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: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 2. AR mRNA levels in cultured CASMCs and intact coro- nary arteries. RT-PCR for detection of the A1R, A2AR (A) and A3R (B) mRNA expression was performed with cultured CASMCs or CASMCs dispersed from freshly isolated porcine right coronary arteries (RCA) denuded of endothelium. Primary cultured por- cine coronary artery endothelial cells (CAEC, B) were used as controls. RT-PCR performed without reverse transcriptase is indicated as “RT.”

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Reverse Transcription

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 5. Sequence, pharmacology, and signaling of the porcine A1R. Deduced amino acid sequence of the cloned A1R was aligned with the human homolog (A). Putative transmembrane-spanning domains are indicated with solid lines and marked as TM I to TM VII. Sequence matches are shaded. F, Potential phosphorylation sites; Œ, consensus sites for N-linked glycosylation; and *Putative palmi- toylation site (A). Saturation binding of [3H]-DPCPX was performed on membranes from stably transfected COS-1 cells or cultured CASMCs (B). Only specific binding is shown. Each point is the mean of triplicate determinations. Binding affinities of various ligands were determined from the competition binding assays on the membranes of transfected COS-1 cells in the presence of 0.5 nmol/L [3H]-DPCPX (C). Data shown are the meanSEM from 3 experiments performed in triplicate. Inhibition of forskolin-stimulated cAMP production by CCPA in transfected COS-1 cells (f) and in cultured CASMCs (▫) (D). Increase in [Ca2]i by CCPA was detected only in transfected COS-1 cells (E). Data for cAMP assay are the meanSEM from 3 independent experiments performed in duplicate and data points in the Ca2 curve indicate the average peak increase in [Ca2]i for 8 to 16 cells on each of 5 coverslips and represent the meanSEM.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Sequencing, Clone Assay, Phospho-proteomics, Glycoproteomics, Binding Assay, Stable Transfection, Transfection, Cell Culture, Inhibition, cAMP Assay

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 6. Effects of A1R antisense OGN and overexpression on the DNA synthesis of CASMCs. CASMCs were pretreated with vehicle (control), transfection reagent (Trans. Reagent), antisense oligonucleotides to porcine A1R (antisense OGN, 0.3 g), or the sense OGN (0.3 g) (A). Western blotting was performed to confirm the knockdown of endogenous A1R. Molecular weights of A1R and actin are 39 and 43 kDa, respectively. In parallel, pretreated CASMCs were stimulated with adenosine (100 mol/L), NECA (10 mol/L), CCPA (0.1 mol/L), or LPA (100 mol/L) for 48 hours, after which cellular DNA content was determined and the data were transformed to the percentage inhibition of the controls (B). In another experiment, CASMCs were transfected with vehicle (control), transfection reagent (Trans. Reagent), pCR3.1 containing the porcine A1R cDNA (Plasmid-A1R), or mock pCR3.1 (C). Western blotting was performed to confirm A1R overexpression. In parallel, the cellular DNA content was determined after stimulation of posttransfected CASMCs with adenosine (100 mol/L), NECA (10 mol/L), CCPA (0.1 mol/L), or LPA (100 mol/L) for 48 hours (D). Data shown are the meanSEM from four independent experiments performed in triplicate. *P0.01 relative to the respective control.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Over Expression, DNA Synthesis, Control, Transfection, Western Blot, Knockdown, Transformation Assay, Inhibition, Plasmid Preparation

Journal: Circulation Research

Article Title: Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells

doi: 10.1161/01.res.0000165800.81876.52

Figure Lengend Snippet: Figure 8. A1R mediate adenosine- and CCPA-induced DNA syn- thesis in an organ culture model of coronary disease. Artery rings of porcine conduit coronary arteries denuded of endotheli- um were stimulated with the indicated concentrations of adeno- sine, CCPA, PDGF, or the vehicle (control) for 24 hours in organ culture, after which the cellular DNA content was determined (A). Alternatively, artery rings were pretreated with DPCPX (0.1 mol/L, 40 minutes) and stimulated by adenosine (10 mol/L), CCPA (10 nmol/L), or PDGF (10 nmol/L), after which the cellular DNA content was determined (B). Data shown are the meanSEM from 4 independent experiments (4 pigs) performed in duplicate. *P0.05 relative to the respective control.

Article Snippet: Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc).

Techniques: Organ Culture, Control