrictor Prostanoids. Front. Pharmacol. 2018, 9, 255. [Google Scholar] [CrossRef] [Green Version]
  47. Buford, T.W. Hypertension and aging. Ageing Res. Rev. 2016, 26, 96–111. [Google Scholar] [CrossRef] [Green Version]
  48. Fujii, N.; Nishiyasu, T.; Sigal, R.J.; Boulay, P.; McGarr, G.W.; Kenny, G.P. Aging attenuates adenosine triphosphate-induced, but not muscarinic and nicotinic, cutaneous vasodilation in men. Microcirculation 2018, 25, e12462. [Google Scholar] [CrossRef]
  49. McCluskey, C.; Mooney, L.; Paul, A.; Currie, S. Compromised cardiovascular function in aged rats corresponds with increased expression and activity of calcium/calmodulin dependent protein kinase IIdelta in aortic endothelium. Vasc. Pharmacol. 2019, 118–119, 106560. [Google Scholar] [CrossRef]
  50. Smith, A.R.; Visioli, F.; Frei, B.; Hagen, T.M. Age-related changes in endothelial nitric oxide synthase phosphorylation and nitric oxide dependent vasodilation: Evidence for a novel mechanism involving sphingomyelinase and ceramide-activated phosphatase 2A. Aging Cell 2006, 5, 391–400. [Google Scholar] [CrossRef]
  51. Nishimura, A.; Sunggip, C.; Tozaki-Saitoh, H.; Shimauchi, T.; Numaga-Tomita, T.; Hirano, K.; Ide, T.; Boeynaems, J.M.; Kurose, H.; Tsuda, M.; et al. Purinergic P2Y6 receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension. Sci. Signal. 2016, 9, ra7. [Google Scholar] [CrossRef] [Green Version]
  52. Fujii, N.; Meade, R.D.; McNeely, B.D.; Nishiyasu, T.; Sigal, R.J.; Kenny, G.P. Type 2 diabetes specifically attenuates purinergic skin vasodilatation without affecting muscarinic and nicotinic skin vasodilatation and sweating. Exp. Physiol. 2018, 103, 212–221. [Google Scholar] [CrossRef]
  53. Handa, P.; Tateya, S.; Rizzo, N.O.; Cheng, A.M.; Morgan-Stevenson, V.; Han, C.Y.; Clowes, A.W.; Daum, G.; O’Brien, K.D.; Schwartz, M.W.; et al. Reduced vascular nitric oxide-cGMP signaling contributes to adipose tissue inflammation during high-fat feeding. Arterioscler. Thromb. Vasc. Biol. 2011, 31, 2827–2835. [Google Scholar] [CrossRef] [Green Version]
  54. Ishida, K.; Matsumoto, T.; Taguchi, K.; Kamata, K.; Kobayashi, T. Mechanisms underlying reduced P2Y(1) -receptor-mediated relaxation in superior mesenteric arteries from long-term streptozotocin-induced diabetic rats. Acta Physiol. 2013, 207, 130–141. [Google Scholar] [CrossRef] [PubMed]
  55. Zhou, R.; Dang, X.; Sprague, R.S.; Mustafa, S.J.; Zhou, Z. Alteration of purinergic signaling in diabetes: Focus on vascular function. J. Mol. Cell Cardiol. 2020, 140, 1–9. [Google Scholar] [CrossRef] [PubMed]
  56. Rizzo, N.O.; Maloney, E.; Pham, M.; Luttrell, I.; Wessells, H.; Tateya, S.; Daum, G.; Handa, P.; Schwartz, M.W.; Kim, F. Reduced NO-cGMP signaling contributes to vascular inflammation and insulin resistance induced by high-fat feeding. Arterioscler. Thromb. Vasc. Biol. 2010, 30, 758–765. [Google Scholar] [CrossRef]
  57. Danila, M.D.; Privistirescu, A.; Duicu, O.M.; Ratiu, C.D.; Angoulvant, D.; Muntean, D.M.; Sturza, A. The effect of purinergic signaling via the P2Y11 receptor on vascular function in a rat model of acute inflammation. Mol. Cell Biochem. 2017, 431, 37–44. [Google Scholar] [CrossRef]
  58. Pendyala, S.; Usatyuk, P.V.; Gorshkova, I.A.; Garcia, J.G.; Natarajan, V. Regulation of NADPH oxidase in vascular endothelium: The role of phospholipases, protein kinases, and cytoskeletal proteins. Antioxid. Redox Signal. 2009, 11, 841–860. [Google Scholar] [CrossRef] [Green Version]
  59. Diaz-Vegas, A.; Campos, C.A.; Contreras-Ferrat, A.; Casas, M.; Buvinic, S.; Jaimovich, E.; Espinosa, A. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells. PLoS ONE 2015, 10, e0129882. [Google Scholar] [CrossRef] [Green Version]
  60. Santillo, M.; Colantuoni, A.; Mondola, P.; Guida, B.; Damiano, S. NOX signaling in molecular cardiovascular mechanisms involved in the blood pressure homeostasis. Front. Physiol. 2015, 6, 194. [Google Scholar] [CrossRef]
  61. Nguyen Dinh Cat, A.; Montezano, A.C.; Burger, D.; Touyz, R.M. Angiotensin II, NADPH oxidase, and redox signaling in the vasculature. Antioxid. Redox Signal. 2013, 19, 1110–1120. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Kalwa, H.; Sartoretto, J.L.; Martinelli, R.; Romero, N.; Steinhorn, B.S.; Tao, M.; Ozaki, C.K.; Carman, C.V.; Michel, T. Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium. Proc. Natl. Acad. Sci. USA 2014, 111, 3383–3388. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Riegel, A.K.; Faigle, M.; Zug, S.; Rosenberger, P.; Robaye, B.; Boeynaems, J.M.; Idzko, M.; Eltzschig, H.K. Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation. Blood 2011, 117, 2548–2555. [Google Scholar] [CrossRef] [Green Version]
  64. Agca, Y.; Qian, S.; Agca, C.; Seye, C.I. Direct Evidence for P2Y2 Receptor Involvement in Vascular Response to Injury. J. Vasc. Res. 2016, 53, 163–171. [Google Scholar] [CrossRef] [Green Version]
  65. Yang, Y.; Delalio, L.J.; Best, A.K.; Macal, E.; Milstein, J.; Donnelly, I.; Miller, A.M.; McBride, M.; Shu, X.; Koval, M.; et al. Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium. J. Immunol. 2020, 204, 2995–3007. [Google Scholar] [CrossRef]
  66. Gidlof, O.; Sathanoori, R.; Magistri, M.; Faghihi, M.A.; Wahlestedt, C.; Olde, B.; Erlinge, D. Extracellular Uridine Triphosphate and Adenosine Triphosphate Attenuate Endothelial Inflammation through miR-22-Mediated ICAM-1 Inhibition. J. Vasc. Res. 2015, 52, 71–80. [Google Scholar] [CrossRef]
  67. Horckmans, M.; Esfahani, H.; Beauloye, C.; Clouet, S.; di Pietrantonio, L.; Robaye, B.; Balligand, J.L.; Boeynaems, J.M.; Dessy, C.; Communi, D. Loss of mouse P2Y4 nucleotide receptor protects against myocardial infarction through endothelin-1 downregulation. J. Immunol. 2015, 194, 1874–1881. [Google Scholar] [CrossRef] [Green Version]
  68. Feliu, C.; Peyret, H.; Poitevin, G.; Cazaubon, Y.; Oszust, F.; Nguyen, P.; Millart, H.; Djerada, Z. Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs. Int. J. Mol. Sci. 2019, 20, 1446. [Google Scholar] [CrossRef] [Green Version]
  69. Strassheim, D.; Karoor, V.; Nijmeh, H.; Weston, P.; Lapel, M.; Schaack, J.; Sullivan, T.; Dempsey, E.C.; Stenmark, K.R.; Gerasimovskaya, E. c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells. Cells 2020, 9, 416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Danila, M.D.; Piollet, M.; Aburel, O.M.; Angoulvant, D.; Lefort, C.; Chadet, S.; Roger, S.; Muntean, M.D.; Ivanes, F. Modulation of P2Y11-related purinergic signaling in inflammation and cardio-metabolic diseases. Eur. J. Pharmacol. 2020, 876, 173060. [Google Scholar] [CrossRef] [PubMed]
  71. Kuang, Y.; Liu, H.; Guo, S.; Wang, Y.; Zhang, H.; Qiao, Y. The antagonist of P2Y11 receptor NF157 ameliorates oxidized LDL-induced vascular endothelial inflammation. Artif. Cells Nanomed. Biotechnol. 2019, 47, 1839–1845. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Ng, P.Y.; McIntosh, K.A.; Hargrave, G.; Ho, K.H.; Paul, A.; Plevin, R. Inhibition of cytokine-mediated JNK signalling by purinergic P2Y11 receptors, a novel protective mechanism in endothelial cells. Cell Signal. 2018, 51, 59–71. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Zylberg, J.; Ecke, D.; Fischer, B.; Reiser, G. Structure and ligand-binding site characteristics of the human P2Y11 nucleotide receptor deduced from computational modelling and mutational analysis. Biochem. J. 2007, 405, 277–286. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Hapil, F.Z.; Copuroglu, F.E.; Ertosun, M.G.; Mert, U.; Ozes, D.; Ozes, O.N. Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1. J. Interferon Cytokine Res. 2020, 40, 225–235. [Google Scholar] [CrossRef]
  75. Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; et al. Activation of P2 × 7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling. Sci. Rep. 2016, 6, 32602. [Google Scholar] [CrossRef]
  76. Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; et al. Erratum: Activation of P2 × 7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling. Sci. Rep. 2016, 6, 35897. [Google Scholar] [CrossRef] [Green Version]
  77. Lezoualc’h, F.; Fazal, L.; Laudette, M.; Conte, C. Cyclic AMP Sensor EPAC Proteins and Their Role in Cardiovascular Function and Disease. Circ. Res. 2016, 118, 881–897. [Google Scholar] [CrossRef]
  78. Qian, S.; Hoggatt, A.; Jones-Hall, Y.L.; Ware, C.F.; Herring, P.; Seye, C.I. Deletion of P2Y2 receptor reveals a role for lymphotoxin-alpha in fatty streak formation. Vasc. Pharmacol. 2016, 85, 11–20. [Google Scholar] [CrossRef] [Green Version]
  79. Eun, S.Y.; Park, S.W.; Lee, J.H.; Chang, K.C.; Kim, H.J. P2Y(2)R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production. Free Radic. Biol. Med. 2014, 69, 157–166. [Google Scholar] [CrossRef]
  80. Jin, H.; Ko, Y.S.; Park, S.W.; Kim, H.J. P2Y2R activation by ATP induces oxLDL-mediated inflammasome activation through modulation of mitochondrial damage in human endothelial cells. Free Radic. Biol. Med. 2019, 136, 109–117. [Google Scholar] [CrossRef]
  81. Chen, X.; Qian, S.; Hoggatt, A.; Tang, H.; Hacker, T.A.; Obukhov, A.G.; Herring, P.B.; Seye, C.I. Endothelial Cell-Specific Deletion of P2Y2 Receptor Promotes Plaque Stability in Atherosclerosis-Susceptible ApoE-Null Mice. Arterioscler. Thromb. Vasc. Biol. 2017, 37, 75–83. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Stachon, P.; Peikert, A.; Michel, N.A.; Hergeth, S.; Marchini, T.; Wolf, D.; Dufner, B.; Hoppe, N.; Ayata, C.K.; Grimm, M.; et al. P2Y6 deficiency limits vascular inflammation and atherosclerosis in mice. Arterioscler. Thromb. Vasc. Biol. 2014, 34, 2237–2245. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Jiang, T.; Jiang, D.; You, D.; Zhang, L.; Liu, L.; Zhao, Q. Agonism of GPR120 prevents ox-LDL-induced attachment of monocytes to endothelial cells. Chem. Biol. Interact. 2020, 316, 108916. [Google Scholar] [CrossRef] [PubMed]
  84. SenBanerjee, S.; Lin, Z.; Atkins, G.B.; Greif, D.M.; Rao, R.M.; Kumar, A.; Feinberg, M.W.; Chen, Z.; Simon, D.I.; Luscinskas, F.W.; et al. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J. Exp. Med. 2004, 199, 1305–1315. [Google Scholar] [CrossRef] [Green Version]
  85. Ding, L.; Ma, W.; Littmann, T.; Camp, R.; Shen, J. The P2Y(2) nucleotide receptor mediates tissue factor expression in human coronary artery endothelial cells. J. Biol. Chem. 2011, 286, 27027–27038. [Google Scholar] [CrossRef] [Green Version]
  86. Liu, Y.; Zhang, L.; Wang, C.; Roy, S.; Shen, J. Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR. J. Biol. Chem. 2016, 291, 1553–1563. [Google Scholar] [CrossRef] [Green Version]
  87. Johnson, E.R.; Matthay, M.A. Acute lung injury: Epidemiology, pathogenesis, and treatment. J. Aerosol Med. Pulm. Drug Deliv. 2010, 23, 243–252. [Google Scholar] [CrossRef]
  88. Rubenfeld, G.D.; Caldwell, E.; Peabody, E.; Weaver, J.; Martin, D.P.; Neff, M.; Stern, E.J.; Hudson, L.D. Incidence and outcomes of acute lung injury. N. Engl. J. Med. 2005, 353, 1685–1693. [Google Scholar] [CrossRef] [Green Version]
  89. Butt, Y.; Kurdowska, A.; Allen, T.C. Acute Lung Injury: A Clinical and Molecular Review. Arch. Pathol. Lab. Med. 2016, 140, 345–350. [Google Scholar] [CrossRef] [Green Version]
  90. Corrales-Medina, V.F.; Musher, D.M.; Wells, G.A.; Chirinos, J.A.; Chen, L.; Fine, M.J. Cardiac complications in patients with community-acquired pneumonia: Incidence, timing, risk factors, and association with short-term mortality. Circulation 2012, 125, 773–781. [Google Scholar] [CrossRef] [Green Version]
  91. Katzan, I.L.; Cebul, R.D.; Husak, S.H.; Dawson, N.V.; Baker, D.W. The effect of pneumonia on mortality among patients hospitalized for acute stroke. Neurology 2003, 60, 620–625. [Google Scholar] [CrossRef] [PubMed]
  92. Katzan, I.L.; Dawson, N.V.; Thomas, C.L.; Votruba, M.E.; Cebul, R.D. The cost of pneumonia after acute stroke. Neurology 2007, 68, 1938–1943. [Google Scholar] [CrossRef] [PubMed]
  93. Stein, E.; Ramakrishna, H.; Augoustides, J.G. Recent advances in chronic thromboembolic pulmonary hypertension. J. Cardiothorac. Vasc. Anesth. 2011, 25, 744–748. [Google Scholar] [CrossRef] [PubMed]
  94. Wettschureck, N.; Strilic, B.; Offermanns, S. Passing the Vascular Barrier: Endothelial Signaling Processes Controlling Extravasation. Physiol. Rev. 2019, 99, 1467–1525. [Google Scholar] [CrossRef]
  95. Wang, Z.; Ginnan, R.; Abdullaev, I.F.; Trebak, M.; Vincent, P.A.; Singer, H.A. Calcium/Calmodulin-dependent protein kinase II delta 6 (CaMKIIdelta6) and RhoA involvement in thrombin-induced endothelial barrier dysfunction. J. Biol. Chem. 2010, 285, 21303–21312. [Google Scholar] [CrossRef] [Green Version]
  96. Eckle, T.; Faigle, M.; Grenz, A.; Laucher, S.; Thompson, L.F.; Eltzschig, H.K. A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood 2008, 111, 2024–2035. [Google Scholar] [CrossRef] [Green Version]
  97. Kovacs-Kasa, A.; Kim, K.M.; Cherian-Shaw, M.; Black, S.M.; Fulton, D.J.; Verin, A.D. Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role. J. Cell. Physiol. 2018, 233, 5736–5746. [Google Scholar] [CrossRef]
  98. Umapathy, N.S.; Fan, Z.; Zemskov, E.A.; Alieva, I.B.; Black, S.M.; Verin, A.D. Molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement. Vasc. Pharmacol. 2010, 52, 199–206. [Google Scholar] [CrossRef] [Green Version]
  99. Umapathy, S.N.; Kaczmarek, E.; Fatteh, N.; Burns, N.; Lucas, R.; Stenmark, K.R.; Verin, A.D.; Gerasimovskaya, E.V. Adenosine A1 receptors promote vasa vasorum endothelial cell barrier integrity via Gi and Akt-dependent actin cytoskeleton remodeling. PLoS ONE 2013, 8, e59733. [Google Scholar] [CrossRef]
  100. Verin, A.D.; Batori, R.; Kovacs-Kasa, A.; Cherian-Shaw, M.; Kumar, S.; Czikora, I.; Karoor, V.; Strassheim, D.; Stenmark, K.R.; Gerasimovskaya, E. Extracellular adenosine enhances pulmonary artery vasa vasorum endothelium cell barrier function via the Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms. Am. J. Physiol. Cell Physiol. 2020, 319, C183–C193. [Google Scholar] [CrossRef]
  101. Kolosova, I.A.; Mirzapoiazova, T.; Adyshev, D.; Usatyuk, P.; Romer, L.H.; Jacobson, J.R.; Natarajan, V.; Pearse, D.B.; Garcia, J.G.; Verin, A.D. Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement. Circ. Res. 2005, 97, 115–124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  102. Smyth, L.M.; Bobalova, J.; Mendoza, M.G.; Lew, C.; Mutafova-Yambolieva, V.N. Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J. Biol. Chem. 2004, 279, 48893–48903. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  103. Billington, R.A.; Bruzzone, S.; De Flora, A.; Genazzani, A.A.; Koch-Nolte, F.; Ziegler, M.; Zocchi, E. Emerging functions of extracellular pyridine nucleotides. Mol. Med. 2006, 12, 324–327. [Google Scholar] [CrossRef] [PubMed]
  104. Ziegler, M.; Niere, M. NAD+ surfaces again. Biochem. J. 2004, 382, e5–e6. [Google Scholar] [CrossRef]
  105. Koch-Nolte, F.; Fischer, S.; Haag, F.; Ziegler, M. Compartmentation of NAD+-dependent signalling. FEBS Lett. 2011, 585, 1651–1656. [Google Scholar] [CrossRef] [Green Version]
  106. Horckmans, M.; Robaye, B.; Leon-Gomicronmez, E.; Lantz, N.; Unger, P.; Dol-Gleizes, F.; Clouet, S.; Cammarata, D.; Schaeffer, P.; Savi, P.; et al. P2Y(4) nucleotide receptor: A novel actor in post-natal cardiac development. Angiogenesis 2012, 15, 349–360. [Google Scholar] [CrossRef]
  107. Visovatti, S.H.; Hyman, M.C.; Goonewardena, S.N.; Anyanwu, A.C.; Kanthi, Y.; Robichaud, P.; Wang, J.; Petrovic-Djergovic, D.; Rattan, R.; Burant, C.F.; et al. Purinergic dysregulation in pulmonary hypertension. Am. J. Physiol. Heart Circ. Physiol. 2016, 311, H286–H298. [Google Scholar] [CrossRef] [Green Version]
  108. Rumjahn, S.M.; Yokdang, N.; Baldwin, K.A.; Thai, J.; Buxton, I.L. Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis. Br. J. Cancer 2009, 100, 1465–1470. [Google Scholar] [CrossRef] [Green Version]
  109. Roedersheimer, M.; Nijmeh, H.; Burns, N.; Sidiakova, A.A.; Stenmark, K.R.; Gerasimovskaya, E.V. Complementary effects of extracellular nucleotides and platelet-derived extracts on angiogenesis of vasa vasorum endothelial cells in vitro and subcutaneous Matrigel plugs in vivo. Vasc. Cell 2011, 3, 4. [Google Scholar] [CrossRef] [Green Version]
  110. Davie, N.J.; Crossno, J.T., Jr.; Frid, M.G.; Hofmeister, S.E.; Reeves, J.T.; Hyde, D.M.; Carpenter, T.C.; Brunetti, J.A.; McNiece, I.K.; Stenmark, K.R. Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: Contribution of progenitor cells. Am. J. Physiol. Lung Cell Mol. Physiol. 2004, 286, L668–L678. [Google Scholar] [CrossRef]
  111. Herrmann, J.; Lerman, L.O.; Rodriguez-Porcel, M.; Holmes, D.R., Jr.; Richardson, D.M.; Ritman, E.L.; Lerman, A. Coronary vasa vasorum neovascularization precedes epicardial endothelial dysfunction in experimental hypercholesterolemia. Cardiovasc. Res. 2001, 51, 762–766. [Google Scholar] [CrossRef]
  112. Davie, N.J.; Gerasimovskaya, E.V.; Hofmeister, S.E.; Richman, A.P.; Jones, P.L.; Reeves, J.T.; Stenmark, K.R. Pulmonary artery adventitial fibroblasts cooperate with vasa vasorum endothelial cells to regulate vasa vasorum neovascularization: A process mediated by hypoxia and endothelin-1. Am. J. Pathol. 2006, 168, 1793–1807. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  113. Langheinrich, A.C.; Kampschulte, M.; Buch, T.; Bohle, R.M. Vasa vasorum and atherosclerosis—Quid novi? Thromb. Haemost. 2007, 97, 873–879. [Google Scholar] [PubMed]
  114. Mulligan-Kehoe, M.J. The vasa vasorum in diseased and nondiseased arteries. Am. J. Physiol. Heart Circ. Physiol. 2010, 298, H295–H305. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  115. Voelkel, N.F.; Douglas, I.S.; Nicolls, M. Angiogenesis in chronic lung disease. Chest 2007, 131, 874–879. [Google Scholar] [CrossRef] [Green Version]
  116. Sakao, S.; Tatsumi, K.; Voelkel, N.F. Endothelial cells and pulmonary arterial hypertension: Apoptosis, proliferation, interaction and transdifferentiation. Respir. Res. 2009, 10, 95. [Google Scholar] [CrossRef] [Green Version]
  117. Nijmeh, H.; Balasubramaniam, V.; Burns, N.; Ahmad, A.; Stenmark, K.R.; Gerasimovskaya, E.V. High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization. Am. J. Physiol. Lung Cell Mol. Physiol. 2014, 306, L661–L671. [Google Scholar] [CrossRef] [Green Version]
  118. Stenmark, K.R.; Yeager, M.E.; El Kasmi, K.C.; Nozik-Grayck, E.; Gerasimovskaya, E.V.; Li, M.; Riddle, S.R.; Frid, M.G. The adventitia: Essential regulator of vascular wall structure and function. Annu. Rev. Physiol. 2013, 75, 23–47. [Google Scholar] [CrossRef] [Green Version]
  119. Billaud, M.; Hill, J.C.; Richards, T.D.; Gleason, T.G.; Phillippi, J.A. Medial Hypoxia and Adventitial Vasa Vasorum Remodeling in Human Ascending Aortic Aneurysm. Front. Cardiovasc. Med. 2018, 5, 124. [Google Scholar] [CrossRef]
  120. Mulligan-Kehoe, M.J.; Simons, M. Vasa vasorum in normal and diseased arteries. Circulation 2014, 129, 2557–2566. [Google Scholar] [CrossRef]
  121. Numano, F. Vasa vasoritis, vasculitis and atherosclerosis. Int. J. Cardiol. 2000, 75 (Suppl. 1), S1–S8. [Google Scholar] [CrossRef]
  122. Hamaoka-Okamoto, A.; Suzuki, C.; Yahata, T.; Ikeda, K.; Nagi-Miura, N.; Ohno, N.; Arai, Y.; Tanaka, H.; Takamatsu, T.; Hamaoka, K. The involvement of the vasa vasorum in the development of vasculitis in animal model of Kawasaki disease. Pediatr. Rheumatol. Online J. 2014, 12, 12. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  123. Kawabe, J.; Hasebe, N. Role of the vasa vasorum and vascular resident stem cells in atherosclerosis. Biomed. Res. Int. 2014, 2014, 701571. [Google Scholar] [CrossRef]
  124. Sedding, D.G.; Boyle, E.C.; Demandt, J.A.F.; Sluimer, J.C.; Dutzmann, J.; Haverich, A.; Bauersachs, J. Vasa Vasorum Angiogenesis: Key Player in the Initiation and Progression of Atherosclerosis and Potential Target for the Treatment of Cardiovascular Disease. Front. Immunol. 2018, 9, 706. [Google Scholar] [CrossRef] [Green Version]
  125. Xu, J.; Lu, X.; Shi, G.P. Vasa vasorum in atherosclerosis and clinical significance. Int. J. Mol. Sci. 2015, 16, 11574–11608. [Google Scholar] [CrossRef] [Green Version]
  126. Mitzner, W.; Wagner, E.M. Vascular remodeling in the circulations of the lung. J. Appl. Physiol. 2004, 97, 1999–2004. [Google Scholar] [CrossRef]
  127. Gerasimovskaya, E.V.; Woodward, H.N.; Tucker, D.A.; Stenmark, K.R. Extracellular ATP is a pro-angiogenic factor for pulmonary artery vasa vasorum endothelial cells. Angiogenesis 2008, 11, 169–182. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  128. Albert, J.L.; Boyle, J.P.; Roberts, J.A.; Challiss, R.A.; Gubby, S.E.; Boarder, M.R. Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase. Br. J. Pharmacol. 1997, 122, 935–941. [Google Scholar] [CrossRef] [Green Version]
  129. Cha, S.H.; Hahn, T.W.; Sekine, T.; Lee, K.H.; Endou, H. Purinoceptor-mediated calcium mobilization and cellular proliferation in cultured bovine corneal endothelial cells. Jpn. J. Pharmacol. 2000, 82, 181–187. [Google Scholar] [CrossRef] [Green Version]
  130. Van Daele, P.; Van Coevorden, A.; Roger, P.P.; Boeynaems, J.M. Effects of adenine nucleotides on the proliferation of aortic endothelial cells. Circ. Res. 1992, 70, 82–90. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  131. Hirakawa, M.; Oike, M.; Karashima, Y.; Ito, Y. Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium. J. Physiol. 2004, 558, 479–488. [Google Scholar] [CrossRef] [PubMed]
  132. Yamamoto, K.; Sokabe, T.; Ohura, N.; Nakatsuka, H.; Kamiya, A.; Ando, J. Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells. Am. J. Physiol. Heart Circ. Physiol. 2003, 285, H793–H803. [Google Scholar] [CrossRef] [Green Version]
  133. Morello, F.; Perino, A.; Hirsch, E. Phosphoinositide 3-kinase signalling in the vascular system. Cardiovasc. Res. 2009, 82, 261–271. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  134. Vanhaesebroeck, B.; Waterfield, M.D. Signaling by distinct classes of phosphoinositide 3-kinases. Exp. Cell Res. 1999, 253, 239–254. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  135. Guba, M.; von Breitenbuch, P.; Steinbauer, M.; Koehl, G.; Flegel, S.; Hornung, M.; Bruns, C.J.; Zuelke, C.; Farkas, S.; Anthuber, M.; et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: Involvement of vascular endothelial growth factor. Nat. Med. 2002, 8, 128–135. [Google Scholar] [CrossRef] [PubMed]
  136. Jiang, B.H.; Zheng, J.Z.; Aoki, M.; Vogt, P.K. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc. Natl. Acad. Sci. USA 2000, 97, 1749–1753. [Google Scholar] [CrossRef] [Green Version]
  137. Wullschleger, S.; Loewith, R.; Hall, M.N. TOR signaling in growth and metabolism. Cell 2006, 124, 471–484. [Google Scholar] [CrossRef] [Green Version]
  138. Carr, E.L.; Kelman, A.; Wu, G.S.; Gopaul, R.; Senkevitch, E.; Aghvanyan, A.; Turay, A.M.; Frauwirth, K.A. Glutamine uptake and metabolism are coordinately regulated by ERK/MAPK during T lymphocyte activation. J. Immunol. 2010, 185, 1037–1044. [Google Scholar] [CrossRef] [Green Version]
  139. Abbracchio, M.P.; Burnstock, G.; Boeynaems, J.M.; Barnard, E.A.; Boyer, J.L.; Kennedy, C.; Knight, G.E.; Fumagalli, M.; Gachet, C.; Jacobson, K.A.; et al. International Union of Pharmacology LVIII: Update on the P2Y G protein-coupled nucleotide receptors: From molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 2006, 58, 281–341. [Google Scholar] [CrossRef]
  140. Wang, L.; Jacobsen, S.E.; Bengtsson, A.; Erlinge, D. P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells. BMC Immunol. 2004, 5, 16. [Google Scholar] [CrossRef] [Green Version]
  141. Phung, T.L.; Hochman, M.; Mihm, M.C. Current knowledge of the pathogenesis of infantile hemangiomas. Arch. Facial Plast. Surg. 2005, 7, 319–321. [Google Scholar] [CrossRef] [PubMed]
  142. Yegutkin, G.G. Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim. Biophys. Acta 2008, 1783, 673–694. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  143. Marteau, F.; Communi, D.; Boeynaems, J.M.; Suarez Gonzalez, N. Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte-derived dendritic cells. J. Leukoc. Biol. 2004, 76, 796–803. [Google Scholar] [CrossRef] [PubMed]
  144. Seye, C.I.; Kong, Q.; Erb, L.; Garrad, R.C.; Krugh, B.; Wang, M.; Turner, J.T.; Sturek, M.; Gonzalez, F.A.; Weisman, G.A. Functional P2Y2 nucleotide receptors mediate uridine 5′-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries. Circulation 2002, 106, 2720–2726. [Google Scholar] [CrossRef] [Green Version]
  145. Seye, C.I.; Yu, N.; Gonzalez, F.A.; Erb, L.; Weisman, G.A. The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1). J. Biol. Chem. 2004, 279, 35679–35686. [Google Scholar] [CrossRef] [Green Version]
Figure 1. P2Y4 and P2Y12 mediate lung vascular barrier function. (A): Quantitative real-time polymerase chain reaction (qPCR) analysis of Gαi in lung microvascular endothelial cells; (B): depletion of Gαi2 attenuated ATPγS-induced increase of transendothelial electrical resistance (TER). Human lung microvascular endothelial cells (HLMVECs) were transfected with non-specific siRNA (nsRNA) or Gαi-specific siRNAs (50 nM, 72 h) and were treated with ATPγS (100 µM, arrow). Inset: Western blot analysis shows the efficiency of Gαi depletion.
Figure 1. P2Y4 and P2Y12 mediate lung vascular barrier function. (A): Quantitative real-time polymerase chain reaction (qPCR) analysis of Gαi in lung microvascular endothelial cells; (B): depletion of Gαi2 attenuated ATPγS-induced increase of transendothelial electrical resistance (TER). Human lung microvascular endothelial cells (HLMVECs) were transfected with non-specific siRNA (nsRNA) or Gαi-specific siRNAs (50 nM, 72 h) and were treated with ATPγS (100 µM, arrow). Inset: Western blot analysis shows the efficiency of Gαi depletion.
Ijms 21 06855 g001
Figure 2. P2Y-mediated mechanisms of lung endothelial barrier protection. P2Y agonists (ATP, ATPγS, or β-NAD) enhance endothelial barrier properties via consequential activation of heterotrimeric G-proteins and enzymes, like myosin light chain phosphatase (MLCP) and PKA, followed by cytoskeletal rearrangement, increased cell-cell contacts, and tightening of endothelial cell (EC) barrier, thus opposing acute lung injury progression.
Figure 2. P2Y-mediated mechanisms of lung endothelial barrier protection. P2Y agonists (ATP, ATPγS, or β-NAD) enhance endothelial barrier properties via consequential activation of heterotrimeric G-proteins and enzymes, like myosin light chain phosphatase (MLCP) and PKA, followed by cytoskeletal rearrangement, increased cell-cell contacts, and tightening of endothelial cell (EC) barrier, thus opposing acute lung injury progression.
Ijms 21 06855 g002
Figure 3. P2Y1 and P2Y13 receptor expression in pulmonary artery vasa vasorum endothelial cells (VVECs). Cells were isolated from the pulmonary arteries of control and chronically hypoxic hypertensive calves. (A,B): WB analysis of P2Y1R and P2Y13R expression in VVEC-Co and VVEC-Hx. Blue arrows indicate increased expression of P2Y1R compared to P2Y13R in some VVEC-Co populations; red arrows indicate increased expression of P2Y13 compared to P2Y1R in some VVEC-Hx populations; (C): Immunofluorescent analysis of P2Y13 (red) co-expression with CD31/PECAM, CD34, and CD133 (green) in VVEC-Hx.
Figure 3. P2Y1 and P2Y13 receptor expression in pulmonary artery vasa vasorum endothelial cells (VVECs). Cells were isolated from the pulmonary arteries of control and chronically hypoxic hypertensive calves. (A,B): WB analysis of P2Y1R and P2Y13R expression in VVEC-Co and VVEC-Hx. Blue arrows indicate increased expression of P2Y1R compared to P2Y13R in some VVEC-Co populations; red arrows indicate increased expression of P2Y13 compared to P2Y1R in some VVEC-Hx populations; (C): Immunofluorescent analysis of P2Y13 (red) co-expression with CD31/PECAM, CD34, and CD133 (green) in VVEC-Hx.
Ijms 21 06855 g003
Figure 4. P2Y1, P2Y11, and P2Y13 purinergic receptors are expressed in angiogenic pulmonary artery vasa vasorum. Immunofluorescent analysis of frozen acetone/methanol-fixed tissue sections of control and chronically hypoxic rats (4 weeks, (barometric pressure) PB 430 mmHg) revealed pulmonary artery adventitial vasa vasorum expansion (white arrows) and the expression of P2Y1, P2Y11, and P2Y13 receptors (green), and α-actin (red) in chronically hypoxic, but not control animals; scale bar = 100 μm.
Figure 4. P2Y1, P2Y11, and P2Y13 purinergic receptors are expressed in angiogenic pulmonary artery vasa vasorum. Immunofluorescent analysis of frozen acetone/methanol-fixed tissue sections of control and chronically hypoxic rats (4 weeks, (barometric pressure) PB 430 mmHg) revealed pulmonary artery adventitial vasa vasorum expansion (white arrows) and the expression of P2Y1, P2Y11, and P2Y13 receptors (green), and α-actin (red) in chronically hypoxic, but not control animals; scale bar = 100 μm.
Ijms 21 06855 g004
Figure 5. P2Y13 and P2Y1 receptor expression in proliferating and involuting infantile hemangiomas. (a,c,e,g): Double immunofluorescent analysis for P2Y13 and P2Y1 receptors (Alexa 594, red) and PECAM/CD31 (Alexa 488, green); nuclei were counterstained with DAPI (blue; Carl Zeiss, x40 magnification; (b,d,f,h): Immunohistochemical analysis for P2Y13 and P2Y1 receptors (AEC substrate, Olympus, X40 magnification). Tissue sections were kindly provided by Dr. Sule Cataltepe (Children’s Hospital, Boston, MA, USA).
Figure 5. P2Y13 and P2Y1 receptor expression in proliferating and involuting infantile hemangiomas. (a,c,e,g): Double immunofluorescent analysis for P2Y13 and P2Y1 receptors (Alexa 594, red) and PECAM/CD31 (Alexa 488, green); nuclei were counterstained with DAPI (blue; Carl Zeiss, x40 magnification; (b,d,f,h): Immunohistochemical analysis for P2Y13 and P2Y1 receptors (AEC substrate, Olympus, X40 magnification). Tissue sections were kindly provided by Dr. Sule Cataltepe (Children’s Hospital, Boston, MA, USA).
Ijms 21 06855 g005
Figure 6. Overview of the molecular mechanisms and endpoints on the involvement of endothelial P2Y receptors in cardiovascular disease (CVD). Pathological responses to CVD include the release of extracellular purines at the sites of injury, following by activation of P2Y receptors and specific P2YR-mediated intracellular pathways leading to either injury progression, resolution, or both in tissue- and agonist-specific manner.
Figure 6. Overview of the molecular mechanisms and endpoints on the involvement of endothelial P2Y receptors in cardiovascular disease (CVD). Pathological responses to CVD include the release of extracellular purines at the sites of injury, following by activation of P2Y receptors and specific P2YR-mediated intracellular pathways leading to either injury progression, resolution, or both in tissue- and agonist-specific manner.
Ijms 21 06855 g006

Share and Cite

MDPI and ACS Style

Strassheim, D.; Verin, A.; Batori, R.; Nijmeh, H.; Burns, N.; Kovacs-Kasa, A.; Umapathy, N.S.; Kotamarthi, J.; Gokhale, Y.S.; Karoor, V.; et al. P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases. Int. J. Mol. Sci. 2020, 21, 6855. https://doi.org/10.3390/ijms21186855

AMA Style

Strassheim D, Verin A, Batori R, Nijmeh H, Burns N, Kovacs-Kasa A, Umapathy NS, Kotamarthi J, Gokhale YS, Karoor V, et al. P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases. International Journal of Molecular Sciences. 2020; 21(18):6855. https://doi.org/10.3390/ijms21186855

Chicago/Turabian Style

Strassheim, Derek, Alexander Verin, Robert Batori, Hala Nijmeh, Nana Burns, Anita Kovacs-Kasa, Nagavedi S. Umapathy, Janavi Kotamarthi, Yash S. Gokhale, Vijaya Karoor, and et al. 2020. "P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases" International Journal of Molecular Sciences 21, no. 18: 6855. https://doi.org/10.3390/ijms21186855

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop