Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis

Abstract

Adenosine/adenosine receptor-mediated signaling has been implicated in the development of various ischemic diseases, including ischemic retinopathies. Here, we show that the adenosine A2a receptor (ADORA2A) promotes hypoxia-inducible transcription factor-1 (HIF-1)-dependent endothelial cell glycolysis, which is crucial for pathological angiogenesis in proliferative retinopathies. Adora2a expression is markedly increased in the retina of mice with oxygen-induced retinopathy (OIR). Endothelial cell-specific, but not macrophage-specific Adora2a deletion decreases key glycolytic enzymes and reduces pathological neovascularization in the OIR mice. In human primary retinal microvascular endothelial cells, hypoxia induces the expression of ADORA2A by activating HIF-2α. ADORA2A knockdown decreases hypoxia-induced glycolytic enzyme expression, glycolytic flux, and endothelial cell proliferation, sprouting and tubule formation. Mechanistically, ADORA2A activation promotes the transcriptional induction of glycolytic enzymes via ERK- and Akt-dependent translational activation of HIF-1α protein. Taken together, these findings advance translation of ADORA2A as a therapeutic target in the treatment of proliferative retinopathies and other diseases dependent on pathological angiogenesis.Pathological angiogenesis in the retina is a major cause of blindness. Here the authors show that adenosine receptor A2A drives pathological angiogenesis in the oxygen-induced retinopathy mouse model by promoting glycolysis in endothelial cells via the ERK/Akt/HIF-1α pathway, thereby suggesting new therapeutic targets for disease treatment.

Fig. 1

Localization and expression of Adora2a in rodent proliferative retinopathy. a Schematic illustration of mouse OIR model. Neonatal mice with nursing mothers were exposed to 75% O₂ from postnatal day (P) 7 to P12, followed by room air (RA) with maximum neovascularization at P17. b Real-Time PCR analysis of Adora1, Adora2a, Adora2b, and Adora3 mRNA expression in the whole retina. Retinas were from RA or OIR mice at P17. ***P < 0.001 vs. RA group (n = 7 mice per group). c, d Real-Time PCR analysis of adenosine receptor mRNA expression in the whole retina. Retinas were obtained from mice at the times indicated. Data were normalized to both the expression of internal control and to gene mRNA expression of each RA control at each time point. **P < 0.01 vs. P12 (n = 4 mice per group for c and n = 7 mice per group for d). e, f Localization and expression of Adora2a in the RA and OIR retinas. Retinopathy was induced in wild-type mice. P17 RA and OIR retinas were stained with Adora2a (green), isolectin B4 (Lectin, red, vessel, e), or IBa1 (red, macrophages/microglias, f) and DAPI (blue, nuclei). In all, 2nd and 4th rows are magnification of the boxed regions in the 1st and 3rd rows, respectively. Scale bar: 50 μm (1st and 3rd rows) and 20 μm (2nd and 4th rows). g Real-Time PCR analysis of Adora2a mRNA expression in laser-capture microdissected pathological neovessels (tufts) from OIR mice compared with normal vessels from control mice raised in RA at P17. ***P < 0.001 vs. RA (n = 4 per group). Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for b, g) and one-way ANOVA followed by Bonferroni test (for c, d)

Fig. 2

Endothelial Adora2a deficiency significantly decreases formation of pathological neovascularization in OIR retinas. a, b Quantification of pathological neovascularization and vessel dropout area (within the white borders) in postnatal day (P)17 OIR retinas. Adora2a ^flox/flox (Adora2a ^WT), Adora2a ^flox/floxLysm^cre/cre (Adora2a ^Mφ-KO), and Adora2a ^flox/floxCdh5^cre (Adora2a ^VEC-KO) mice were exposed to 70% O₂ a or 75% O₂ b. Areas of pathological neovascularization and vessel dropout are quantified as percentage of total retinal area. n = 16, 20, 15 retinas for a; n = 13, 10, 12 retinas for b; ***P < 0.001 vs. Adora2a ^WT group. Scale bars: 1000 μm. c Histological analysis of infiltration of neovascular nuclei from inner limiting membrane into vitreous in the OIR retinas. Nuclei on the vitreal side of the inner limiting membrane are indicated by asterisk. Scale bars: 100 μm. d Quantitative analysis of the number of neovascular nuclei in the OIR retinas. *P < 0.001 (n = 6 mice for Adora2a ^WT group; n = 8 for Adora2a ^Mφ-KO and Adora2a ^VEC-KO groups). e Ki-67 immunofluorescent staining on OIR retinas. Representative green (Ki-67), red (ERG), blue (nuclei, DAPI), and merged images were captured with confocal fluorescent microscopy. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bars: 50 μm. f Quantitative analysis of the Ki-67 and ERG double-positive cells in each group. *P < 0.001 (n = 9 mice for each group). Data are represented as means ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Bonferroni test

Fig. 3

Hypoxia upregulates ADORA2A expression by activating HIF-2α in HRMECs. a Real-Time PCR analysis of mRNA expression for adenosine receptors in HRMECs. HRMECs were exposed to normoxia (21% O₂) or hypoxia (0.5% O₂) for 6 h. n = 4. ***P < 0.001 vs. normoxia. b, c Real-Time PCR analysis of ADORA2A mRNA expression in HRMECs. HRMECs were exposed to normoxia (21% O₂) or hypoxia (0.5% O₂) for indicated times b or with indicated O₂ concentrations for 6 h c. n = 4. *P < 0.05; **P < 0.01, ***P < 0.001 vs. normoxia. d Western blot analysis of ADORA2A protein expression in HRMECs exposed to hypoxia (0.5% O₂) for indicated times. n = 4. HIF-1α and HIF-2α were used as positive controls for hypoxia, and β-actin was used as loading control. e Real-Time PCR analysis of ADORA2A mRNA expression in HRMECs. HRMECs were infected with 10 ( + ) or 30 ( +  + ) pfu per cell of either Ad-mutHIF-1α, Ad-mutHIF-2α, or Ad-Ctrl. n = 3. *P < 0.001. f, g Real-Time PCR analysis of ADORA2A mRNA expression in HRMECs. HRMECs were transfected with siHIF-1α f, siHIF-2α g, or siCtrl. Forty-eight hours later, cells were exposed to hypoxia (0.5% O₂) or air (21% O₂) for an additional 12 h. n = 4. **P < 0.01. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test

Fig. 4

ADORA2A regulates HRMEC proliferation, sprouting and tube formation. a, b Bromodeoxyuridine (BrdU) staining of HRMECs transfected with siRNAs targeting human ADORA2A (siA_2AR) or with a non-targeting negative control (siCtrl) under hypoxia conditions (0.5% O₂). n = 6. *P < 0.05. c Cell proliferation measured by WST-1 cell proliferation assay. n = 6. **P < 0.01. d Growth curves of transfected cultures. n = 6. *P < 0.05, ***P < 0.001 vs. siCtrl. e–g Ki-67 and BrdU staining of HRMECs. HRMECs were infected with a recombinant adenovirus vector expressing human ADORA2A (Ad-A_2AR) or a negative control adenovirus (Ad-Ctrl) in the presence of adenosine. Scale bars: 50 μm. n = 6. ***P < 0.001. h Cell proliferation measured by WST-1 cell proliferation assay. n = 6. ***P < 0.001. i Growth curves of HRMECs over 72 h following infection with Ad-Ctrl or Ad-A_2AR in the presence of adenosine. n = 6. ***P < 0.001 vs. Ad-Ctrl. j–u HRMECs were transfected with siA_2AR or siCtrl, or infected with Ad-Ctrl or Ad-A_2AR, and then were cultured in collagen gel to grow into 3D multicellular spheroids, or on a 2D matrix to form a tube network in the presence or absence of VEGF or adenosine. j, m Representative images of spheroidal sprouting after culturing for 24 h in collagen matrix under hypoxia (0.5% O₂) or normoxia (21% O₂). Scale bars: 100 μm. Morphometric quantification of spheroid sprouting by calculating the number of sprouts per spheroid k, n as well as total sprout length l, o. n = 10 per group. n is number of spheroids quantified. **P < 0.01; ***P < 0.001. Representative fluorescence photographs of angiogenic tube formation p, s. Scale bars: 200 μm. Cumulative tube length quantified using the Image J software q, t, and branch points calculated from five experiments in each case r, u. *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for b, c, f, g, h, k, l, n, o, q, r, t, u) and two-way ANOVA followed by Bonferroni test (for d, i)

Fig. 5

Adenosine-ADORA2A signaling regulates glycolysis in HRMECs and mouse retinal ECs. a Scheme showing the glycolytic pathway and associated enzymes. b Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs transfected with siA_2AR or siCtrl under normoxia (21% O₂) or hypoxia (0.5% O₂). n = 5. *P < 0.05, **P < 0.01, ***P < 0.001 vs. siCtrl normoxia group; ^& P < 0.05, ^&& P < 0.01, ^&&& P < 0.001 vs. siCtrl hypoxia group. c Real-Time PCR analysis of the mRNA levels of glycolytic genes in retinal blood vessels isolated with laser-capture microdissection from OIR-Adora2a ^WT and Adora2a ^VEC-KO mice at P17. n = 4. *P < 0.05 vs. Adora2a ^WT group. d Levels of secreted lactate of HRMECs transfected with siA_2AR or siCtrl under normoxia or hypoxia for indicated times. n = 3. ***P < 0.001. e ECAR profile showing glycolytic function in siCtrl- and siA_2AR-transfected cells under hypoxia (0.5% O₂) or normoxia (21% O₂). Vertical lines indicate the time of addition of glucose (10 mmol/l), oligomycin (2 μmol/l), and 2-DG (50 mmol/l). f Quantification of glycolytic function parameters from e. n = 8 for normoxic groups and n = 16 for each of CoCl₂ treatment groups. *P < 0.05; ***P < 0.001. g Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs infected with Ad-Ctrl or Ad-A_2AR, with or without adenosine treatment. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ad-Ctrl; ^& P < 0.05; ^&& P < 0.01; ^&&& P < 0.001 vs. Ad-Ctrl + Ado. h Levels of secreted lactate of HRMECs infected with Ad-A_2AR or Ad-Ctrl with or without adenosine treatment. n = 3. ***P < 0.001 vs. Ad-Ctrl. i ECAR profile showing glycolytic function in Ad-Ctrl- and Ad-A_2AR-infected cells, with or without adenosine treatment. j Quantification of glycolytic function parameters from i. n = 8 per group. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ad-Ctrl. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for b, c, g), one-way ANOVA followed by Bonferroni test (for f, j), and two-way ANOVA followed by Bonferroni test (for d, h)

Fig. 6

Glycolysis is involved in ADORA2A activation-mediated HRMEC proliferation and sprouting. a–g Results of Ki-67 and BrdU staining a–c, WST-1 cell proliferation assay d and EC spheroid sprouting assay e–g in HRMECs. HRMECs were infected with Ad-Ctrl or Ad-A_2AR adenoviruses, and then treated with adenosine in the presence or absence of two different glycolytic inhibitors (3PO, 10 μM, and 2-DG, 5 mM). n = 6. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 50 μm for a; Scale bars: 100 μm for e. h–j HRMECs were cultured as 3D multicellular spheroids in the presence or absence of 3PO or 2DG. Representative images of spheroidal sprouting after culturing for 24 h in collagen matrix under hypoxia (0.5% O₂) or normoxia (21% O₂) h. Morphometric quantification of spheroid sprouting by calculating the number of sprouts per spheroid i and total sprout length j. n = 10 per group (n is number of spheroids quantified). *P < 0.05, **P < 0.01; ***P < 0.001. Scale bars: 100 μm. Data are represented as means ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Bonferroni test

Fig. 7

ADORA2A regulates HRMEC tip cell formation. a Real-Time PCR analysis of the mRNA levels of the sprouting-governing genes in HRMECs transfected with siA_2AR or siCtrl, in the presence or absence of DAPT (n = 3; *P < 0.05; **P < 0.01; ***P < 0.001 vs. siCtrl; ^& P < 0.05, ^&& P < 0.01 vs. siCtrl + DAPT). b Real-Time PCR analysis of the mRNA levels of the sprouting-governing genes in 2-DG-treated HRMECs in the presence or absence of DAPT (n = 4; *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle control; ^& P < 0.05, ^&& P < 0.01 vs. DAPT). c, d Morphometric quantification of spheroid sprouting from cells transfected with siCtrl or siA_2AR in the presence or absence of DAPT. n = 10 per group. n is number of spheroids quantified. *P < 0.05; **P < 0.01; ***P < 0.001. e Representative fluorescence photographs of EC spheroids containing a 1:1 mixture of siCtrl^GFP and siCrl^RED ECs, or a 1:1 mixture of siCtrl^GFP ECs and ECs with siA_2AR^RED in the presence of DAPT. Scale bars, 100 μm. f Quantification of the fraction of tip cells with the indicated genotypes shown in e (n = 10; ***P < 0.001 vs. siCtrl^GFP). g Representative fluorescence photographs of mosaic EC spheroids containing a 1:1 mixture of siCtrl^GFP ECs and siA_2AR^RED ECs under hypoxia conditions. Cells were stained with DRAQ5 (blue) to mark EC nuclei. Tip cells are indicated by “^“; Stalk cells are indicated by “*“. The 2nd and 4th rows are magnification of the boxed regions in the 1st and 3rd rows, respectively. Scale bar: 50 μm (1st and 3rd rows) and 20 μm (2nd and 4th rows). h Quantification of the fraction of tip cells with the indicated genotypes shown in g (n = 10 per group). ***P < 0.001 vs. siCtrl^GFP. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for a, b, f, h) and one-way ANOVA followed by Bonferroni test (for c, d)

Fig. 8

ADORA2A activation mediates increase in glycolysis via a HIF-1α-dependent pathway. a–d Western blot analysis of HIF-1α mRNA and protein expression in HRMECs transfected with siA_2AR or siCtrl under hypoxia (0.5% O₂) or normoxia (21% O₂) a, b, or infected with Ad-Ctrl or Ad-A_2AR under normoxia c, d. n = 4. ***P < 0.001. e, f HIF-1α immunofluorescent staining of OIR retinas from Adora2a ^WT and Adora2a ^VEC-KO mice at postnatal day (P)15. Representative green (HIF-1α), red (CD31), blue (nuclei, DAPI), and merged images captured with confocal fluorescent microscopy. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer. Scale bar, 50 μm. The fluorescence intensity of HIF-1α staining was calculated by Image J software then normalized to that of WT control (Adora2a ^WT). n = 6 mice for each group. **P < 0.01. g Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs transfected with siHIF-1α or siCtrl under normoxia (21% O₂) or hypoxia (0.5% O₂). n = 3. *P < 0.05; **P < 0.01; ***P < 0.001 vs. siCtrl normoxia group; ^& P < 0.05; ^&& P < 0.01 vs. siCtrl hypoxia group. h Real-Time PCR analysis of the mRNA levels of glycolytic genes in HRMECs. Cells were transfected with HIF-1α siRNA or siCtrl for 24 h under normoxia, and further infected with Ad-A_2AR or Ad-Ctrl for an additional 24 h, followed by adenosine treatment for another 12 h. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ad-A_2AR + Ado + siCtrl group. i Quantification of glycolytic function in HRMECs transfected with siCtrl or siHIF-1α in the presence of CoCl₂ (200 μM). n = 8 per group. *P < 0.05; ***P < 0.001. j Quantification of glycolytic function in HRMECs infected with Ad-Ctrl or Ad-A_2AR, with or without adenosine treatment, in the presence or absence of the HIF-1α inhibitor CAY10585. n = 7, 7, 6, respectively. *P < 0.05; ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by unpaired Student’s t-test (for d, f, g, h) and one-way ANOVA followed by Bonferroni test (for b, i, j)

Fig. 9

ADORA2A regulates HIF-1α protein level through activation of the PI3K/Akt and MEK/ERK-mediated translational machinery. a Western blot analysis of HIF-1α at protein level in HRMECs. Cells were transfected with siA_2AR or the siCtrl for 48 h, and then treated with MG-132 (10 µM) for 1, 2 and 4 h. b Western blot analysis of HIF-1α at protein level in HRMECs. Ad-Ctrl and Ad-A_2AR-infected HRMECs were first treated with CoCl₂ (200 μM) for 8 h to increase HIF-1α protein level and then further treated with CHx (50 µM) for the indicated time periods. c, d The relative protein levels of HIF-1α were quantified by comparing the intensities of protein bands at the indicated times to that at time 0. n = 3, ***P < 0.001. Data are represented as means ± s.e.m. Statistical significance was determined by two-way ANOVA followed by Bonferroni test. e Western blot analysis of the levels of total-ERK (t-ERK), phospho-ERK1/2 (p-ERK1/2), phospho-p38 (p-p38), total-p38 (t-p38), phospho-JNK1/2 (p-JNK1/2), total-JNK1/2 (t-JNK1/2), phospho-p70^S6K (p-p70^S6K), total-p70^S6K (t-p70^S6K), phospho-eIF-4E (p-eIF-4E), and total-eIF-4E (t-eIF-4E) in HRMECs transfected with siA_2AR or siCtrl, with or without adenosine treatment under hypoxia. n = 3. f Western blot analysis of HIF-1α and phosphoproteins in HRMECs infected with Ad-A_2AR, with or without adenosine treatment. n = 3. g Western blot analysis of HIF-1α and phosphoproteins in HRMECs infected with Ad-Ctrl and Ad-A_2AR in the presence of U0126 (10 μM) or LY294002 (10 μM). n = 4

Fig. 10

Schematic diagram illustrating the molecular mechanisms underlying the angiogenic effect of adenosine-ADORA2A-mediated signaling cascade