Figure 2.

Expression of adenosine receptors in female and male pulmonary endothelial cells (mPEC) isolated from WT and A_2AKO mice. Pulmonary endothelial cells were isolated using collagenase-mediated tissue digestion and immune-selection with CD31-coated Dynabeads. (A) Representative blots of endothelial cell markers. VEGF receptor 2 (KDR, ~230 kDa), CD34 (~80 kDa) and β-actin (~43 kDa) were identified in cell extraction of immunoselected (CD31⁺, Waltham, MA, USA) cells derived from wild type (WT) and A_2A-deficient mice (A_2AKO). Cells that were immunoselected are identified with a plus sign (+). (B) Semiquantitative densitometry of KDR/β-actin and CD34/β-actin ratio. (C) Confirmation of gender and genetic background of mPEC isolated from females (single band in the PCR for Jarid gene) or male mice (double band). A_2AKO cells were identified by positive amplification of neomycin cassette (NEO). mlp37 gene was used as housekeeping. DNA Ladder 100 bp. (D) In vitro angiogenesis assay at 4 h of incubation with bovine serum (1%). (E) Quantification of tube length of angiogenesis in vitro. (F) QPCR analysis of mRNA levels of A₁, A_2B, and A₃ adenosine receptors in male and female WT and A_2AKO mice. See Table S1 for details about primers and PCR amplicons. In (B) * p < 0.05 versus CD31^- cells in WT mice. † p < 0.05 versus CD31^- cells in A_2AKO. In (F). * p < 0.05 versus respective value in female WT mice. † p < 0.05 versus respective value in male WT mice. Values were expressed as mean ± SEM. n = 3–5 per group. All experiments were performed in duplicate.