CCN1 stimulated angiogenesis in vivo. (A-C) Matrigel (0.5 mL) was injected into C57BL/6 mice subcutaneously. Angiogenesis after 6 days was measured by the hemoglobin content of the Matrigel plug and by ingrowing vascular structures confirmed by staining for VWF. Pictures represent H&E sections of Matrigel plugs, upper left insets show the total Matrigel plug and the upper right insets shows sections stained with antibodies against VWF (A) Matrigel in the absence (control) and in the presence of 50 ng/mL CCN1 or 50 ng/mL VEGF. Increase in angiogenesis was significant different from control levels (*P < .05). (B) Matrigel (0.5 mL) in the absence (control) and in the presence of 20 ng/mL CCN1 or 2.5 × 10⁵ CD34⁺ cells. In addition, 2.5 × 10⁵ CD34⁺ cells were injected into Matrigel plugs already containing 20 ng/mL CCN1. Increase in angiogenesis was significant different from CCN1 alone and CD34⁺ cells alone (*P < .05). (C) Matrigel in the absence (control) or in the presence of supernatants (SN) from CD34⁺ cells unstimulated (control) or stimulated with 100 ng/mL CCN1 for 24 hours. Increase in angiogenesis was significant different from control levels and supernatants from unstimulated CD34⁺ cells (*P < .05). Hemoglobin content is given in nanograms per milliliter per plug weight in milligrams. Vascular structures are expressed as percentage vascular area per field. Data represent the mean (± SEM) of at least 3 independent experiments.