Journal: Purinergic Signalling

Article Title: Blockage of the adenosine A 2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension

doi: 10.1007/s11302-023-09952-z

Figure Lengend Snippet: Cardiac fibroblasts (CFs) isolated from MCT-treated rats overexpress the adenosine A 2B receptor subtype. Shown are representative immunofluorescence confocal microscopy images of RV myocardium sections ( A ) and isolated CFs from the RV and LV myocardium ( B ) of CTRL and MCT-treated rats stained against A 2B AR (green) and vimentin (red). Nuclei are stained in blue with DAPI. Micrographs are representative of at least five different individuals and were obtained with a laser-scanning confocal microscope using the same acquisition settings. Scale bar: 30 μm. In C , shown are representative immunoblots to document the relative amounts of the A 2B AR in CFs isolated from the RV myocardium of CTRL and MCT-treated rats allowed to grow in culture for 28 days; gels were loaded with 150 μg protein amounts. Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype. Please note that the naturally occurring A 2B AR isotype is highly enriched in CFs isolated from the RV myocardium of MCT-treated rats compared to their CTRL littermates. Both bands fully disappeared after pre-adsorption of the A 2B AR primary antibody with a 10-fold molar excess of the A 2B AR blocking peptide (#BLP-AR003, Alomone Inc., Jerusalem, Israel) corresponding to amino acid residues 147–166 of the human A 2B AR second extracellular loop (negative control). The rat urinary bladder (RB) was used as a positive control for the A 2B AR. β-Tubulin (55 kDa) was used as a reference protein. Each bar represents pooled data from three different individuals; three replicas were performed in each experiment. The vertical bars represent SEM. * p < 0.05 (Student’s unpaired t -test) represents significant differences compared to the CTRL group

Article Snippet: Two protein species were recognized by the A 2B AR antibody (#AAR-003, Alomone Inc., Jerusalem, Israel) corresponding to a peptide near the predicted molecular weight of the A 2B AR (~37 kDa) and a higher molecular mass (~45 kDa) isotype.

Techniques: Isolation, Immunofluorescence, Confocal Microscopy, Staining, Microscopy, Western Blot, Molecular Weight, Adsorption, Blocking Assay, Negative Control, Positive Control

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 1. Signaling schematic depicting our hypothesis of how adenosine regulates human coronary artery smooth muscle cell cell-cycle progression. Extracellular mitogens activate classical signal transduction pathways that ultimately phosphorylate (and thus activate) ERK1/2 and Akt. ERK1/2 is well known to increase expression of cyclin D (G1 phase cyclin). Phosphorylated Akt activates a signal transduction pathway that stabilizes S-phase kinase–associated protein-2 (Skp2), which is the F-box protein of SCFSkp2 ubiquitin ligase that polyubiquitinates p27Kip1 and thus accelerates the degradation of p27Kip1. Removal of p27Kip1 deinhibits cyclin D activity. Cyclin D activates cyclin-dependent kinases 4 and 6 to hyper-phosphorylate retinoblastoma protein (Rb), thus releasing the transcription factor elongation 2 factor (E2F) and allowing increased expression of G1/S and S phase cyclins. G1/S and S phase cyclins then drive the cell cycle forward to complete mitogenesis and cytokinesis (cell proliferation). Adenosine stimulates A2B receptors that are positively coupled to adenylyl cyclase, which increases the formation cAMP and activates protein kinase A. Protein kinase A decreases expression of Skp2 and inhibits phosphorylation of Akt, resulting in increased levels of p27Kip1 which reduce cyclin D activity. Protein kinase A also inhibits phosphorylation of ERK1/2, which reduces cyclin D expression. This protein kinase A (PKA)–induced signaling converges at cyclin D. This results in inhibition of Rb hyperphosphorylation, and hypophosphorylated Rb can now bind E2F and prevent this transcription factor from increasing the expression of G1/S and S phase cyclins.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Transduction, Expressing, Ubiquitin Proteomics, Activity Assay, Phospho-proteomics, Inhibition

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 2. Adenosine A2B receptor mediated inhibition of human coronary artery smooth muscle cell growth. A, Representative northern blots (upper) and western blots (lower) showing the presence of A1 and A2B receptors, with low expression of A2A receptors and minimal expression of A3 receptors. B, Concentration–response relationships for the inhibition of 3H-thymidine incorporation (DNA synthesis) by 2-chloroadenosine (Cl-Ad), 5′-N-methylcarboxamidoadenosine (MECA), 1-deoxy-1- [6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d- ribofuranuronamide (IB-MECA), 5′-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA), and CGS21680 (CGS). C, The effects of Cl-Ad (1 μmol/L) on DNA synthesis in the presence and absence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; selective A1 antagonist, 100 nmol/L); SCH442416 (SCH; selective A2A antagonist, 100 nmol/L); VUF5574 (VUF; selective A3 antagonist, 100 nmol/L); and MRS1754 (MRS; selective A2B antagonist, 100 nmol/L). D, The inhibitory effects of Cl-Ad (100 nmol/L) on cell-cycle distribution. *P<0.05 vs no treatment; §P<0.05 significant reversal of Cl-Ad effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates or quadruplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Inhibition, Northern Blot, Western Blot, Expressing, Concentration Assay, DNA Synthesis

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 3. A, Bar graphs show the effects of 2-chloroadenosine (Cl-Ad; 1 μmol/L) and 5′-N-methylcarboxamidoadenosine (MECA; 1 μmol/L) on cell number in human coronary artery smooth muscle cells (HCASMCs). The inhibitory effects of Cl-Ad were reversed by MRS1754 (MRS; A2B receptor antagonist), but not by SCH442416 (SCH; A2A receptor antagonist), 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX; A1 antagonist), or VUF5574 (VUF; A3 antagonist). Similar to Cl-Ad, the effects of MECA were blocked by MRS1754. *P<0.05 vs control; §significant reversal of the inhibitory effects. B, Bar graph demonstrates the effects of Cl-Ad (1 μmol/L) and MECA (1 μmol/L) on cell migration in HCASMCs. The inhibitory effects of Cl-Ad were mimicked by MECA, but not by N6-cyclopentyladenosine (CPA; A1 agonist), CGS21680 (CGS; A2A agonist), or 1-deoxy-1-[6-[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA; IB-M: A3 adenosine receptor agonist). Moreover, the effects of Cl-Ad and MECA were reversed by MRS1754 (MRS; A2B receptor antagonist). *P<0.05 vs control; §significant reversal of the inhibitory effects. C, Effects of erythro-9-(2-hydroxy-3- nonyl)adenine (EHNA; 5 μmol/L; adenosine deaminase inhibitor) and 5-iodotubercidin (IDO; 0.1 μmol/L; adenosine kinase inhibitor) on cell number in HCASMCs. The inhibitory effects were significantly enhanced when the adenosine catabolism inhibitors EHNA+IDO were combined. Moreover, the effects of EHNA+IDO were reversed by MRS1754 (MRS; A2B receptor antagonist), but not by SCH442416 (SCH; A2A receptor antagonist), DPCPX (A1 antagonist), or VUF5574 (VUF; A3 antagonist), suggesting that endogenous adenosine inhibits HCASMC growth via A2B receptors. *P<0.05 vs control; §significant reversal of the inhibitory effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates or quadruplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Control, Migration

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 5. A (Top), Western blot depicting the downregulation of the expression of A2B receptors in human coronary artery smooth muscle cells (HCASMCs) by siRNA against A2B receptors. No treatment with siRNA (Con); treated with negative-control siRNA (si-Con); treated with siRNA against A2B receptor (siRNA). Bar graph for the western blot represents change in optical density ratio of A2B to β-actin. (Bottom), Depicts the effects of siRNA against A2B receptors on the stimulatory effects of 2-chloroadenosine (Cl-Ad; 1 μmol/L) on cAMP levels in HCASMCs. No treatment with siRNA (Control); treated with negative-control siRNA (siControl); treated with siRNA against A2B receptor (siRNA). *P<0.05 vs no Cl-Ad. B, Inhibitory effects of Cl-Ad, 5′-N-methylcarboxamidoadenosine (MECA), 5′-N- ethylcarboxamidoadenosine (NECA), N6-cyclopentyladenosine (CPA), 8-bromo-cAMP (cAMP), and erythro-9-(2-hydroxy-3- nonyl)adenine (EHNA; 10 μmol/L) plus 5-iodotubercidin (IDO; 0.1 μmol/L) on DNA synthesis in the absence and presence of A2B receptor siRNA in HCASMCs. No treatment with agonists (Control); treated with negative-control siRNA (si-Control); treated with siRNA against A2B receptor (A2B-siRNA)]. *P<0.05 vs no agonist; §significant reversal of the inhibitory effects. Values represent mean±SEM from 3 separate experiments, each conducted in triplicates.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Western Blot, Expressing, Negative Control, Control, DNA Synthesis

Journal: Hypertension

Article Title: Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D

doi: 10.1161/hypertensionaha.115.05912

Figure Lengend Snippet: Figure 11. A, Concentration (0, 0.01, 0.1, 1 μmol/L)–response relationships for the inhibition of DNA synthesis (3H-thymidine incorporation) by 2-chloroadenosine (Cl-Ad) and adenosine (Ad) in human coronary artery smooth muscle cells (HCASMCs). B, The effects of 100 nmol/L of 2-chloroadenosine (Cl-Ad) and adenosine (Ad) on cell number in the presence and absence of MRS1754 (MRS; selective A2B antagonist, 100 nmol/L) or inhibitory effects of adenosine in presence or absence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 1 μmol/L) plus 5-iodotubercidin (IDO; 0.1 μmol/L) and with or without MRS1754 (100nmol/L). C, The effects of adenosine on p27Kip1 and S-phase kinase–associated protein-2 (Skp2) expression in HCASMCs treated with or without adenosine (Ad; 100 nmol/L) for 48 hours. *P<0.05 vs no treatment; §P<0.05 significant reversal of inhibitory effects; #P<0.05 significant increase of inhibitory effects. Values represent mean±SEM from 3 separate experiments using separate HCASMCs, each conducted in triplicate. The optical density (OD) ratio in the bar graphs represents Skp2 or p27Kip1 to β-actin ratio.

Article Snippet: Primary Antibody (Source) Dilution of Primary Antibody (Time and Temperature of Incubation) Anti-Rb hypo/hyperphosphorylated (BD Biosciences) 1:1000 (overnight at 4°C) Anti-cyclin D1 (Upstate Biotechnology) 1:1000 (1 hour at room temperature; RT) Anti-β actin (Sigma) 1:10000 (40 min at RT) Anti-ERK1/2 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-ERK1/2 phosphorylated (Calbiochem) 1:1000 (1 hour at RT) Anti-Akt (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-Akt phosphorylated (Cell Signaling Technology) 1:1000 (1 hour at RT) Anti-cyclin A1 (Upstate Biotechnology) 1:1000 (1 hour at RT) Anti-Skp2 (Cell Signalling) 1:1000 (2 hours at RT) Anti-p27 (Pharmigen) 1:250 (1 hour at RT) Anti-Adenosine Receptor A1 (Santa Cruz) 0.5-5 ug/ml (1 hour at RT) Anti A2A Adenosine receptor (Chemicon) 1:200 (1 hour at RT) 3 Anti A2B Adenosine receptor (Santa Cruz) 1:200 (1 hour at RT) Anti A3 Adenosine receptor (Santa Cruz) 1:200 (overnight at 4°C) Table S2: Effects of administration of 2-chloroadenosine (20 μmol/L in 25% pluronic acid) peri-arterially for 7 days on myointimal proliferation after balloon injury of carotid arteries of intact male Wistar-Kyoto rats.

Techniques: Concentration Assay, Inhibition, DNA Synthesis, Expressing