Eng+/− HHT-like vascular malformation and perfusion defects in hindlimb after ischemic injury. (A) MR multi-intensity plot reveals the presence of vascular malformations. Eng+/− vascular malformations not seen in wild-type eng+/+ vessels include (arrows): (a) focal dilatations; (b) dilated and thickened vessels; and (c) tortuous vessels. (B) MR surface projection shows collateral vessels formed in response to injury (arrows). (C) A representative axial view of 8-week-old eng+/+ and heterozygous endoglin-targeted (eng+/−) mice (n = 5/group) imaged using MR imaging (RARE8 images). Arrows indicate ischemic muscle with edematous water accumulation; brightness is proportional to degree of residual injury. (D) Percentage of muscle area with edema. *P < .05. n = 4 animals. (E) Anti–VE-cadherin and anti-SDF1 immunofluorescence show colocalization of SDF1 expression in endothelial cells (day 10 after injury; overlay color: yellow, arrows). Smooth muscle cell staining (red, SMA) highlights adjacent endothelial cell location of anti-SDF1 staining (green). SDF1 staining appeared less prominent in eng+/−–injured tissues. EC indicates endothelial cells. Nuclei were stained with TOPRO. (F) Mutant eng+/− and wild-type eng+/+ mice subjected to hindlimb ischemic injury; sections at day 10 were stained using anti-SDF1 antibody (or IgG-matched control) and visualized by horseradish peroxidase–conjugated secondary antibody. LacZ response is the result of the mutated eng allele,52 which is absent in wild-type sections. Strongest SDF1 response is in injured eng+/+ tissues (arrows). (G) SDF1-positive stained cell counts/field for noninjured and injured eng+/+ and eng+/− hindlimb ischemia tissue sections measured as previously described.35 *P < .05. n = 3 fields counted/section.
