Soluble and membrane-bound adenylate kinase and nucleotidases augment ATP-mediated inflammation in diabetic retinopathy eyes with vitreous hemorrhage
ATP and adenosine are important signaling molecules involved in vascular remodeling, retinal function, and neurovascular coupling in the eye. Current knowledge on enzymatic pathways governing the duration and magnitude of ocular purinergic signaling is incompletely understood. By employing sensitive analytical assays, this study dissected ocular purine homeostasis as a complex and coordinated network. Along with previously characterized ecto-5′-nucleotidase/CD73 and adenylate kinase activities, other enzymes have been identified in vitreous fluids, including nucleoside triphosphate diphosphohydrolase (NTPDase), adenosine deaminase, and alkaline phosphatase. Strikingly, activities of soluble adenylate kinase, adenosine deaminase, ecto-5′-nucleotidase/CD73, and alkaline phosphatase, as well as intravitreal concentrations of ATP and ADP, were concurrently upregulated in patients suffering from diabetic retinopathy (DR) with non-clearing vitreous hemorrhage (VH), when compared to DR eyes without VH and control eyes operated due to macular hole or pucker. Additional histochemical analysis revealed selective distribution of key ecto-nucleotidases (NTPDase1/CD39, NTPDase2, ecto-5′-nucleotidase/CD73, and alkaline phosphatase) in the human sensory neuroretina and optic nerve head, and also in pathological neofibrovascular tissues surgically excised from patients with advanced proliferative DR. Collectively, these data provide evidence for specific hemorrhage-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and pro-angiogenic ATP-regenerating phenotype. In the future, identifying the exact mechanisms by which a broad spectrum of soluble and membrane-bound enzymes coordinately regulates ocular purine levels and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative DR and other vitreoretinal diseases will be an area of intense interest.
NTPDase, alkaline phosphatase, and adenosine deaminase circulate in human vitreous.
Purinergic enzymes are up-regulated in diabetic eyes with vitreous hemorrhage.
Soluble adenylate kinase maintains high ATP levels in diabetic retinopathy eyes.
Ecto-nucleotidases are co-expressed in the human retina and optic nerve head.
Alkaline phosphatase is expressed on neovascular tissues excised from diabetic eyes.
KeywordsDiabetic vitreous hemorrhage Intravitreal ATP Retina Optic nerve head Soluble adenylate kinase Alkaline phosphatase Ecto-nucleotidases
Clear signaling roles for extracellular ATP, ADP, and adenosine have been established in virtually all organs and tissues , including the eye [2, 3]. ATP released from damaged neurons and activated microglia generally acts as a pro-inflammatory molecule initiating immunomodulatory, neurodegenerative, and hyperemic processes in the eye, which are mediated via activation of P2X7, P2Y1, and other ligand-gated (P2X) and G protein-coupled (P2Y) receptor subtypes co-expressed in the sensory retina and other ocular structures [2, 4, 5, 6, 7, 8, 9]. Another mechanism of ATP action comprises its ectoenzymatic breakdown into adenosine, which in turn, mediates multiple effects (often counteracting to ATP) via binding to its own nucleoside-selective receptors. A2A and other subtypes of adenosine receptors were shown to be implicated in the modulation of the light responses of photoreceptors, retinal hyperemia, pathological retinal angiogenesis, protection of neurons from hyper-excitation and glutamate toxicity [3, 10, 11, 12, 13]. Previous research from our and other laboratories revealed the ability of VF and aqueous humor to maintain ATP and adenosine at certain characteristic nanomolar levels, which increase in pathological conditions, such as dry eye disease , glaucoma [2, 12, 15], age-related macular degeneration with subretinal hemorrhage , and diabetic retinopathy (DR) [16, 17].
DR is the most common microvascular complication of diabetes worldwide, characterized by visual impairment, vascular leakage, vessel occlusion, neuronal and glial dysfunctions, and cell death in retinal capillaries. The growth of newly formed abnormally differentiated vessels and non-clearing vitreous hemorrhage (VH) are clinical hallmarks in the proliferative form of DR (PDR) [18, 19, 20, 21, 22, 23]. Treatments for the vision-threatening complications of DR have been greatly improved over the past decade. However, further development of preventional and interventional strategies against DR requires a better understanding of the exact mechanisms underlying diabetes-induced microvascular, neurodegenerative and inflammatory complications.
This study aims to further elaborate the role of purinergic mechanisms in the pathogenesis of DR. We have identified a broad spectrum of soluble and membrane-bound enzymes, which are co-expressed in the human VF, sensory retina and optic nerve head and coordinately regulate ocular nucleotide and nucleoside levels via two counteracting, purine-inactivating and ATP-regenerating, pathways. Furthermore, data on marked upregulations of intravitreal adenylate kinase (AK), ecto-5′-nucleotidase/CD73 (eN/CD73), adenosine deaminase (ADA), and alkaline phosphatase (ALP) activities and concurrently elevated ATP/adenosine ratio in DR eyes with VH provide a sufficient justification for reexamination of the role of purine homeostasis in the pathogenesis of DR.
Materials and methods
Surgical collection of vitreous samples and PDR neovascular tissues
Diabetes mellitus, age and gender distribution among the patients studied
DM1 / DM2
67.6 ± 0.8
45.3 ± 2.4
64.0 ± 2.3
Duration of DM (years)
24.0 ± 2.0 (43)
15.5 ± 2.0 (35)
0 / 56
Blood HbA1c (%)
9.3 ± 0.3 (34)
7.6 ± 0.4 (32)
Preparation of human eye and PDR neovascular tissue sections
Cadaver eyeballs were enucleated 48 h postmortem from a female donor without apparent eye diseases. The use of human tissues and organs has been approved by the Ethical Committee of Turku University Hospital. Transverse sections of the retrobulbar optic nerve close to the optic nerve head, as well as neovascular tissues (excised surgically from PDR patients as described above), were embedded in the cryo-mold with Tissue-Tek® O.C.T. compound (Sakura Finetek Europe B.V. The Netherlands), cut using a cryostat and stored at − 80 °C. Eyeballs were additionally enucleated from a 68-year-old patient having a periocular tumor which was estimated to be invading to the bulbus. Immediately after the enucleation, the eyeballs were fixed in 10% formalin for 2–3 days followed by embedding in paraffin according to a routine protocol. Experienced pathologists confirmed that the tumor invasion had not reached the eyeball. The tenets of the Declaration of Helsinki and Kuopio University Hospital ethical rules were followed.
Measurement of soluble purine-converting activities
Soluble NTPDase/ADPase, eN/CD73, ADA, and AK were assayed by incubating the VF (or serum) with [3H]ADP, [3H]AMP, [3H]adenosine, and [3H]AMP plus ATP, as respective enzyme substrates. 3H-labeled nucleotides and nucleosides were separated by thin-layer chromatography (TLC) and quantified by scintillation β-counting [17, 25] or developed by autoradiography, as described in the Supplementary Material. In competitive assays, the samples were pretreated for 30 min with inhibitors of NTPDases sodium polyoxotungstate-1 (POM-1) (Tocris Bioscience, Bristol, UK) and POM-144 , selective eN/CD73 inhibitors α,β-methylene ADP (APCP, Sigma) and its derivative N6-phenylethyl-[(phosphonomethyl)-phosphonic acid], (PSB-12379 ), inhibitor of AK diadenosine pentaphosphate (Ap5A), and ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, Tocris Bioscience). Soluble ALP was assayed by incubating VF (8 μl) on clear 96-well plates in 180 μl of basal salt solution (BSS; comprising 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgSO4, 25 mM HEPES and 5 mM glucose, pH 9.3) in the absence or presence of a specific ALP inhibitor tetramisole (5 mM). After a 60-min pre-treatment, 20 μl of chromogenic substrate for ALP, p-nitrophenyl phosphate (p-NPP), was added to the microwells at a final concentration of 3 mM, followed by incubation at 37 °C for 20 h and subsequent measurement of the absorbance intensity at 405 nm (Tecan Infinite M200, Salzburg, Austria). Likewise, serum ALP activity was determined after a 3-h incubation of human serum (2 μl) with 3 mM p-NPP.
Determination of ATP, ADP, adenosine, and HbA1c
Intravitreal purine concentrations were determined by using bioluminescent (ATP and ADP) and fluorometric (adenosine) enzyme-coupled purine-sensing assays , under the conditions delineated experimentally for VF . After defrosting, VF samples were immediately heat-inactivated for 5 min at 65 °C, thus preventing further metabolism of endogenous purines through soluble purinergic enzymes. The protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL). Glycated hemoglobin A1c (HbA1c) was measured in the blood using the Tina-quant® HbA1c assay kit with the Cobas Integra-800 analyzer (Roche Diagnostics, Rotkreuz, Switzerland).
Immunohistochemical analysis of ecto-nucleotidases in the human eye
Formalin-fixed, paraffin-embedded sections of the human retina were stained for ecto-nucleotidases using UltraView DAB Detection Kit and BenchMark XT automated slide staining system (Ventana Medical Systems Inc., Roche, USA), according to manufacturer’s instructions. For immunofluorescence staining, cryosections of the optic nerve head and neovascular tissues were sequentially incubated with primary and secondary fluorescently labeled antibodies. Additional details are given in the Supplementary Material.
For localization of ecto-nucleotidase activities, a modification of the lead nitrate method was employed [29, 30], as described in the Supplementary Material. ALP activity was additionally evaluated by measuring dark purple precipitate after incubating the tissues at alkaline pH with artificial chromogenic enzyme substrates 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT, 0.35 mM each) .
Two-tailed paired Student’s t test was used for single comparisons and one-way ANOVA with Dunnett’s multiple comparison post-hoc tests for multiple comparisons. Nonparametric Spearman’s correlation coefficients were computed to investigate the inter-correlations of continuous variables. In the case of comparative studies, the researcher(s) analyzed the encoded vitreous samples provided by the surgeon in a masked way, without knowing the clinical status of the patients. The results were analyzed with Prism GraphPad 7 software (San Diego, CA) and presented either as column bars (mean ± SEM) or as box-and-whiskers plots, where the box extends from the 25th to the 75th percentile with a horizontal line at the median and with whiskers showing the range of the data. The statistical significance levels were denoted as *P < 0.05 and **P < 0.01.
A broad network of soluble enzymes co-exists in human vitreous and regulates intravitreal purine levels via counteracting, purine-inactivating and ATP-regenerating, pathways
Consistent with the autoradiographic data, a direct quantitative analysis confirmed the presence of low but clearly detectable ADPase activity in most of the VF studied (Fig. 1b). Pre-treatment of the samples with a non-selective NTPDase inhibitor POM-144 , but not with another inhibitor POM-1, diminished the rate of [3H]ADP hydrolysis by ~ 50%. Taking into account the different ATP:ADP hydrolysis ratios for NTPDase1 and other members of this family, NTPDase2 and NTPDase3 (~ 1:1, 10–40:1 and 3–4:1, respectively) [31, 32] and by drawing an analogy with soluble NTPDase1/CD39 freely circulating in the human and murine blood , it may be speculated that NTPDase1/CD39 represents the major enzyme responsible for the measured [3H]ADP hydrolysis in human vitreous. The employment of [3H]AMP as another tracer substrate demonstrated the presence of AMPase activity, which can be blocked by eN/CD73 inhibitors, APCP and PSB-12397 (Fig. 1b). In the presence of γ-phosphate-donating ATP, part of [3H]AMP was phosphorylated into [3H]ADP/ATP, while Ap5A prevented this phosphotransfer reaction. For direct quantification of ADA activity, we used a particular solvent mixture that enabled better TLC separation of [3H]adenosine and its 3H-metabolites . Incubation of VF with [3H]adenosine caused its deamination into [3H]inosine, which can be blocked by a specific ADA inhibitor EHNA (Fig. 1b). Soluble ALP was also determined spectrophotometrically. Due to the low sensitivity of this colorimetric technique, the assay settings have been modified and optimized accordingly, particularly by incubating the microplate for up to 20 h at alkaline pH and 37 °C. Long-term incubation of VF with p-NPP was accompanied by the development of a soluble yellow reaction product that may be detected at 405 nm, and this catalytic reaction was prevented in the presence of a specific ALP inhibitor, tetramisole (Fig. 1b). Collectively, the identification of a broad spectrum of purine-converting enzymes in human VF forms a solid background for further elucidation of the role of the ATP-adenosine axis in the pathogenesis of vitreoretinal diseases in humans.
Soluble eN/CD73, ADA, AK, and ALP activities are selectively upregulated in diabetic retinopathy eyes with vitreous hemorrhage
High intravitreal AK correlates with concurrently upregulated ADA activity and increased levels of ATP and ADP in DR eyes with VH
Immunohistochemical analysis reveals the selective distribution of key ecto-nucleotidases and ALP within the human sensory retina, PDR neovascular tissues, and optic nerve head
Cryosections of the neovascular tissues surgically excised from two PDR eyes during vitrectomy, as well as the prelaminar region of the cadaver optic nerve head were also analyzed using enzyme (immuno)histochemistry. Measurement of ecto-nucleotidase and ALP activities, in combination with immunofluorescence staining, allowed us to pinpoint the distribution of key nucleotide-inactivating enzymes within the tissues. Incubation of neovascular tissues with BCIP/NBT (known as preferred chromogenic substrates for ALP ) revealed the development of dark blue precipitates in the connective tissues (Fig. 5f). Likewise, fairly similar staining patterns were observed by staining the tissues with an anti-ALP antibody (Fig. 5f; rightmost panels). Though, lead nitrate-based enzyme histochemistry did not detect any ecto-nucleotidase activities in the tissues studied (data not shown).
We have recently identified soluble intravitreal enzymes, eN/CD73 and AK (mainly comprising of the AK1 isoform) and further demonstrated that AK activity was selectively up-regulated in PDR eyes, accompanied by concurrently elevated concentrations of ATP, ADP, angiopoietin-2, TGFβ1, MMP-9, and other inflammatory and angiogenic factors . The current study extends the earlier findings by showing that, along with eN/CD73 and AK, other soluble enzymes NTPDase/ADPase, ADA, and ALP circulate in the human vitreous. Strikingly, by dividing the VF based on non-clearing VH as additional clinical enrollment criteria for diabetic vitrectomy, we revealed highly specific upregulations of soluble AK, ADA, eN/CD73, and ALP activities in DR eyes with VH (see Fig. 3a).
Another salient finding of this study is the identification of an extensive network of ocular membrane-bound ectoenzymes. Previous histochemical analyses of human, canine and rodent eyes have shown a selective distribution of ecto-nucleotidases in the retinal vasculature (NTPDase1/CD39) and parenchyma (NTPDase2) [7, 34, 35], as well as inner Müller cell processes (eN/CD73) [7, 17, 34, 36]. Here, we have also demonstrated differential localizations of key ecto-nucleotidases in the human ganglion cells (NTPDase2), rod-and-cone-containing photoreceptor layer (eN/CD73), the outer segments of the photoreceptors, retinal vasculature and the adjacent RPE and choriocapillaris layers (NTPDase1/CD39) (see Fig. 5). Abundant expression of nucleotide-inactivating enzymes in the plane where the retina is cleaved in the living eye during pathological retinal detachment might be of particular relevance in terms of preventing excessive inflammatory responses and maintaining the choroidal and retinal blood supplies via rapid inactivation of locally released ATP. In this context, it is relevant to mention that P2X7 receptors expressed on photoreceptor cells can be implicated in ATP-mediated subretinal hemorrhage, choroidal neovascularization, and photoreceptor cell death [4, 7], while macrophage/microglial P2X7 seems to act as a scavenger receptor by clearing debris around the RPE and removing metabolic end products from photoreceptors .
Data on the specific distribution of ecto-nucleotidases in the optic nerve head further extend our knowledge on the regulatory mechanisms of purinergic signaling in the human eye. In particular, NTPDase1/CD39 was shown to be expressed in the central retinal artery and other ocular blood vessels. These findings are consistent with data on the abundant expression of NTPDase1/CD39 on vascular endothelial cells and its implication in the control of hemostasis through the termination of pro-inflammatory and pro-thrombotic effects of intravascular ATP and ADP [31, 32, 37]. By contrast, nerve bundle-surrounding microglial cells displayed strong immunoreactivity for NTPDase2. Due to the high preference of NTPDase2 for the hydrolysis of ATP over ADP [31, 32], this ectoenzyme presumably has functionality in the rapid scavenging of mitogenic extracellular nucleoside triphosphates in a neuronal environment, while the subsequent degradation of ATP-derived ADP into AMP will occur with considerable delay . The identification of eN/CD73 in the connective tissue septa surrounding NTPDase2-positive microglia and NTPDase1/CD39-positive blood vessels suggest the efficient crosstalk between these ecto-nucleotidases, mediating tuned regulation of the ATP-adenosine axis within the optic nerve-fiber bundles and vasculature.
Tissue-nonspecific ALP is known to play a crucial role in bone and cartilage mineralization via the maintenance of the proper concentrations of a powerful mineralization inhibitor PPi [32, 39] and it has also been identified among the top calcification-related genes overexpressed in the human trabecular meshwork . Therefore, the selective expression of ALP in the neurosensory retina and optic nerve head (mainly restricted to the choriocapillaris, pia mater, and small blood vessels and capillaries) may be considered as an important and heretofore unrecognized mechanism controlling blood supply and soft tissue calcification. Furthermore, the presence of ALP in the pathological neofibrovascular tissues surgically excised from PDR eyes (see Fig. 5f), together with data on significantly upregulated soluble ALP activity in DR eyes without and with VH (see Fig. 3a) provide a novel insight into the role of this enzyme in the pathogenesis of advanced DR. Though, given a broad substrate specificity of ALP towards different phosphate-containing compounds with a pH optimum for this catalytic reaction lying in the alkaline range [31, 32], further studies are required to validate the contribution of ALP to the ocular nucleotide homeostasis.
Data on selective compartmentalization of ecto-nucleotidases in certain ocular structures, together with the identification of a wide spectrum of soluble enzymes co-expressed to a variable extent in human VF, provide novel insights into the regulatory mechanisms governing the duration and magnitude of purinergic signaling in a healthy and diseased eye. Taking into account the fact that the vitreous is the largest structure within the eye occupying about 80% of the ocular volume , the constitutive presence of soluble purine-converting enzymes might represent an important auxiliary effector system for tuned control of balanced nucleotides and adenosine levels in the VF. For instance, eN/CD73 is highly expressed on the photoreceptor cells in human (see Fig. 5a), canine [36, 42] and rodent  retinas, while A2A receptors are known to be predominantly localized on the inner nuclear layer and ganglion cells facing towards the vitreous lumen [11, 36, 43]. These data indirectly imply an important physiological contribution of the soluble forms of eN/CD73 and ADA in controlling local adenosine levels at the vitreoretinal interface. The identification of soluble AK suggests that regulation of ocular nucleotide homeostasis extends beyond the inactivating pathways. AK is abundantly present in the cytosols of well-differentiated tissues with a high-energy demand [44, 45], including the brain [31, 46], and rod outer segments of the retina , where it regulates adenine nucleotide pools by catalyzing reversible phosphoryl transfers ATP + AMP ↔ 2ADP. Along with intracellular localization, this ATP-regenerating enzyme is expressed on the surface of vascular endothelial and other cells [31, 33] and it also freely circulates in the human and murine bloodstream [25, 48], human VF (; current study), and rat pancreatic juice . Strikingly, the dramatic activation of AK-mediated phosphotransfer reactions in DR eyes with VH correlates with concurrently elevated concentrations of ATP and ADP (see Fig. 4b), thus allowing to consider this soluble enzyme as an unfavorable factor triggering spatial propagation of ATP-mediated inflammatory responses into the surrounding retina far distant from the site of focal detachment.
These findings prompted important questions regarding the origins of the measured activities and mechanisms underlying their appearance in the vitreous. It may be speculated that certain portions of ocular (ecto)enzymes are co-released into the vitreous cavity, along with ATP and other transmitters, in response to light-evoked neuronal stimulations, mechanical perturbations caused by incessant eye movements and variations in the vascular tone and intraocular pressure. Cellular mechanisms underlying the release of purinergic enzymes might particularly involve the shedding of membrane-bound eN/CD73 and ALP via cleavage of their glycosyl-phosphatidylinositol anchors, microvesicular shedding, exocytosis, and other secretory pathways [31, 32]. Therefore, in principle, high soluble activities in the DR eyes might reflect a global upregulation of membrane-bound ectoenzymes and their partial shedding into the vitreous lumen. Indeed, the activities of ecto-nucleotidases and other components of the purinergic signaling cascade are known to be activated in the retina under all pathophysiological conditions investigated so far [4, 5, 7, 13]. For instance, the expression of A2A receptors and the activity of eN/CD73 are upregulated in the inner Müller cell processes during the vasculoproliferative stage of an experimental model of oxygen-induced retinopathy triggered by a 4-day exposure of neonatal dogs to high oxygen and their subsequent return to normal air . Though, our data on markedly activated intravitreal purine homeostasis only in the DR eyes with severe VH suggest the implication of more specific mechanisms triggering the release of soluble enzymes and/or endogenous ATP from the extravascular blood cells and abnormal blood vessels in the course of retinal degeneration, blood clotting, and neovascularization.
Collectively, these data provide evidence for selective VH-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and angiogenic ATP-regenerating phenotype. Identifying the exact mechanisms by which the extensive network of membrane-associated and soluble enzymes regulates ocular nucleotide and nucleoside concentrations and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative retinopathies with VH and other vascular and degenerative eye diseases will be an area of intense interest in the future.
Open access funding provided by University of Turku (UTU) including Turku University Central Hospital. This work was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, Erasmus Internship Program, the Finnish Eye Foundation, the Mary and Georg C. Ehrnrooth Foundation, and HUCH Clinical Research Grants (TYH2018127 and TYH2016230). We are grateful to Dr. Janne Liukkonen (Turku University Hospital, Turku, Finland) for providing us with the human optic nerve samples and to Prof. Jean Sevigny (Laval University, Quebec, Canada) for sharing the antibodies. We also thank Sari Mäki and Anne Seppänen for the technical assistance, Seija Rusanen for drawing the schematic eye images, and Ruth Fair-Mäkelä for the revision of the text.
Compliance with ethical standards
The studies on surgical collection of vitreous samples and neovascular tissues were conducted according to the principles of the Declaration of Helsinki and approved by the Institutional Review Board of the Helsinki University Central Hospital and the Ethical Committee. The use of postmortem human tissues has been approved by the Ethical Committee of Turku University Hospital.
Conflict of interest
The authors declare that they have no conflict of interest
- 1.Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492Google Scholar
- 5.Sanderson J, Dartt DA, Trinkaus-Randall V, Pintor J, Civan MM, Delamere NA, Fletcher EL, Salt TE, Grosche A, Mitchell CH (2014) Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Muller cells, lens, trabecular meshwork, cornea and lacrimal gland. Exp Eye Res 127:270–279CrossRefGoogle Scholar
- 9.Vessey KA, Gu BJ, Jobling AI, Phipps JA, Greferath U, Tran MX, Dixon MA, Baird PN, Guymer RH, Wiley JS, Fletcher EL (2017) Loss of function of P2X7 receptor scavenger activity in aging mice: a novel model for investigating the early pathogenesis of age-related macular degeneration. Am J Pathol 187:1670–1685CrossRefGoogle Scholar
- 13.Liu Z, Yan S, Wang J, Xu Y, Wang Y, Zhang S, Xu X, Yang Q, Zeng X, Zhou Y, Gu X, Lu S, Fu Z, Fulton DJ, Weintraub NL, Caldwell RB, Zhang W, Wu C, Liu XL, Chen JF, Ahmad A, Kaddour-Djebbar I, al-Shabrawey M, Li Q, Jiang X, Sun Y, Sodhi A, Smith L, Hong M, Huo Y (2017) Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis. Nat Commun 8:584CrossRefGoogle Scholar
- 15.Lu W, Hu H, Sevigny J, Gabelt BT, Kaufman PL, Johnson EC, Morrison JC, Zode GS, Sheffield VC, Zhang X et al (2015) Rat, mouse, and primate models of chronic glaucoma show sustained elevation of extracellular ATP and altered purinergic signaling in the posterior eye. Invest Ophthalmol Vis Sci 56:3075–3083CrossRefGoogle Scholar
- 21.Duh EJ, Sun JK, Stitt AW (2017) Diabetic retinopathy: current understanding, mechanisms and treatment strategies. JCI Insight 2:e93751Google Scholar
- 23.Rubsam A, Parikh S, Fort PE (2018) Role of inflammation in diabetic retinopathy. Int J Mol Sci 19. https://doi.org/10.3390/ijms19040942
- 24.Loukovaara S, Robciuc A, Holopainen JM, Lehti K, Pessi T, Liinamaa J, Kukkonen KT, Jauhiainen M, Koli K, Keski-Oja J, Immonen I (2013) Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFbeta1 in diabetic eyes undergoing vitrectomy. Acta Ophthalmol 91:531–539CrossRefGoogle Scholar
- 27.Bhattarai S, Freundlieb M, Pippel J, Meyer A, Abdelrahman A, Fiene A, Lee SY, Zimmermann H, Yegutkin GG, Strater N et al (2015) α,β-methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5'-nucleotidase (CD73) inhibitors. J Med Chem 58:6248–6263CrossRefGoogle Scholar
- 42.Lutty GA, Merges C, McLeod DS (2000) 5′ nucleotidase and adenosine during retinal vasculogenesis and oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 41:218–229Google Scholar
- 45.Yan H, Tsai M-D (1999) Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv Enzymol Relat Areas Mol Biol 73:103–134Google Scholar
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