The effect of thalidomide and CC-5013 on angiogenic and cytokine receptor expression in HUVECs. (A-C) RNase protection analyses of HUVEC mRNA are shown. Total RNA was extracted after 18 hours of exposure to 10^-6 M thalidomide, CC-5013, or control medium. Ten micrograms total RNA was hybridized to either (A) an angiogenesis probe set containing templates for angiopoietin (ANG), CD31 (platelet endothelial cell adhesion molecule-1 [PECAM]), endoglin, VEGF receptor-1, fms-like tyrosine kinase (flt-1), Ang receptors (TIE-1 and TIE-2), VEGF, and VEGF-C; or (B) a receptor probe set containing the templates for interleukin-1 receptor I (IL-1RI), IL-1RII, tumor necrosis factor-α receptor p55 (TNFRp55), TNFRp75, IL-6Rα, gp130, TGFβRI, and TGFβRII genes. (C) Three independent sets of experiments yielded similar data. The graph represents the quantification summary for each probe. Gene signals were normalized to that from L32 (mRNA for a ribosomal protein subunit). White bars represent gene expression in untreated HUVECs, while gray and black bars represent gene expression in HUVECs treated with CC-5013 and thalidomide, respectively. Comparisons between relative PhosphorImager units for control and experimental samples for each gene were made by 2-tailed Student t test. ^*P < .05. (D) Western blot analysis of HUVECs and primary EPC whole-cell lysates. HUVECs and primary EPCs outgrown from PBMCs isolated from 2 untreated patients were cultured to confluence in gelatin- and laminin-coated 6-well plates, respectively. Data from 1 of 2 experiments with similar results are shown. Lysates from control and treated ECs are shown. Following electrophoresis and blotting, the membranes were developed by means of antibodies specific for either rabbit anti-TGFβRI or mouse anti-TR, followed by chemiluminescence and autoradiography. The membranes were stripped and developed similarly with rabbit anti-actin to control for equal loading.