Fig. 1.
EGKO mice displayed defective reperfusion and collateralization in response to limb ischemia. Genotypic analysis of EGKO mice were conducted using PCR (A) and immunoblot (B). (A) Genomic DNA from EGKO and control mice were subjected to PCR with the use of primers for genotyping. The larger fragment (flox, 630 bp) indicates Gab1flox/flox allele, and the smaller fragment (Δ, 150 bp) indicates WT allele. Left: Representative PCR results with DNA from organs and purified ECs of EGKO mice. Right: Representative PCR results with DNA from livers, purified hepatocytes (Hep), and liver sinusoid ECs (LSEC) of EGKO and control mice. (B) ECs isolated from EGKO and control mice were lysed and the expression of Gab1∼3 were analyzed using specific antibodies with GAPDH as a loading control. (C–E) Phenotypic analysis of EGKO and control mice at 4 wk after femoral artery ligation of left hindlimbs. (C) Left: The ischemic hindlimb of EGKO mice displayed necrosis (arrow), whereas that of control mice did not. Right: Clinical scores as an index of severity of limb ischemia, based on published standard (26): Control, n = 14; EGKO, n = 10. **P < 0.01 compared with control. (D) Left: Serial laser Doppler analysis of blood perfusion in hindlimbs of EGKO and control mice. Note that ischemic hindlimbs of EGKO displayed a poor blood perfusion (arrow). Right: Quantitative analysis of blood flow using percentage of the ischemic limb relative to the control limb. Control, n = 10; EGKO, n = 7. **P < 0.01 compared with control. (E) Angiographic analysis of ischemic (Ligation) and nonischemic (Non-Ligation) hindlimbs in EGKO and control mice. Asterisks indicate sites of femoral artery ligation. Arrows indicate newly established arteries and recovered collateral circulation in ischemic hindlimb of control but not EGKO mice.