α2β1 integrin expression and endothelial cell proliferation in vivo and in vitro. (A) Immunofluorescence analysis demonstrated colocalization of α2β1 integrin expression (red) and CD31 (green) on the vessels within the tumors, but not quiescent vessels in the skin of wild-type mice. Nuclei are stained with DAPI (blue; 20×/0.75 NA objective). (B) Immunofluorescence analysis of proliferation (anti-Ki67 [red]), endothelial cells (anti-CD31 [green]) and nuclei (DAPI [blue]). Ki67-positive, α2-null tumor endothelial cell nuclei or Ki67-negative, wild-type tumor endothelial cell nuclei are indicated by arrows; 20×/0.75 NA objective. (C) The percentage of the Ki67 and CD31 double-positive cells in nonnecrotic tumor tissue of wild-type or α2-null mice was quantitated by counting the number of CD31⁺ cells that were Ki67 positive or negative in 10 high-power fields. The data are presented as mean plus or minus SEM (*P < .001). Nine mice of each genotype were included in the analysis. (D) Proliferation of primary pulmonary microvascular endothelial cells from wild-type and α2-null animals on matrices of either type I collagen, type IV collagen, fibronectin (10 μg/mL of each), or tissue-culture plastic was determined. Bars and errors indicate the mean plus or minus SEM (3 separate experiments performed in quadruplicate; *P < .01 for each pair). (E) Expression of the α1β1 integrin on tumor endothelial cells was not significantly up-regulated on tumor endothelial cells from tumors in the α2-null mice. Quantitative RT-PCR measurement of α1 integrin subunit mRNA by primary tumor endothelial cells isolated by flow cytometric sorting from tumors implanted into wild-type and α2-null mice harboring tumors for 3 weeks is shown. Data indicate mean plus or minus SEM of 2 pairs of animals (P = .14).