Abstract Body: Background: Fatty acids are broadly classified as long, medium and short chain based on the number of carbons. Among the long-chain fatty acids, Palmitate has been shown to be associated with cardiovascular disease burden. Our current study aims to determine whether Palmitate is a critical determinant of PAD severity. In contrast to Palmitate, we studied the role of short chain fatty acids (SCFAs) in regulating ischemic revascularization in PAD
Methods: Hind limb ischemia was used as a preclinical PAD model. Hypoxia serum starvation was used as an in vitro PAD model.
Results: Palmitate showed a dramatic inhibitory effect on ischemic-EC survival and angiogenic capacity in vitro, whereas SCFAs significantly induced their angiogenic capacity. Based on the LC-MSMS analysis that showed a decrease in the SCFA content in ischemic muscle, we treated ischemic muscle with SCFAs and examined perfusion recovery. Laser Speckle perfusion imaging showed that SCFAs significantly induced perfusion recovery, whereas Palmitate showed a modest but significant impairment in perfusion recovery. Western blot analysis showed that SCFAs preferentially induce Free fatty acid receptor (FFAR)-3, but not FFAR2. Accordingly, silencing FFAR3 decreased ischemic-EC angiogenic capacity whereas inhibiting FFAR2 induced ischemic angiogenesis. To determine whether the enhanced perfusion recovery achieved by SCFA is dependent on FFAR3, beta-hydroxy butyrate (BHB), the only known inhibitor of FFAR3 was delivered into ischemic muscle followed by SCFA treatment. Laser speckle showed that SCFA treatment further decreased perfusion recovery in BHB treated ischemic muscle. Mechanistically, inhibiting FFAR3 induced FFAR2 levels that inhibited ischemic-EC angiogenic capacity by decreasing NO levels and inducing ROS levels in SCFA treated ischemic-ECs. P<0.05 considered significant in all experiments.
Conclusions: Our data shows that Palmitate alone is not sufficient to drive the PAD severity observed in preclinical Diabetic-PAD models. Increased FFAR3 levels in ischemic muscle allows SCFAs to activate AKT-NO axis to induce ischemic angiogenesis and perfusion recovery. However, loss of FFAR3 in ischemic muscle promotes FFAR2 activation that blocks SCFA induced NO production and redox balance thereby inhibiting perfusion recovery experimental-PAD models.