Figure 1.

ADK expression and adenosine level in proliferative VSMCs. (A) Quantitative RT–PCR analysis of ADK mRNA levels in HCSMCs treated with vehicle or PDGF-BB (20 ng/mL) for 12 h (n = 6 independent cultures). (B) Representative western blots showing ADK expression in HCSMCs treated with vehicle or PDGF-BB (20 ng/mL) for time periods as indicated. Images are representative from three independent experiments. (C and D) Intracellular and extracellular adenosine levels of VSMCs. HCSMCs were treated with vehicle or PDGF-BB (20 ng/mL) for 24 h (n = 4 independent cultures). (E and F) Western blot detection (E) and densitometric quantification (F) of the indicated proteins normalized to Vinculin in HCSMCs treated with vehicle or PDGF-BB (20 ng/mL) for 48 h. Expression of proteins in vehicle-treated control group was set as 1 (red dashed line). Images are representative from four independent experiments. (G) Representative IF staining of Adk on sections of mouse carotid arteries from mice without or with ligation injury. Arterial neointima is indicated within the white line. (H) The Adk fluorescence relative optical density values in carotid arterial neointimal or medial layer from mice with ligation injury (n = 4 mice per group). (I) Quantification of adenosine levels in carotid arteries from mice without or with ligation injury (n = 4 mice per group). (J and K) Intracellular and extracellular adenosine levels in control and ADK KD HCSMCs (n = 3 independent cultures). For all bar graphs, data are expressed as the means ± SEM, *P < 0.05 and **P < 0.01 (unpaired, two-tailed Student’s t-test).