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The Impact of Purinergic System Enzymes on Noncommunicable, Neurological, and Degenerative Diseases.

1. Introduction

Noncommunicable, neurological, and degenerative diseases are characterized by cell loss, ultimately leading to deterioration in quality or function of tissues or organs and possible failure of vital organs [1]. Although the etiology and pathogenesis of these diseases remain unclear, recent advances indicate that the processes of organ deterioration share common core features, including cell injury and dysfunction that contribute to functional and morphological impairment of cells. Despite considerable progress in understanding the molecular mechanisms of these diseases, current therapeutic options are limited, and no effective pharmacological treatment has emerged to date. Elucidation of common and unique mechanisms responsible for the deterioration present in these pathologies may facilitate the identification and development of effective targets and therapies [2]. Furthermore, the search for specific (bio) markers for each human condition--physiological and pathological--is becoming critical.

Elements of the purinergic signaling system are involved in many processes in health and disease conditions [3]. Therefore, a complete understanding of purinergic system could potentially unveil possible markers or relevant pathways for pathological processes, mainly related to human degeneration. Briefly, the purinergic system consists of three main components: (i) the extracellular nucleotides and nucleosides, which mediate signaling; (ii) the receptors through which these nucleotides and nucleosides exert their effects; (iii) and the ectoenzymes, responsible for the control of extracellular levels of these molecules [4]. The control of the levels of extracellular nucleotides adenine and adenosine and the consequent signaling by purinergic receptors induced by them is critical in maintaining the physiological processes [5]. This control is performed by ectonucleotidases, which are enzymes anchored to the cell surface or located in the interstitial medium (soluble form) [6].

2. Purinergic System

Purines' extracellular role was first demonstrated in 1929 by Drury and Szent-Gyorgyi [7], which described its actions in mammary hearts [8-10]. Although, only in 1970, Burnstock proposed the term "purinergic" and presented his hypothesis about ATP as an independent neurotransmitter released from nonadrenergic noncholinergic neurons in the intestines, bladder, and gut [11, 12]. Two years later, Burnstock described adenosine triphosphate (ATP) as an extracellular signaling molecule and its effects [13]. However, the purinergic system and ATP had an arduous path to be accepted by the scientific community. Only in 2006, ATP was finally recognized as a cotransmitter in both the peripheral and central nervous systems (CNS) [9, 10, 14], and the purinergic signaling was recognized as a system involved in many nonneuronal and neuronal mechanisms [12].

ATP is the most versatile nucleotide and the primary energy source for cellular functions. Hundreds of reactions in the cell, from metabolic transformations to signaling events, are coupled to the hydrolysis of ATP [15]. Intracellularly, ATP is stored at very high levels (from 5 to 10mmol/l), which can quickly be degraded by ubiquitous extracellular nucleotidases after connecting to specific receptors under physiological conditions. In fact, extracellular ATP has an extremely short half-life before it is degraded to adenosine--milliseconds to seconds. This rapid breakdown results in the activation of a multiplicity of receptor subtypes, which can mediate physiological processes such as proliferation, differentiation, migration, and cell death [16]. On the other hand, the excess of ATP in the brain extracellular space can induce neurotoxicity [17].

ATP stores energy by losing a phosphate group and forming ADP. It has been shown that the ADP molecule can have an important role in platelet aggregation (platelet granules contain high concentrations of ADP), blood vessel tone, cardioprotection, and vascular wall integrity [18].

The critical functions of ATP and its subsequent hydrolysis are initiated upon binding to purinergic receptors, such as P2 nucleotide and P1 adenosine receptors [19]. Abbracchio and Burnstock divided P2 receptors into two families: P2X family of ligand-gated ion channel receptors and the P2Y family of G protein-coupled receptors, based on their molecular structure, induced mechanism of action, and the sequence analysis of cloned P2 receptors [20].

Currently, thirteen human P2X receptor subtypes can be distinguished: 6 homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7) and 7 heteromeric (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6 [21], and P2X4/7) [22]. P2X receptors are nonselective ligand-gated ion channels that mediate sodium influx, potassium efflux, and at some extent calcium influx, leading to cell membrane depolarization [23]. The P2X2/3 receptors are located in the nodose ganglia [24], P2X4/6/7 in the CNS [25, 26], P2X1/5 in the blood vessels, and P2X2/6 receptors are mainly located in the brain stem [24, 27, 28]. P2X receptors have important functions in the central and peripheral nervous systems, such as slow neuromodulatory function, rapid synaptic transmission, neurotransmitter release, and the generation of pain signals. Furthermore, these receptors have pathophysiological role in injury, inflammation, anxiety, dementia, epilepsy [26], and neurodegenerative disorders such as Alzheimer's and Huntington's diseases [29-32].

P2Y receptors are G protein-coupled receptors, virtually present in all cells, and mainly activated by adenine and uridine nucleotides. There are eight subtypes of P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14), which are ubiquitously expressed in body, including the CNS [32]. They activate intracellular signaling cascades to regulate a variety of cellular and peripheral pathophysiological processes, including inflammation, ischemia, and pain [32, 33]. In the brain, P2Y receptors exert important roles in neurotransmission, glia cell communication, and neurogenesis due to their localization on neurons, oligodendrocytes, microglia, and astrocytes [13, 34-36].

After release and binding to specific receptors, ATP and other nucleotides undergo rapid enzymatic degradation by ectonucleotidases [4]. Ectonucleotidase families include the ectonucleoside triphosphate diphosphohydrolases (E-NTPDase/CD39/NTPDase 1), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPP), and ecto-5'-nucleotidase (E-5'-nucleotidase/CD73). E-NTPDases and E-NPPs hydrolyze ATP and ADP to adenosine monophosphate that is further hydrolyzed by E-5'-nucleotidase to adenosine [6,37,38].

Since 1929, adenosine has been recognized as a biologically significant molecule, responsible for regulating multiple systems including cardiac conduction, arterial pressure, and intestinal motility [7]. Adenosine is produced both intracellularly and extracellularly through enzymatic degradation of adenine nucleotides [39, 40]. The maintenance of its levels in the extracellular fluids is the result of the balance between its production and consumption [19]. Indeed, adenosine synthesis is controlled by ectonucleotidases located on the cytoplasmatic membrane, and its concentrations vary according to physiological and pathological stimuli such as hypoxia and inflammation [41, 42]. Extracellular adenosine is involved in many cytoprotective functions of the body, including conditioning the heart against ischemia, counteracting the damaging effects of excitotoxicity and seizure activity in the brain, and suppressing excessive immune and inflammatory responses. Finally, extracellular adenosine can be deaminated to inosine by adenosine deaminase (ADA) [43].

Critically, the understanding of the physiological and pharmacological roles of adenosine was greatly facilitated by localizing the different P1 receptor subtypes in target tissues and identifying its four subtypes--A1, A2A, A2B, and A3 receptors [44-46]. These are G protein-coupled receptors, which present inhibitory action upon adenosine binding [20]. P1 receptors are widely located in the brain and in peripheral tissues, such as the heart, kidney, and adrenals of different species [45, 46].

After the binding to receptors, the nucleotides and nucleosides are rapidly degraded by specific hydrolase enzymes, which occurs via different ways [47]. The E-NTPDase catalyzes ATP and ADP hydrolyses, which culminates into AMP formation. E-5'-nucleotidase is responsible for AMP degradation leading to adenosine formation. Alkaline phosphatase removes inorganic phosphate (Pi) of a broad range of substrates, including nucleotides (ATP, ADP, and AMP) producing adenosine (Figure 1) [48, 49]. The producing adenosine is converted into inosine by adenosine deaminase (ADA) [38].

The E-NTPDase family is very efficient in controlling the bioavailability of ATP [49]. This family consists of eight members, E-NTPDases 1-8, which differ in substrate specificity, tissue distribution, and cellular localization [50]. Members 1, 2, 3, and 8 are the principal enzymes responsible for the hydrolysis of tri- and diphosphate nucleotides on the cell surface under physiological conditions. E-NTPDases 4, 5, 6, and 7 are associated with intracellular organelles [50]. These ectoenzymes are anchored to the plasma membrane via hydrophobic domains with the active site facing the extracellular medium [5, 38, 48].

The E-5'-nucleotidase family is comprised of seven isolated and characterized isoforms with different nomenclature, depending on the subcellular localization. Five isoforms are cytosolic, one is located in the mitochondrial matrix, and one is related to the outer plasma membrane [47]. This enzyme generates extracellular adenosine from AMP, as previously mentioned, and its activity is the rate-limiting step of adenosine formation from adenine nucleotides in most tissues [49, 51].

The E-NPPs are transmembrane glycoprotein type II enzymes, which are able to catalyze many different reactions: 3'-5'-cyclic adenosine monophosphate (5'-cAMP) to AMP; ATP to AMP and inorganic diphosphate (PPi); AMP to ADP and inorganic phosphate (Pi); nicotinamide adenine dinucleotide oxidized (NAD+) to AMP and mononucleotide nicotinamide [6]. Although this family of enzymes is composed by 7 members, only E-NPPs 1, 2, and 3 are able to hydrolyze nucleotides [5].

ADA activity has been found altered in various pathological conditions including acquired immunodeficiency syndrome (AIDS), anemia, lymphomas, tuberculosis, and leukemia [47]. In humans, different ADA isoforms have been identified: adenosine deaminase-1 (ADA-1), adenosine deaminase-2 (ADA-2), and adenosine deaminase-3 (ADA-3) [52]. ADA-1 plays a metabolic role not only as a key cytosolic enzyme in the purine pathway but also as an ectoenzyme by regulating extracellular adenosine levels. In contrast, ADA-2 is specifically designed to act in the extracellular environment according to its presence in the serum [53]. Thus, ADA can be considered as a multifunctional protein, playing several roles and importantly regulating biological systems, which consequently impacts on human health [53].

3. Noncommunicable, Neurological, and Degenerative Diseases

According to the World Health Organization (WHO), chronic disorders account for 38 million deaths each year, more than 80% of all deaths worldwide. Cardiovascular diseases cause most of the deaths (17.5 million per annum), followed by cancers (8.2 million), respiratory diseases (4 million), and diabetes (1.5 million) [54, 55]. In the last two decades, this profile of morbidity and mortality causes around the world has significantly been changed, and, increasingly, chronic neurological and degenerative diseases are becoming more prominent [54].

Among the better-known chronic diseases are the noncommunicable, such as atherosclerosis, hypertension, diabetes, and cancer; the neurological, such as epilepsy; and the neurodegenerative, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). The study and control of these diseases are exceedingly difficult due to their multiplicity and diversity, for instance, the interconnection network of risk and protective factors, diverse onsets followed by multistep pathogenesis, and, in some cases, multifocal localization [56]. A common feature among many of the neurological, neurodegenerative, and several aspects of cardiovascular diseases may be a component of degeneration. It means that at some stage occurs loss of cells, subcellular function, or tissue elements or function [57]. In fact, these diseases may also share other pathological features or events, such as evidence of membrane damage, oxidative stress, mitochondrial dysfunction, and upregulation of autophagy [58].

Once purinergic signaling is involved in virtually all body functions, the roles of purines might be altered in pathological states in different body systems [59]. Indeed, purinergic mechanisms and specific receptor subtypes have been shown to be involved in several pathologies including brain trauma and ischemia, neurodegenerative diseases involving neuroimmune and neuroinflammatory reactions [60], diabetes [61], vascular diseases including atherosclerosis [62, 63], and hypertension [9, 10]. Importantly, studies have shown the potential of purinergic mechanisms as therapeutic targets for the treatment of neurological [36] and degenerative disorders [9, 64, 65]. Moreover, the currently used strategies in searching for novel purinergic targeting drugs include the development of (i) selective agonist and antagonist ligands for the P2X and P2Y receptors, (ii) inhibitors of extracellular catabolism of purines, and (iii) modulators of nucleotide and nucleoside transport [66]. Adenosine signaling manipulation may also have therapeutic potential in neurodegenerative diseases such as AD, PD, and Huntington's diseases and in neurological and psychiatric disorders such as epilepsy [67], schizophrenia, and autism [68].

Taking that into account, the next section provides an overview of recent findings regarding the role of purinergic system enzymes in the pathophysiology of noncommunicable, neurological, and degenerative diseases. Considerable attention has been directed towards diseases related to the cardiovascular system (atherosclerosis and hypertension), cancer, and central nervous system (epilepsy, Alzheimer's disease, Parkinson's disease, and multiple sclerosis).

3.1. Atherosclerosis. Atherosclerosis is popularly defined as an artery wall thickening triggered by accumulation of foam cells and proliferation of intimal smooth muscle cell, which results in a fibrofatty plaque. Atherosclerotic disease still is the worldwide leading cause of cardiovascular complications and death [69]. The drivers for atherosclerotic plaque development include fluid shear forces, lipid milieu, cells of the vascular wall, and cells recruited from the circulation. However, one of the central events that results in the atherosclerosis development is the disturbance in blood flow in human arteries. In addition, some cells, such as endothelial cells, platelets, monocytes, and T cells, can be involved and/or activated in response to changes in blood flow [70].

Arteries presenting constant flow are further resistant to plaque formation when compared to arteries presenting disturbance in blood flow. Taking this into account, it is important to point out that the aspects that control the blood flow, such as genes, microRNAs, and epigenomic processes, initiate responses that can result in activated or quiescent endothelial phenotypes and, in turn, susceptibility to or protection from atherosclerosis [70-72]. Corroborating with this, Nam and collaborators demonstrated that disturbed flow directly causes atherosclerosis within 14 days, under hyperlipidemic conditions, after partial carotid ligation surgery [71].

It is well stablished that E-NTPDase 1 is the major ectonucleotidase expressed in blood vessels. Several studies have been carried out in order to elucidate the role of this enzyme in controlling the blood flow, atherosclerosis development, and inflammation related to vascular disorders [70, 73] (Table 1). It has been widely shown that E-NTPDase 1 expressed in both endothelial and smooth muscle cells, along with autonomic nervous system, can control the contraction of arteries, which directly influences the regulation of blood circulation [49, 74-77]. Moreover, E-NTPDase 1 plays a critical role limiting the activation of some P2 receptors, thus, preventing its desensitization in endothelial and smooth muscle cells, consequently, preventing excessive artery contraction [76-79]. This fact can be explained because ATP, released from sympathetic nerves, along with norepinephrine (NE) and neuropeptide Y, can activate P2X receptors at postjunctional membranes evoking vascular smooth muscle contraction, which generally potentiate the effects of NE at [alpha]1-adrenoceptors [51, 80]. Accordingly, Cardoso et al. have shown that E-NTPDase 1 expression and activity have the ability to control the effects of sympathetic nervous system in the vasculature [81].

Regarding to the vascular injury related to atherogenesis, it has been shown that E-NTPDase 1 naturally results in protection from the action of nucleotides released from the injured cell in vasculature or activated platelets by eliminating these prothrombotic and proinflammatory stimuli, that is, by metabolizing extracellular ATP and ADP. In a recent work, Kanthi et al. have evaluated the role of E-NTPDase 1 in the development of atherogenesis, in an apolipoprotein E-deletion mouse model of atherosclerosis (ApoE-deficient) [70]. ApoE-deficient group, fed with a high-fat diet, showed evidently augmented and plaque formation along with platelet activation that was verified by some circulating markers. In this work, the authors also showed that in regions of stable flow, E-NTPDase 1 was markedly present in ApoE-deficient mice. In contrast, E-NTPDase 1 was reduced in atheroprone as subject to disturbed flow. Moreover, in this same work, it has been demonstrated that the deregulated flow triggered by a partial carotid artery ligation quickly suppressed endothelial E-NTPDase 1 expression. Furthermore, unidirectional laminar shear stress induced atheroprotective ENTPDase 1 expression in human endothelial cells. In these conditions, the vascular transcription factor involved in the E-NTPDase 1 is the Kruppel-like factor 2 (KLF2), which can bind near the transcriptional start site of E-NTPDase 1.

Mercier and collaborators have shown that ApoE knockout mice displayed decreased E-NTPDase 1 expression and activity in the thoracic aorta, which correlates with reduced vascular reactivity and presence of atherosclerotic plaques in aortic roots and arches. The authors have speculated that the accumulation of ATP and ADP in ApoE knockout mice may cause desensitization of P2 receptors, which in turn may contribute to decreased blood flow and may predispose an individual to atheroma formation [82]. In a carotid artery wire injury model, E-NTPDase 1 knockout mice showed decreased migration of vascular smooth muscle cells and reduced neointimal formation, indicating that CD39 contributes to a harmful neointima formation [83]. Taken together, these data establish that the key regulator of atherosclesosis development driven by shear stress is the E-NTPDase 1 enzyme. Kanthi et al. have unveiled what is now believed to be a previously unrecognized role for E-NTPDase 1 as an endogenous regulator of endovascular purine levels, serving as a modulator of crucial cellular drivers of atherogenesis [70]. Emphasizing the role of E-NTPDase 1 in providing anti-inflammatory and antithrombotic mediator adenosine, it has been shown that therapeutic strategies targeting CD39 offer promising opportunities in the management of vascular thromboinflammatory diseases [73].

CD73 also has been reported playing a key role in the atherogenic process by driving purinergic signaling [84, 85]. In a work developed by Buchheiser et al., it was demonstrated that the inactivation of CD73 promotes atherogenesis in apolipoprotein E-deficient mice [86]. The ablation of CD73 in ApoE knockout animals triggered an augmentation in atheroma formation, which was probably generated by the reduced inhibitory control exerted by adenosine on immune cells, which are dynamically related to the atherogenic process [86]. The role of CD73 in protection against atherosclerosis was also observed after injury of carotid arteries. This condition results in augmented expression of vascular cell adhesion molecule- (VCAM-) 1 and increased nuclear factor-kappa B activity, which triggers the formation of neointimal plaque and continuous infiltration of macrophage in CD73 knockout animals when compared to control groups [87].

Jalkanen and collaborators have evaluated 226 patients with stable peripheral artery disease admitted for nonurgent invasive imaging and treatment. They verified that the progression of atherosclerosis is associated with low ENTPDase 1 activity and high CD73 [88]. Same authors also found high levels of ATP and ADP in the plasma of those patients, suggesting that low E-NTPDase 1 activity is associated with ATP- and ADP-induced platelet aggregation and trombus formation [88].

Since adenosine is the main molecule that presents antithrombotic effects, ADA also has a key role in atherogenesis process. This enzyme has been suggested as an inflammatory marker [89], and its high expression and activity were associated with the risk of atherosclerosis development [89, 90]. Kalvegren et al. have verified changes in metabolism of extracellular nucleotides in the atherosclerotic vessel wall from aortoiliac bifurcation of apoliprotein E- and LDL-deficient animals (ApoE/LDLr (-/-)). They observed that the ADA activity was decreased as well as the levels of adenosine in ApoE/LDLr (-/-) mice when compared to littermate control, reinforcing the idea that low ADA activity is a predictor of atherogenesis. Another mechanism that can contribute to the development of atherosclerosis is that high levels of ADA can increase the release of reactive oxygen species from neutrophils, through a downregulation of the inhibitory adenosine/cAMP system and an enhanced activation of A1 receptors. This sustained neutrophil activation could also contribute to inflammatory disorders and atherogenesis [91].

Genetic studies have been performed regarding the relationship between ADA and atherosclerosis, and two main points can be highlighted: (1) single-nucleotide polymorphisms of the RNA-specific gene of ADA are associated with metabolic disorders in general and atherosclerosis [92] and (2) high ADA gene expression is related to the augmentation of cathepsin S mRNA, which encodes a cysteine protease associated with angiogenesis and atherosclerosis [93].

Taking the above information into account, it is possible to conclude that a high expression and activity of E-NTPDase 1 and CD73, in order to generate adenosine, and a low expression and activity of ADA, in order to keep the levels and let the adenosine molecule exerts its antithrombotic effects, may exert a positive effect in preventing atherosclerosis.

3.2. Hypertension. Hypertension is a highly prevalent disease, which is estimated to affect 26% of the worldwide adult population [94, 95]. Hypertension is defined by a systolic blood pressure (BP) of 140 mmHg or higher, a diastolic BP of 90 mmHg or higher, or currently using BP-lowering drugs [95]. The disease represents a major risk factor for the development of kidney failure, coronary events, cerebrumvascular disease, heart failure, and peripheral vascular disease [94, 95]. Hypertension is the leading cause of morbidity and mortality worldwide while cigarette-smoking is the major preventable cause of death [96]. Nowadays, most patients require more than one drug to achieve BP target, and monotherapy would only be sufficient in about 20-30% of patients [95]. This fact highlights the unmet need to unveil other mechanisms that can be associated with hypertension development and maintenance.

Among the events related to hypertension, we can cite platelet activation and aggregation, vasoconstriction, and low-grade inflammatory status. It is well known that the purinergic system enzymes are tightly involved regulating nucleotides that can trigger or protect against platelet aggregation. Moreover, the control of extracellular circulating nucleotides by ectonucleotidases is related to both anti- and proinflammatory status [49]. Recently, Fabbiano and collaborators have demonstrated that regulatory T (TReg) cell-expressing E-NTPDase 1 require an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II- (AngII-) dependent hypertension [97]. Tissue-resident neutrophils suffer apoptosis by ATP hydrolysis of E-NTPDase 1. The same group also stated that genetic alterations in the number of TReg and TH cells has an influence on tissue-resident neutrophil number, cardiomyocyte hypertrophy, cardiorenal fibrosis, and elevation of arterial pressure during AngII-induced hypertension [97]. Thus, TReg cell-expressing E-NTPDase 1 can protect against hypertension-driven fibrosis in tissue.

In pulmonary arterial hypertension (PAH), two independent studies have been performed with different outcomes. Using human samples, in vitro strategies, and a rat model, Helenius et al. have demonstrated that the suppression of E-NTPDase 1 is linked to the pathogenesis of PAH [98]. Furthermore, they stated that the accumulation of extracellular ATP and ADP is strictly linked to vascular dysfunction and remodeling in PAH and can modulate the disease course in multiple levels. On the other hand, Visovatti et al. have observed that microparticles from platelets and endothelial cells of patients with PAH display increased E-NTPDase 1 activity and expression [99]. Therefore, more studies are crucial to elucidate whether high or low levels of ENTPDase 1 can be related to PAH development.

Taking advantage of a preeclampsia mouse model, McRae et al. have evaluated the impact of E-NTPDase 1 overexpression. In this study, the authors injected Th1-polarized cells on pregnancy days 10 and 12 of wild-type and E-NTPDase 1 transgenic mice and measured the systolic blood pressure (SBP) until pregnancy day 15, when mice were sacrificed. Following transfer of Th1-polarized cells, SBP of E-NTPDase 1 transgenic mice remained unchanged, without evidence of renal lesions, while an increase was observed in pregnant wild-type mice. Thus, the authors have concluded that E-NTPDase 1 overexpression can be protective in a mouse model of preeclampsia [100].

Sympathetic system has a crucial link with hypertension development by controlling vascular tonus. E-NTPDase 2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space in the heart [75]. Later, Rucker et al. have shown that E-NTPDase 2 is the most expressed ectonucleotidase in synaptosomes prepared from rat heart left ventricles, indicating that this enzyme may be essential for modulating ATP and NE responses on heart fibers [101]. At the same time, the presence of E-NTPDase 2 in the vascular murine adventitial cells [75] also suggests that this enzyme plays a role in hydrolyzing the ATP released from sympathetic nerves and, thus, can help to control vascular tone and hypertension development.

E-5-nucleotidase is also highly expressed in the left ventricle, and its activity seems to be important for the control of the nucleotide/nucleoside ratio in the vicinity of nerve endings in the heart [101]. Moreover, in human coronary arteries, sympathetic nerves are one of the sources of adenine nucleotides and coronary vasodilation was found to be associated with the endothelial expression of E-5'-nucleotidase. Inhibition of E-5'-nucleotidase and the usage of P1 antagonists have been associated with a marked reduction of the relaxation of coronary arteries [102]. The same occurs in mesenteric arteries [103] and other vascular beds [51]. Data from Sousa and colleagues have revealed that the increase in the sympathetic tonus in spontaneously hypertensive rats can be associated to a higher NE/ATP release ratio from sympathetic nerves and to deficits in the endogenous inhibitory tonus mediated by prejunctional adenosine A1 receptors [103]. This fact reinforces the important role of E-NTPDase 1, E-NTPDase 2, and CD73 in producing adenosine and preventing hypertension development caused by augmented sympathetic tonus. Indeed, adenosine produced by E-5'-nucleotidase activates P1 receptors, which can cause hyperpolarization and relaxation of the underlying vascular smooth muscle cells [102].

Increased E-NTPDase and CD73 activities have been observed in both animal models of hypertension, in human studies, and in platelets and lymphocytes [81, 104-107]. These specific platelet and lymphocyte responses can be understood as a mechanism to ameliorate hypertension through the elevation of adenosine levels, by combined actions of E-NTPDase and CD73.

Taken together, these results indicate that E-NTPDase 1 and CD73 expressed in lymphocytes, TReg cells, endothelial cells, and platelets constitute a protective barrier against hypertension-driven tissue fibrosis. In addition, the results suggest new therapeutic avenues to prevent hypertension and hypertension-linked pathologies.

Changes in ADA expression and activity are also related to hypertension development and maintenance. Human studies have shown increase in ADA activity and expression in response to hypertension and hypertension-associated pathologies, such as metabolic syndrome [108]. A genetic study with hypertensive patients showed that a common polymorphism (C34T) of the ADA gene (isoform 1) is strongly correlated with essential hypertension [109]. ADA activity is also increased in both platelets and lymphocytes in an animal model of hypertension induced by L-NAME administration [81, 104-106].

Moreover, Tofovic et al. have suggested that the inhibition of ADA may provide beneficial effects in old hypertensive animals, and an inhibitor of this enzyme could be designed and used to offer cardiovascular protection in hypertension [110]. Franco and colleagues have analyzed the activity of nucleotidases and ADA in cytosolic and membrane fractions of renal tissue, in an angiotensin-II model of hypertension [111]. They observed a decrease in the membrane ADA activity and expression, in AngII-treated rats. Furthermore, despite the adenosine elevation, A1 and A2B receptor protein expression did not change; in contrast, a downregulation was observed in A2A receptors and an upregulation in A3 receptor levels. A similar pattern was found in the cortex and in the medulla--the expression of A3 receptor decreased in both segments. These results suggest that the elevation of renal tissue and interstitial adenosine contributes to the renal vasoconstriction observed in the AngII-induced hypertension. This can be either mediated by a decrease in the activity and expression of ADA, increased production of adenosine, or an imbalance in adenosine receptors [111].

Original studies have shown increased platelet aggregation as a result of high ADA activity and expression and lack of adenosine in pregnancy hypertensive [112, 113]. It was indeed suggested that ADA activity as well as platelet aggregation could serve as peripheral markers for the development of therapy for the maintenance of homeostasis and inflammatory processes in hypertension and hypertension associated pathologies [108, 113]. In contrast, Iriyama et al. have analyzed adenosine metabolism using two different animal models of preeclampsia [114]. They have demonstrated that adenosine levels were high in preeclampsia, and this increase was enough to induce hallmark features of preeclampsia including hypertension, proteinuria, small fetuses, and impaired placental vasculature. This study has also revealed that besides the high levels of adenosine, the receptor A2B was excessively activated, contributing to the development of preeclampsia-related features, although the key finding of this study was that the placental adenosine increase is triggered by the elevation of placental CD73, thereby contributing to preeclampsia [114]. Discrepancies between those two studies can be explained by experimental design differences, for instance, site of analysis (platelets versus placenta). Finally, Iriyama et al. did not evaluate the ADA enzyme. Therefore, more studies are necessary to elucidate whether adenosine causes or protects against preeclampsia and to unveil the actual role of ADA in preeclampsia. This information is summarized in Table 2.

3.3. Cancer. Cancers are considered heterogeneous pathologies that vary by tissue of origin and genomic, proteomic, and metabolic alterations [115]. The inflammation plays an important role in the steps required for cancer metastasis because a wide variety of cytokines and other proinflammatory markers contributes to both the extrinsic and intrinsic pathways of inflammation associated by cancer [116].

The enzymatic chain responsible for ATP hydrolysis and adenosine production, present in all immune and vascular cells, has an important role in the control or promotion of inflammation in tumors [117]. This is because of an exuberant immune/inflammatory response, not adequately balanced by endogenous mechanisms of homeostatic control that can lead to persistent and abnormal forms of collateral tissue damage [118]. Adenosine can accumulate in the tumor environment and stroma and generate an immunosuppressed microenvironment that favors the development and metastasis of neoplasias [119].

Multiple mechanisms are involved in adenosine effects, which include inhibition of T helper 1 cell (TH1 cell) cytokine production, deregulation of mononuclear phagocyte cell differentiation and maturation, suppression of effector T cells, and generation of an angiogenic and matrix remodelling environment that is suitable for cancer growth [120, 121]. Most of the signaling actions of extracellular adenosine are mediated by G protein-coupled cell surface receptors that are divided into four subtypes: A1, A2A, A2B, and A3 [122].

The extracellular adenosine acts as a local modulator and exerts effects by protecting cells and tissues from an excessive inflammatory response and as favorable to the onset and cancer growth, by favoring angiogenesis and matrix remodeling [122, 123]. When adenosine active A2A, A2B, and A3 receptors, macrophages promotes the release of the anti-inflammatory cytokine like as TNF, IL-6, IL-10, IL-12, nitric oxide (NO) and macrophage inflamatory protein-(MIP)-1[alpha] [52, 120].

The A2A receptor activity, a T-cell surface immune checkpoint protein, could lead to the discovery that adenosine in the tumor microenvironment interferes with antitumor immunity, suggesting that antagonism of the A2A could be an effective cancer immunotherapeutic [124].

3.4. Epilepsy. Epilepsy is a debilitating neurological disease, characterized by recurring, spontaneous, unprovoked seizures. Temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy, affecting people from all ages [125]. The World Health Organization (WHO) reports that epilepsy affects approximately 50 million people worldwide, with about a third of patients either resistant to current antiepileptic drugs (AED) or experiencing unacceptable side effects.

Neuroinflammation is increasingly recognized as one of the key players in seizure generation and propagation and in the maintenance of the epileptic phenotype [126]. During pathological conditions with increased neuronal firing or cell death nucleotides, ATP is released into the extracellular space in the brain. Although studies show discrepancies about ATP release during seizures and chronic epileptic state, this is possibly due to ATP rapid hydrolysis, by ectonucleotidases in the extracellular space, making it difficult to directly measure its release [127]. Once released, ATP activates P2 receptors (P2X and P2Y), mediating the release of gliotransmitters, which triggers neuronal hyperexcitability and neuroinflammation. Taking advantage of experimental models of epilepsy, the expression and function of P2X receptors have been well established in research field [36, 128]. However, only recently has been suggested that the P2Y receptor subfamily plays a relevant role in experimental and human epilepsy [67,129]. Furthermore, while adenosine anticonvulsant properties are well established [130], the contribution of extracellular nucleotides to seizures and epilepsy pathology is an area to be further explored [36]. Therefore, not only the targeting of molecules involved in inflammatory pathways but also the targeting of purinergic signaling or their combination seems relevant approaches for developing novel therapies for epilepsy [36, 126].

3.5. Alzheimer's Disease. Alzheimer's disease (AD), the leading cause of dementia worldwide, is a progressive neurodegenerative disease associated with the deposition of [beta]-amyloid peptide (A[beta]) within the brain, along with intracellular neurofibrillary tangles (NFTs) mainly formed by hyperphosphorylated tau protein [131]. The impairment of mitochondrial function and reduction of ATP levels are pathological conditions found in AD, which is closely linked to the decline of cognitive processes [132, 133]. It is well known that in the brain, ATP is secreted by the neurons, glia, and endothelial cells that constitute the blood-brain barrier. Coincident with its release is the secretion of soluble ectonucleotidases which control the effective ATP concentration by breaking it down to adenosine [134]. The involvement of ectonucleotidases on the process of learning and memory in rats has already been described [135-137]. It has also been shown that P2X7 purinergic receptors are upregulated in the brain of patients with AD and in animal models [138, 139]. Interestingly, inhibition of P2X7 in mice, transgenic for mutant human amyloid precursor protein, reduced the number of amyloid plaques in the hippocampus [140] and stimulation of P2X7 receptors on human macrophages and microglia enhanced the degenerative lesions observed in AD [141] (Tables 3 and 4). Furthermore, direct alterations of purine metabolism have been detected in AD by metabolomics, in the ventricular cerebrospinal fluid at postmortem [142], cerebrospinal fluid in living individuals [142-144], and AD brains [145].

Ectonucleotidase activities may be involved in the early events related to memory acquisition and consolidation of an aversively motivated learning task [135, 136]. Previous studies performed in animals subjected to scopolamine model of dementia, which mimics the memory deficit observed in diseases characterized by impairment in cholinergic neurotransmission, such as AD [146-148], have shown a marked reduction in the ATP levels and changes in ectonucleotidase activities (E-NTPDase, E-5 -nucleotidase, and ADA), in the cerebral cortex and hippocampus of rats [149, 150].

Tissue-nonspecific alkaline phosphatase (TNAP) is one of four alkaline phosphatase isozymes that has been described as an ectonucleotidase being able to cleave all forms of adenosine phosphates, influencing purinergic signaling [48]. Preclinical assays tested on more than 100 AD patients have demonstrated that TNAP activity is significantly increased in the hippocampus of AD patients compared with age-related controls [151]. In fact, it has been shown that the extracellular hyperphosphorylated tau protein coming from damaged neurons must be dephosphorylated to become an agonist of a muscarinic receptor [152]. This event has been associated with increased TNAP expression and unbalances of the intracellular calcium homeostasis and phosphorylation levels of intracellular tau [153]. Increased TNAP levels in the plasma of the AD patients have also been reported in literature [151, 154], suggesting that TNAP is a good biomarker of disease progression. On the other hand, the activity of the purinergic enzyme ADA did not present any significant differences in the serum of patients with AD, compared to unaffected controls [155].

3.6. Parkinson's Disease. Parkinson's disease (PD) is considered the most frequent movement disorder. Clinically, "movement disorder" is defined by its cardinal motor symptoms: bradykinesia, rigidity, and tremor at rest [156]. Furthermore, most patients will also experience nonmotor symptoms that include mood, cognitive, speech, sensation, and sleep disturbances. The major cause of these symptoms is the progressive loss of dopaminergic neurons in the substantia nigra pars compacta projecting to the striatum, leading to a severe deficiency of dopamine in the putamen and the caudate nucleus. The neuropathological hallmark of PD is cytosolic Lewy bodies, which are characterized by aggregated a-synuclein ([alpha]-Syn) [157].

Activated microglia is a common feature observed in individuals with neurodegenerative disorders, including PD, and it possibly contributes to neuronal death. Jiang et al. have shown that stimulation of the microglial P2X7 receptor by extracellular [alpha]-Syn, with PI3K/AKT activation and increased oxidative stress, could be an important mechanism and a potential therapeutic target for PD [158]. Furthermore, release of ATP from disrupted cells might cause cell death in neighboring cell-expressing P2X7 receptors, leading to a necrotic volume increase, which has also been implicated in the pathogenesis of PD [159]. Recently, several reports have suggested potential effects of the adenosine A2A receptor antagonist on cognitive dysfunction in PD [160, 161]. Indeed, in rodent models of PD, A2A antagonism exerts antiparkinsonian actions [162-164]. Similarly, this treatment proved to be effective against experimentally induced tremor [165].

Despite several studies provide strong evidence that purinergic receptors are linked to the pathogenesis of PD, the evidence available on a potential involvement of purinergic enzymes is still very scarce. To date, we have only found a few articles concerning this issue. Medeiros et al. have studied the levels of ATP and the activity of the enzymes ADP ENTPDases, ecto-5'-nucleotidase, and ADA in patients with PD [166]. The results show higher E-NTPDase ATP/ADP activities in PD patients, suggesting an important inflammatory activity in this disease. Another study has analyzed the mRNA levels of E-NTPDase and E-5 -nucleotidase from striatal slices, in a unilaterally lesioned 6-OHDA rat model [167]. The classical method of intracerebral infusion of 6-OHDA involving a massive destruction of nigrostriatal dopaminergic neurons is largely used to investigate motor and biochemical dysfunctions in PD [168]. However, it was found no alteration in the mRNA levels of the enzymes tested [167] (Tables 3 and 4).

3.7. Multiple Sclerosis. Multiple sclerosis (MS) is a chronic inflammatory disease, of progressive character with relapsing-remitting phases, that represents the major disabling neurological illness of young adults [176]. This disease affects the myelin sheath of axons and is characterized by demyelination of neurons in the central and peripheral nervous system. Furthermore, the establishment of tissue oxidative stress associated with a neuroinflammatory process culminates in the loss of myelin content and subsequent death of oligodendrocytes and Schwann cells [177, 178].

The mechanisms implicated in the pathogenesis of MS include autoimmunity, inflammation, demyelination, and neurodegeneration. Since the beginning of demyelination processes, the appearance of multiple plates in the white matter of brain and spinal cord can occur [179]. These lesions lead to the disability or complete loss of nerve impulses that result in the appearance of clinical manifestations such as motor impairment [180], blurred vision, hyperalgesia in the upper and lower limbs, excessive tiredness, weakness, anxiety, depression, and irreversible neurological deficits [179, 181, 182]. The remission is a subsequent phase characterized by the remyelination and tissue repair, with partial or complete disappearance of symptoms [176].

The relationship between inflammation and degeneration during the progression of MS is due to successive outbreaks and remissions. There is a higher frequency of events accompanied inflammatory demyelination of the white matter and damage to axons. In this step, it is possible to tissue repair capacity in which there is a remyelination and axonal in the disappearance of clinical manifestations. As these events are repeated, the tissue recovery becomes ineffective. The secondary progressive phase initiates with the establishment of neurodegenerative tissue followed by cerebral gray matter atrophy. At this stage, there is a progressive increase over the score disabilities that can lead to incapacitation of the patient [183].

The MS does not have etiology and cure defined. Therefore, several types of animal models that produce demyelination have been used for scientific research, in order to discover pharmacological tools for assessing compounds as potential drugs or search for evidence of cellular mechanisms to better understand this disease. Among these, it is possible to highlight the chemically induced CNS lesions, such as those generated with cuprizone feeding, ethidium bromide injection, and lysolecithin, or the experimental autoimmune encephalomyelitis (EAE) model, widely used in drug screening as it most faithfully represents the pathology seen in MS [184]. As described by Merrill, the advantages of these models are the dissociation of the demyelination event, from the complexities introduced into the tissue pathology by chronic inflammatory cells and their soluble mediators, reproducibly timed spontaneous remyelination, and robust demyelination and remyelination in anatomically distinct areas facilitating focused, quantitative assessment of lesion generation and repair [185-187].

Recent reports have highlighted the association between receptors and signaling molecules of the purinergic system with demyelinating diseases, including MS [188-191]. Furthermore, ATP and adenosine also play a significant role in the pathophysiology of numerous acute and chronic disorders that include events related with CNS demyelination and remyelination [23, 192-194].

Over the last decade, the ectonucleotidases have become target of study by modulating purinergic signaling and contributing to the fine-tuning of inflammatory and immune responses. The overwhelming evidence indicates that extracellular ATP acting through specific cell surface receptors is involved in proinflammatory functions such as stimulation and proliferation of lymphocytes and microglial cell and cytokine production and secretion [195, 196]. However, its breakdown product, adenosine, exhibits potent anti-inflammatory and immunosuppressive action by inhibiting proliferation of T cells and secretion of cytokines [197, 198].

The association of the purinergic receptors in the demyelinating diseases has been explored in some previously published studies [199, 200]. An overview of the main findings relating the purinoreceptors with MS in experimental models and postmortem tissue of humans with this pathology can be found in Table 5.

Based on the evidence reported in Table 2, the four purinergic receptors especially known to be involved in MS are P2X7, P2Y12, A1, and A2A receptors. Although the role of purinoreceptors is not well elucidated, it is important to note that the number of published evidence and studies for MS is superior to investigations into the role of ectonucleotidases in this pathology. In this way, in Table 2, we also reviewed the activity and expression of ectonucleotidase enzymes ecto-NTPDase, E-5 -nucleotidase, and adenosine deaminase in experimental models and in patients with MS (see Table 6).

4. Conclusions

The main causes of death and sickness around the world have changed significantly in the last years, with chronic neurologic and degenerative diseases becoming more and more important. Several evidences show that purinergic signaling system is involved in processes associated with health and disease. In this way, the role of the purinergic receptors in the pathophysiology of different degenerative diseases has been extensively studied. However, the role of the purinergic system enzymes is not fully understood yet. This review summarizes the most current knowledge on the role of families of nucleotide metabolizing enzymes, the ectonucleotidases, on degenerative diseases. Considerable attention was directed towards diseases related to the cardiovascular system (atherosclerosis and hypertension) and central nervous system (epilepsy, Alzheimer's disease, Parkinson's disease, and multiple sclerosis). Firstly, it has been shown that a reduction in the activity of ectonucleotidases can be associated with progression of arteriosclerosis. Furthermore, high expression and activity of CD73 and E-NTPDase 1 may have beneficial effects contributing to the production of extracellular adenosine. In parallel, the low expression and activity of ADA are an alternative maintenance of levels of adenosine, in order to increase antithrombotic and anti-inflammatory effects. In relation to hypertension, a suppression of E-NTPDase 1 is linked to the pathogenesis of this disease and an increase in the activity of ectonucleotidases was reported in animal models of hypertension and in human. On the other hand, P2 receptor modulation seems to be a promising tool for a novel therapeutic approach for epilepsy. Lastly, a reduction of ATP levels in experimental models for AD and increase in the ectonucleotidases activities, especially E-NTPDase in patients with Parkinson's disease and multiple sclerosis, were found.

In conclusion, we verified that the activity and expression of the ectonucleotidases investigated in this review may be altered in some degenerative diseases. Taken together, the data discussed in this paper collaborate for a better understanding of the molecular mechanisms involved in these highly complex diseases, suggesting a number of directions for future research. In fact, more studies should be conducted to better understand the changes in metabolism and maintenance of adenine nucleotide and nucleoside levels in degenerative diseases. In addition, the study of the ectonucleotidase role may contribute to a better understanding of the molecular mechanisms of highly complex diseases and with major impact on patients' quality of life.

5. Future Prospects

Ectonucleotidases are nucleotide-metabolizing enzymes and play an essential role in regulating and controlling extracellular nucleotides. These nucleotides and nucleosides are responsible for the well functioning of normal cells. A wrong signaling mediated by these molecules triggers pathophysiological disorders like as atherosclerosis, hypertension, cancer, epilepsy, Alzheimer's disease, Parkinson's disease and multiple sclerosis. In this way, understanding the role of these molecules can be an important key factor to discover pharmacological targets and mechanisms that minimize deleterious effects of these diseases.

Until now, the investigations describes which ectonucleotidases have their activity or expression changes in different biological tissues in experimental models and patients with the pathologies reviewed in this study. From these findings, the next step is to know how the catabolic activity of ectonucleotidases can regulate the production of proinflammatory mediators and the immune response, both physiologically and pathologically.

Evidences have pointed out the important role of inhibitors of ectonucleotidases, highlighting their potential as novel drugs [220-222]. It is suggested that inhibitors of ectonucleotidases might be an ideal drug target for many therapeutic applications such as anticancer and immunomodulatory for the treatment of central nervous system disorders and cardiovascular diseases [220]. An important perspective would be to explore the use of these drugs in experimental biology. Such investigations would reveal whether up- or downregulation of the ectonucleotidases would restore immune markers and physiological functions compromised in experimental models and cell cultures. Since inhibitors of ectonucleotidases might be an ideal drug target for many therapeutic applications such as anticancer and immunomodulatory for the treatment of central nervous system disorders and cardiovascular diseases [220], more research is needed to elucidate the role of these enzymes in these pathologies and their role potential targets for drug discovery.

Conflicts of Interest

All authors report no conflicts of interest.


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Margarete Dulce Bagatini (ID), (1,2) Alessandra Antunes dos Santos, (3) Andreia Machado Cardoso, (1,2) Aline Manica, (2) Cristina Ruedell Reschke, (4) and Fabiano Barbosa Carvalho (2,5)

(1) Coordenagao Academica, Universidade Federal da Fronteira Sul, Campus Chapeco, Chapeco, SC, Brazil

(2) Programa de Pos-graduagao em Ciencias Biologicas-Bioquimica Toxicologica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil

(3) Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA

(4) Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland

(5) Laboratorio de Pesquisa em Patologia, Universidade Federal de Ciencias da Saude de Porto Alegre, Porto Alegre, RS, Brazil

Correspondence should be addressed to Margarete Dulce Bagatini;

Received 26 January 2018; Revised 3 July 2018; Accepted 22 July 2018; Published 12 August 2018

Academic Editor: Theresa Hautz

Caption: Figure 1: Cell membrane-anchored ectonucleotidases and their respective hydrolysis reactions.
Table 1: Ectonucleotidases (E-NTPDase, E-5'-nucleotidase,
and ADA) and purinergic receptors in experimental models
and patients with atherosclerosis.

Sample                     E-NTPDase        E-5'-nucleotidase

Endothelial cells         [down arrow]              --
and thoracic aorta     enzymatic activity
from mouse model of      and expression

ApoE-deficient mice            --              [down arrow]
                                            enzymatic activity
                                              and expression

Platelets and plasma      [down arrow]         [up arrow]1
from patients          enzymatic activity   enzymatic activity

Plasma of                      --                   --
experimental model

Atherosclerotic                --                   --
vessel wall from
bifurcation of
Apoliprotein E-and

Sample                 Adenosine deaminase    Involved receptors

Endothelial cells               --                    --
and thoracic aorta
from mouse model of

ApoE-deficient mice             --                    --

Platelets and plasma            --                    --
from patients

Plasma of              [up arrow] enzymatic    A1 upregulation
experimental model           activity

Atherosclerotic        [up arrow] enzymatic           --
vessel wall from             activity
bifurcation of
Apoliprotein E-and

Sample                 Reference

Endothelial cells      [72, 84]
and thoracic aorta
from mouse model of

ApoE-deficient mice      [88]

Platelets and plasma     [90]
from patients

Plasma of                [93]
experimental model

Atherosclerotic          [92]
vessel wall from
bifurcation of
Apoliprotein E-and

Table 2: Ectonucleotidases (E-NTPDase, E-5'-nucleotidase, and ADA)
and purinergic receptors in experimental models and patients with

Sample                      E-NTPDase          E-5'-nucleotidase

Treg cells from             [up arrow]                 --
angiotensin II-        expression CD39 [up
dependent                arrow] enzymatic
hypertension           activity to ATP and

Pulmonary arterial          [up arrow]                 --
hypertension           expression CD39 [up
                         arrow] enzymatic
                       activity to ATP and

Preeclampsia                [up arrow]                 --
                       expression CD39 can
                         protect against

Synaptosomes from           [up arrow]             [up arrow]
rat heart                 expression of            expression
                            NTPDase 2

Human coronary                  --                 [up arrow]
arteries                                           expression

Platelets and               [up arrow]        [up arrow] enzymatic
lymphocytes of            expression [up      activity [up arrow]
hypertensive human       arrow] enzymatic      enzymatic activity

Animal membrane                 --                     --
fractions of renal

Platelet of pregnant            --                     --
hypertensive woman

Placenta of pregnant            --                 [up arrow]
hypertensive woman                               expression [up
                                                arrow] enzymatic

Sample                 Adenosine deaminase     Involved receptors

Treg cells from                 --                     --
angiotensin II-

Pulmonary arterial              --                     --

Preeclampsia                    --                     --

Synaptosomes from
rat heart

Human coronary                  --              P1 upregulation

Platelets and                                          --
lymphocytes of
hypertensive human

Animal membrane            [down arrow]        A2A downregulation
fractions of renal      enzymatic activity      A3 upregulation
tissue                    and expression

Platelet of pregnant   [up arrow] enzymatic            --
hypertensive woman           activity

Placenta of pregnant            --                     --
hypertensive woman

Sample                 Reference

Treg cells from           [99]
angiotensin II-

Pulmonary arterial     [100, 101]

Preeclampsia             [102]

Synaptosomes from      [77, 103]
rat heart

Human coronary           [104]

Platelets and          [109-111]
lymphocytes of
hypertensive human

Animal membrane          [113]
fractions of renal

Platelet of pregnant   [114, 115]
hypertensive woman

Placenta of pregnant     [116]
hypertensive woman

Table 3: Role of purinoreceptors in experimental models and
patients with Alzheimer's disease (AD) and Parkinson's disease (PD).

R Sample                  P2X7 receptor           A2A receptor

Transgenic mouse           Upregulated                 --
model of AD (brain

Microglia from AD          Upregulated                 --
patients Human
microglia treated
with amyloid-beta
peptide in vitro Rat
hippocampus after
amyloid-beta peptide

Transgenic mouse       Inhibition of P2X7R             --
model of AD            decreased the number
                          of hippocampal
                         amyloid plaques

Human macrophages       P2X7R stimulation              --
and microglia           enhanced secretion
preactivated with       of proinflammatory
amyloid-beta peptide        cytokines

Rats injected with              --             Inhibition of A2AR
6-OHDA                                           improved motor
                                                performance and

Rats injected with              --             Inhibition of A2AR
haloperidol (DA                                reversed locomotor
antagonist)                                     suppression and
                                                 tremulous jaw

R Sample               Reference

Transgenic mouse         [169]
model of AD (brain

Microglia from AD        [170]
patients Human
microglia treated
with amyloid-beta
peptide in vitro Rat
hippocampus after
amyloid-beta peptide

Transgenic mouse         [171]
model of AD

Human macrophages      [172, 173]
and microglia
preactivated with
amyloid-beta peptide

Rats injected with       [174]

Rats injected with     [174, 175]
haloperidol (DA

Alzheimer's disease (AD), P2X7 receptors (P2X7R), adenosine
A2A receptor (A2AR), 6-hydroxydopamine (6-OHDA), and dopamine (DA).

Table 4: Ectonucleotidases (E-NTPDase, E-5'-nucleotidase, and
adenosine deaminase) in experimental models and patients with
Alzheimer's disease (AD) and Parkinson's disease (PD).

R Sample                    E-NTPDase

Synaptosomes of rats   [up arrow] enzymatic       [down arrow]
injected with            activity to ATP       enzymatic activity
scopolamine              (hippocampus and        (hippocampus)
                       cortex) [down arrow]
                        enzymatic activity
                       to ADP (hippocampus)

Synaptosomes of rats   [up arrow] enzymatic       [down arrow]
injected with            activity to ATP       enzymatic activity
scopolamine              (hippocampus and        (hippocampus)
                        cortex) [up arrow]
                        enzymatic activity
                       to ADP (hippocampus)

Hippocampus and                 --                     --
plasma from patients
with AD

Plasma from patients            --                     --
with AD

Serum from patients    [up arrow] enzymatic            --
with PD                activity to ATP [up
                         arrow] enzymatic
                         activity to ADP

                       Adenosine deaminase          Alkaline
R Sample                                       phosphatase (TNAP)

Synaptosomes of rats       [down arrow]                --
injected with           enzymatic activity
scopolamine               (hippocampus)

Synaptosomes of rats       [down arrow]                --
injected with           enzymatic activity
scopolamine               (hippocampus)

Hippocampus and                 --            [up arrow] enzymatic
plasma from patients                                activity
with AD

Plasma from patients            --            [up arrow] enzymatic
with AD                                             activity

Serum from patients             --                     --
with PD

R Sample               Reference

Synaptosomes of rats     [169]
injected with

Synaptosomes of rats     [170]
injected with

Hippocampus and          [171]
plasma from patients
with AD

Plasma from patients   [172, 173]
with AD

Serum from patients      [174]
with PD

Alzheimer's disease (AD) and Parkinson's disease (PD).

Table 5: Role of purinoreceptors in experimental models and patients
with MS: overview of main findings.

Sample               Receptors                 Main findings

Cerebral cortex    P2Y12 receptor       Reduction in the P2Y12R is
from healthy and                    immunoreactive in the lesions. This
MS patients                         event was directly correlated with
                                     the extent demyelination found in
                                         grey matter cortical and
                                         subcortical white matter.

Tissues of rats    P2X7 receptor       P2X7R is highly expressed in
exposed to EAE     P2Y12 receptor   microglia in MS lesions during the
model and brain                      peak of EAE. P2X7R is associated
tissue from                         with a proinflammatory phenotype of
healthy and MS                         human microglia. In parallel,
patients                            P2Y12R was associated with an anti-
                                      inflammatory phenotype in human
                                    microglia. P2Y12R was expressed at
                                    lower levels in active inflammatory
                                       MS lesions. P2Y12R expression
                                    increased in the remission phase of

Spleen and lymph   P2X7 receptor       Coculture of [P2X7R.sup.-/-]
node cell from                          macrophages with wild/type
[P2X7R.sup.-/-]                      lymphocytes showed that enhanced
mice exposed to                       proliferative activity resided
EAE model                               within the [P2X7R.sup.-/-]
                                    lymphocyte population. Furthermore,
                                      mRNA and protein for IFN/y were
                                    significantly reduced in the CNS of
                                      [P2X7R.sup.-/-]  mice with EAE.
                                        Enhanced susceptibility of
                                        [P2X7R.sup.-/-] mice to EAE
                                       reflects a loss of apoptotic
                                         activity in lymphocytes.

EAE induced in      A1 receptor     Investigation of the role of the A1
rats by guinea                          receptor using antagonists.
pig spinal cord                     Caffeine (10-30 m-kg) decreases the
homogenates                         incidence of EAE and attenuates EAE
(GPSCH model)                            pathology at behavioral,
                                      histological (inflammatory cell
                                     infiltration and demyelination),
                                     and neurochemical (expression of
                                    inflammatory cytokines) levels. In
                                    addition, caffeine also upregulated
                                      A1 receptor and TGF-[beta] mRNAs
                                      and suppressed INF-[gamma] mRNA
                                               in EAE rats.

Brain of female    P2X7 receptor      Enhanced expression of GFAP and
Lewis rats                             S100[beta] is associated with
exposed to EAE                      expression of P2X7R. Brilliant blue
model                                   G, an antagonist of P2X7R,
                                          significantly decreases
                                    astrogliosis (GFAP and S100[beta]).

Brain of rats      P2X7 receptor     The enhancement in the expression
exposed to EAE                       of the P2X7 receptor at the level
model                                  of both mRNA and protein was
                                          observed in the peak of
                                       neurological symptoms and was
                                     connected mostly with neurons (4,
                                             6, 8, and 10 days

Human monocytes    P2X7 receptor    A rare P2X7 variant Arg307Gln with
from                                  absent pore formation function
Australasian                        protects against neuroinflammation
patients with MS                                  in MS.

Two independent     A2A receptor    Upregulation of A2AR in the CNS in
mouse EAE                            EAE, predominantly detected on T
models: by                          cells and macrophages/microglia. A
immunization in                     preventive EAE treatment with A2AR/
C57BL/6 using                       specific agonist inhibited myelin/
[MOG.sub.35/55]                      specific T cell proliferation ex
and by                               vivo and ameliorated disease. In
[PLP.sub.139/                        parallel, the application of the
155] in mouse                        same agonist after disease onset
wild/type and                          exacerbated nonremitting EAE
lacking A2AR                         progression and resulted in more
([A2AR.sup.-/                        severe tissue destruction. A2AR/
-])                                  deficient mice showed accelerated
                                          and exacerbated disease
                                     manifestation with higher numbers
                                      of inflammatory lesions in the
                                         early stage. EAE quickly
                                       ameliorated and myelin debris
                                         accumulation was lower in
                                    [A2AR.sup.-/-] mice. Finally, an in
                                    vitro activation of A2AR inhibited
                                         phagocytosis of myelin by
                                     macrophages and primary microglia
                                    as well as migration of [CD4.sup.+]
                                     T cells, macrophages, and primary

Lymphocyte          A2A receptor       CGS21680 (CGS, A2AR agonist)
isolation from                       significantly suppressed specific
nerve tissue and                     lymphocyte proliferation, reduced
lumbar spinal                          infiltration of [CD4.sup.+] T
cord of female                        lymphocytes, and attenuated the
mice exposed to                         expression of inflammatory
EAE                                 cytokines, which in turn inhibited
                                       the EAE progression. CGS can
                                      increase the [[Ca.sup.2+]]i in
                                     murine lymphocytes, which may be
                                       the mechanism underlying the
                                    suppressive effects of CGS-induced
                                    A2AR activation on EAE progression.

Oligodendrocyte    P2X7 receptor     Sustained activation of P2X7R in
cultures and                           vivo causes lesions that are
postmortem optic                     reminiscent of the major features
nerve samples                          of MS plaques (demyelination,
from MS patients                     oligodendrocyte death, and axonal
                                      damage). In addition, treatment
                                      with P2X7R antagonists reduces
                                     demyelination and ameliorates the
                                     associated neurological symptoms.
                                      The study suggests that ATP can
                                      kill oligodendrocytes via P2X7R
                                        activation and this process
                                     contributes to EAE. Importantly,
                                      P2X7R expression is elevated in
                                    normal-appearing axon tracts in MS

Peripheral blood    A1 receptor      Decreased levels of adenosine and
mononuclear cells                    its A1 receptor modulate TNFa and
from MS patients                     IL-6 levels and may contribute to
                                          the pathogenesis of MS.

Brain and spinal    A2A receptor    A2A receptors participate in anti-
cord of female                            inflammatory effects of
SJL/J mice                              cannabidiol. A2A antagonist
infected with                          ZM241385 partially blocks the
Theiler's virus                      protective effects of cannabidiol
infection                                in the initial stages of

Human microglia    P2Y12 receptor    P2Y12 is expressed on parenchymal
                                    microglia and is stable throughout
                                    human brain development, including
                                        fetal phases. MS result in
                                    decreased P2Y12 immunoreactivity in
                                    plaque-or lesion-associated myeloid
                                    cells. P2Y12 is a useful marker for
                                        the identification of human
                                    microglia throughout the lifespan.

Blood from MS      P2X4 receptor    A rare genetic variant in P2RX4 and
patients           P2X7 receptor         P2RX7 is a major genetic
                                    contributor to disease (description
                                     of the three variant haplotypes:
                                     P2RX7 rs140915863:C>T [p.T205M];
                                     P2RX7 rs201921967:A>G [p.N361S];
                                         and P2RX4 rs765866317:G>A

C57BL6 mice and    P2X7 receptor      The incidence of EAE disease in
P2X7-deficient                         P2X7 mice was reduced 4-fold
mice exposed to                      compared to the wild type. Mouse
EAE model                           splenic T cells isolated from P2X7
                                        null mice produced greater
                                    IFN[gamma] and IL-17 (from 3-to 12-
                                      fold greater levels) than wild-
                                     type cells. Although infiltrating
                                     cells were detected in the brains
                                      of both the P2X7 and wild type,
                                     astroglial activation and axonal
                                      damage were reduced compared to
                                                wild type.

Brain and spinal    A1A receptor      [A1AR.sup.-/-] mice developed a
cord from female                     severe progressive-relapsing form
mice exposed to                       of EAE compared with their wild
EAE model                           type. Demyelination, axonal injury,
                                        and enhanced activation of
                                      microglia and macrophages were
                                    observed in [A1AR.sup.-/-]. Spinal
                                      cords from [A1AR.sup.-/-] mice
                                          demonstrated increased
                                     proinflammatory gene expression.
                                     [A1AR.sup.-/-] macrophage-derived
                                    soluble factors caused significant
                                       oligodendrocyte cytotoxicity
                                     compared with wild-type controls.

Spinal cord from   P2X7 receptor     In control spinal cord, few small
MS patients                          microglial cells/macrophages were
                                     scattered throughout the tissue.
                                         However, MS specimens had
                                     significantly greater density of
                                    such cells with longer processes in
                                       affected regions. MS also had
                                     significantly greater density of
                                    P2X7 and immunoreactive microglial
                                       cells/macrophages in affected

Sample             Reference

Cerebral cortex      [201]
from healthy and
MS patients

Tissues of rats      [202]
exposed to EAE
model and brain
tissue from
healthy and MS

Spleen and lymph     [203]
node cell from
mice exposed to
EAE model

EAE induced in       [204]
rats by guinea
pig spinal cord
(GPSCH model)

Brain of female      [205]
Lewis rats
exposed to EAE

Brain of rats        [206]
exposed to EAE

Human monocytes      [207]
patients with MS

Two independent      [194]
mouse EAE
models: by
immunization in
C57BL/6 using
and by
155] in mouse
wild/type and
lacking A2AR

Lymphocyte           [208]
isolation from
nerve tissue and
lumbar spinal
cord of female
mice exposed to

Oligodendrocyte      [209]
cultures and
postmortem optic
nerve samples
from MS patients

Peripheral blood     [210]
mononuclear cells
from MS patients

Brain and spinal     [211]
cord of female
SJL/J mice
infected with
Theiler's virus

Human microglia      [212]

Blood from MS        [213]

C57BL6 mice and      [214]
mice exposed to
EAE model

Brain and spinal     [215]
cord from female
mice exposed to
EAE model

Spinal cord from     [216]
MS patients

MS (multiple sclerosis), experimental autoimmune encephalomyelitis
(EAE), central nervous system (CNS), and glial fibrillary acid
protein (GFAP).

Table 6: Ectonucleotidases (E-NTPDase, E-5'-nucleotidase,
and ADA) in experimental models and patients with MS: a review.

Sample                      E-NTPDase          E-5'-nucleotidase

Serum of MS patients            --                     --
(RRMS form)

Lymphocytes of MS           [up arrow]                 --
patients (RRMS form)   expression CD39 [up
                         arrow] enzymatic
                       activity to ATP and

Platelets of MS        [up arrow] enzymatic       [down arrow]
patients (RRMS form)   activity to ATP and     enzymatic activity

Platelets from rats    [up arrow] enzymatic       [down arrow]
demyelinated with EB   activity to ATP (on     enzymatic activity
                        days 3, 7, 15, and        (on day 15)
                          21) [up arrow]
                        enzymatic activity
                        to ADP (on day 7)

Platelets from rats    [up arrow] enzymatic   [up arrow] enzymatic
demyelinated with EB   activity to ATP (on     activity (on day7)
                        day 7) [up arrow]
                        enzymatic activity
                        to ADP (on day 7)

Synaptosomes from      [up arrow] enzymatic   [up arrow] enzymatic
cerebral cortex of     activity to ATP (on    activity (on day 7)
rats demyelinated       days 7 and 15) [up
with EB                  arrow] enzymatic
                       activity to ADP (on
                              day 7)

Plasma membrane from   [up arrow] enzymatic   [up arrow] enzymatic
lumbosacral region     activity to ATP (on    activity (on days 15
of spinal cords from   days 7, 15, and 25)          and 25)
EAE-induced rats       [up arrow] enzymatic
                       activity to ADP (on
                             day 15)

Blood serum from       [up arrow] enzymatic       [down arrow]
EAE-induced rats       activity to ATP (on     enzymatic activity
                       day 25) [down arrow]       (on day 15)
                        enzymatic activity
                        to ADP (on days 15
                             and 25)

T cells from             [down arrow] in               --
patients with            number of CD39-
relapsing-remitting    positive Treg cells

Sample                 Adenosine deaminase    Reference

Serum of MS patients   [up arrow] enzymatic     [169]
(RRMS form)                  activity

Lymphocytes of MS          [down arrow]         [170]
patients (RRMS form)    enzymatic activity

Platelets of MS            [down arrow]         [171]
patients (RRMS form)    enzymatic activity

Platelets from rats             --            [172, 173]
demyelinated with EB

Platelets from rats             --              [174]
demyelinated with EB

Synaptosomes from               --            [174, 175]
cerebral cortex of
rats demyelinated
with EB

Plasma membrane from            --              [217]
lumbosacral region
of spinal cords from
EAE-induced rats

Blood serum from                --              [217]
EAE-induced rats

T cells from                    --            [218,219]
patients with

EB (ethidium bromide), relapsing-remitting
multiple sclerosis (RRMS), experimental autoimmune
encephalomyelitis (EAE), and Treg (regulatory T
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Author:Bagatini, Margarete Dulce; Santos, Alessandra Antunes dos; Cardoso, Andreia Machado; Manica, Aline;
Publication:Journal of Immunology Research
Date:Jan 1, 2018
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