CD133-2 and KDR⁺ immature ECs in proliferating hemangioma. (A) Pooled RNA (5 μg) from proliferating, involuting, or involuted hemangioma tissues, neonatal foreskins, or healthy infant skins and 15 μg RNA from cultured WERI-RB-1, hemangioma-derived ECs (HemEC) or human dermal microvascular ECs (HDMECs) were analyzed by Northern blotting using a ³²P-labeled CD133 probe. The blot was exposed for 8 hours (upper left) and 10 days (upper right). RNA levels were verified by ethidium bromide–stained rRNA. (B) CD133-1 and CD133-2 were amplified by RT-PCR from hemangioendothelioma (HEOMA), proliferating hemangiomas designated 14, 19, 26, and 31 from patients 4 to 12 months of age, neonatal foreskin, and WERI-RB-1 retinoblastoma cells. Equivalent mRNA level was indicated by 18s rRNA. Frozen sections of proliferating (C) and involuting (D) hemangioma from patients 5 months and 2 years of age, respectively, were stained with an antihuman KDR mAb and developed with AEC chromagen. Cell nuclei were counterstained with hematoxylin. Interstitial KDR⁺ cells and examples of flat ECs are indicated by arrowheads and arrow, respectively. Scale bar is 20 μm.