Abstract
The growth of endogenous collateral arteries that bypass arterial occlusion(s), or arteriogenesis, is a fundamental shear stress-induced adaptation with implications for treating peripheral arterial disease. MicroRNAs (miRs) are key regulators of gene expression in response to injury and have strong therapeutic potential. In a previous study, we identified miR-146a as a candidate regulator of vascular remodeling. Here, we tested whether miR-146a regulates in vitro angiogenic endothelial cell (EC) behaviors, as well as perfusion recovery, arteriogenesis, and angiogenesis in response to femoral arterial ligation (FAL) in vivo. We found miR-146a inhibition impaired EC tube formation and migration in vitro. Following FAL, Balb/c mice were treated with a single, intramuscular injection of anti-miR-146a or scramble locked nucleic acid (LNA) oligonucleotides directly into the non-ischemic gracilis muscles. Serial laser Doppler imaging demonstrated that anti-miR-146a treated mice exhibited significantly greater perfusion recovery (a 16% increase) compared mice treated with scramble LNA. Moreover, anti-miR-146a treated mice exhibited a 22% increase in collateral artery diameter compared to controls, while there was no significant effect on in vivo angiogenesis or muscle regeneration. Despite exerting no beneficial effects on angiogenesis, the inhibition of mechanosensitive miR-146a enhances perfusion recovery after FAL via enhanced arteriogenesis.
Highlights
Peripheral arterial disease (PAD) is caused by blockage(s) of the arteries, in the lower limbs, due to occlusive atherosclerosis (Norgren et al, 2007)
For Human Umbilical Vein Endothelial Cell (HUVEC) cultured under normal growth condition, culture medium was changed to normal growth medium containing 10% fetal bovine serum (FBS) and cells were incubated for the given experimental time at normoxia conditions (20% O2)
For HUVECs cultured under hypoxia serum starvation (HSS) to simulate ischemia in vitro, culture medium was changed to endothelial serum starvation medium (#209-250, Cell Applications) and cells were incubated in a 2% oxygen chamber (BioSpherix, Lacona, NY, USA) for the given experimental duration
Summary
Peripheral arterial disease (PAD) is caused by blockage(s) of the arteries, in the lower limbs, due to occlusive atherosclerosis (Norgren et al, 2007). There are no medical therapies available to treat PAD and many PAD patients are not amenable to surgical revascularization options or receive little long-term benefit from such surgeries (Annex, 2013) This has led to the rise of new therapeutic strategies that have sought to induce endogenous revascularization via stimulation of new capillary growth from preexisting vessels (i.e., angiogenesis) and/or lumen expansion of preexisting arteries (i.e., arteriogenesis) to restore lower limb perfusion. By applying shear stress waveforms biomimetic of these in vivo hemodynamics to endothelial cells (ECs) in vitro, we identified several mechanosensitive miRs (-100, -199a, and -146a,) as potential negative regulators of arteriogenesis To this end, inhibition of miR-100 has been shown previously to enhance perfusion recovery following FAL (Grundmann et al, 2011), and we have recently determined that miR-199a inhibition is a potent enhancer of perfusion recovery and arteriogenesis following FAL in Balb/c mice (Heuslein and Price, 2017). We tested the hypothesis that miR-146a regulates angiogenesis, arteriogenesis, and perfusion recovery after FAL
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