Figure 5. Hif1β ablation decreases vascular permeability and Vegf expression.
A) Left panels: CD31 immunohistochemistry on whole mounts of subcutaneous fat from control and FH1βKO male mice at 8 weeks of age. The photograph was taken at 100x. Right panels: CD31 immunohistochemistry on whole mounts of subcutaneous fat from control and FH1βKO male mice after 12 weeks of exposure to high fat diet.
B) Quantitation of capillary density in the perigonadal and subcutaneous fat pads after 12 weeks on HFD, starting at 6 weeks of age (n = 4/group). Slides were stained for GSL I - isolectin B4. Capillaries were counted from three digital images (20×) from non-overlapping fields were taken from each slide (total 12 fields per group) from control and FH1βKO males. Data are shown as mean ± SEM of three samples. Asterisks indicate p<0.05.
C) Quantitation of the amount of Evan’s blue in fat pads 10 minutes after intra-cardiac injection and perfusion into control and FH1βKO male mice at 8 weeks of age. Data are normalized by gram of lipid from fat pads and are shown as mean ± SEM of three samples. Asterisks indicate a significant difference in all panels (p<0.05).
D) Levels of Vegf mRNA were compared using qPCR between adipocytes isolated from the subcutaneous fat (SCF) and perigonadal fat (PGF). Mice were 6-8 weeks old. Data are shown as mean ± SEM of five samples. Asterisks indicate p<0.05. Western blot of Vegf from isolated PGF adipocytes of female control and FH1βKO− mice at 12 weeks of age. Western blot for actin is a loading control. Data are representative of four samples.
E) mRNA was isolated from shHif1β-1, shHif1β-2, and shGFP stably transfected adipocytes after eight days of differentiation, and subjected to normoxia, hypoxia (0.1% O2 for 16hrs), or 200 nM CoCl2 for 16 hrs. Expression of Vegf was measured by qPCR. Data shown as mean ± SEM of triplicate samples and repeated three times.
F) Western blot of Vegf from protein extracts from 3T3-L1 adipocytes stably transfected with shHif1β-1, shHif1β-2, and shGFP adipocytes, or media secreted by shHif1β-1, shHif1β-2, and shGFP adipocytes after eight days of differentiation. Actin was used as a loading control.