Regulation of Arteriogenesis by Mechanosensitive MicroRNAs and DNA Methylation: A Potential Epigenetic Approach for Treating Peripheral Arterial Disease

Peripheral arterial disease (PAD) is the leading cause of lower limb amputation and is estimated to affect over 202 million people worldwide. PAD arises when atherosclerotic plaques block arteries in the lower limbs, thereby limiting blood flow to the distal tissue. The lumenal expansion of pre-existing collateral arteries bypassing the occlusion(s) (i.e. arteriogenesis) remains a promising therapeutic option. However, large clinical trials have had limited success to date, highlighting the critical need to better understand the basic mechanisms regulating arteriogenesis.

In this thesis, we report that collateral artery segments in the mouse hindlimb exhibit either “moderate” or “amplified” arteriogenesis, depending on the initiating hemodynamics to which they are exposed (i.e. non-reversed or reversed flow waveforms, respectively) following femoral arterial ligation (FAL). We first determined this reversed flow-mediated amplification of collateral artery growth to be dependent on ICAM-1 mediated macrophage recruitment. Moreover, we were able to apply flow waveforms biomimetic of those quantified in-vivo, to endothelial cells (ECs) in-vitro, and perform genome-wide analyses to comprehensively map EC mechanosensitive signaling to sustained, differential arteriogenesis responses.

We next sought to uncover molecular regulators of arteriogenic capacity and collateral artery maturation. We determined that ECs exposed to a non-reversed flow waveform exhibit increased DNMT1 expression and DNA hypermethylation. Moreover, we determined that DNMT1-dependent EC DNA hypermethylation regulates arteriogenic capacity via adjustments to shear stress set-point in-vivo, identifying a novel role for DNA methylation in arteriogenesis.

Finally, we interrogated our genome-wide analysis of EC mechanosensitive signaling mapped to differential arteriogenesis responses to identify potentially novel microRNA regulators of arteriogenesis. Using this unique approach, we discovered microRNA-199a to be a potent mechanosensitive regulator of perfusion recovery and arteriogenesis after arterial occlusion. To this end, miR-199a inhibition elicited complete foot perfusion recovery, markedly augmented collateral arteriogenesis, and improved gastrocnemius muscle tissue composition following FAL.

Overall, these studies demonstrate the critical roles of EC mechano-signaling, flow mediated inflammation, and epigenetic mechanisms in regulating endogenous shear stress-mediated arteriogenesis. These results may also have important bearing on the development of therapeutic arteriogenesis strategies in patients with peripheral arterial disease.