Informed consent was obtained from all patients. Patients were recruited consecutively from those undergoing evaluation by the Department of Vascular Surgery for lower limb bypass surgery over a 12-month period. These patient data were collected in a blinded fashion without any knowledge of the VEGF response. 8 Measurement of the Fold Induction of VEGF mRNA by RNase Protection Assay Total RNA was isolated from the monocytes with TRI reagent and the quantity of VEGF mRNA was determined by RNase protection assay using riboprobes to VEGF and 18S rRNA as previously described. Analysis between groups for statistically significant differences in the levels of VEGF was performed using Student's unpaired t -tests.
All p values are two-tailed.
In the diabetic group, five of the CLI patients had ischemic ulcers and six had rest pain. In the nondiabetic group, none of the patients had ischemic ulcers and three CLI patients had rest pain. We have previously established the reproducibility and consistency of the measurement of the hypoxic induction of VEGF in monocytes by performing the analysis from the same individual on several different occasions. Our results in nondiabetic patients presented here are consistent with this hypothesis. It is not clear from this preliminary study if a greater hypoxic induction of VEGF is causally related to the development of CLI in the diabetic patient.