Requirement for endogenous apelin for recovery of hind limb perfusion after induction of ischemia. (A) Immunohistochemical staining of muscle sections from sham-operated (sham) or ischemic (ischemia) hind limb with anti-CD31 (green) and anti-APJ (red) antibodies. (B-C) Quantitative real-time PCR analysis of APJ (B) or apelin (C) mRNA expression in CD31⁺CD45⁻ ECs or CD31⁻non-ECs derived from muscle of sham-operated (sham) or ischemic (ischemia) hind limb. Levels were normalized against the level of expression of CD31 as an EC marker. Threshold values for target genes normalized against the level of CD31 (dCt) are shown below the graph. (D) Gross appearance of foot pad from sham-operated (sham) or ischemic WT and apelin^−/− mice 14 days after resection of the femoral artery. (E) Representative laser Doppler images of mouse hind limbs from WT or apelin^−/− mice 14 days after surgery. Red represents greater flow; and blue, less flow. (F) Quantification of laser Doppler-monitored blood flow measurements. Data are from ratio of ischemic right leg versus nonischemic left leg in mice (n > 10). *P < .01.