Peripheral arterial disease (PAD) is a major complication of systemic atherosclerosis which affects approximately 12 million Americans. The lower extremity is the most common site for PAD where current medical treatments target systemic atherosclerosis but are not able to improve perfusion to the ischemic limb and directly treat the problem in PAD. There is a pressing clinical need for therapeutic approaches for PAD. Though only fully recognized as a true biologic entity less than 15 years ago, micro-RNAs (miRs) which are 15 - 23 nucleotide long RNA's, are now appreciated to be key regulators of gene expression;especially in the adaptive response to injury. The best characterized method by which miRs regulate gene expression is by their ability to bind to the 3'un-translated region of a target mRNA and thereby reducing mRNA expression or protein translation. However, a single miR can regulate entire biologic pathways by affecting several functionally related genes thus making miRs attractive therapeutic agents. We found that miR-93 appears to play a critical role in experimental PAD. Following HLI, C57BL/6 (B6) mice have excellent perfusion recovery and little tissue loss. In contrast, in Balb/C mice perfusion recovery is limited and necrosis is common. Utilizing both experimental and computational approaches we identified mir- 93 as the miR that was MOST differentially expressed based on the combination of strain and ischemia. miR- 93 was significantly increased following HLI (p<0.0001 and >4-fold increase) in B6 mice not in Bab/C. Local delivery of a pre-miR93 led to greater perfusion recovery in Balb/C mice and systemic delivery of a miR-93 antagomer impaired perfusion recovery in C57/Bl6 mice. In-vitro, antagomers to miR-93 increased the extent of hypoxia induced apoptosis while treatment with pre-miR-93 reduced the extent of hypoxia-induced apoptosis in both cell types. Antagomers to miR-93 reduced, and administration of pre-miR 93 increased, proliferation. We consistently found that the cycle genes p53, E2F1, and p21 were altered. Collectively, central hypothesis that miR-93, can serve as a potent modulator of post-natal angiogenesis in PAD though its ability to regulate cycle genes alone, or with other potential targets.
Specific Aim 1) : Define the role that miR-93 plays in-vivo in regulating the extent of blood flow recovery post-HLI using gain of function and loss of function approaches by the examination of mice at appropriate pre-selected time points.
Specific Aim 2) : Utilize in-vitro studies to determine the extent to which th gene expression changes induced by miR-93 can be directly linked to miR-93/mRNA interactions and establish the extent to which the gene expression changes induced by miR-93 are cell and condition specific.
Specific Aim 3) : Measure miR-93 in an established bank of human plasma and skeletal human samples from patients with varying severity of PAD as well as a group of non-PAD controls.
Peripheral arterial disease (PAD) is a major health problem caused by atherosclerosis that impairs blood flow to the legs. Currently, there are no medical therapies that have the ability to improve blood flow to the legs of patients suffering from the devastating and debilitating symptoms of PAD. While it has classically been taught that DNA makes RNA and RNA makes protein, a number of other processes alter gene expression. Micro- RNA's are 15 - 23 base (nucleotide) long RNA's that are now appreciated to be key regulators of gene expression;especially in the adaptive response to injury such as occurs in PAD. We have identified a microRNA that plays a critical role in PAD and the goal of this project is to rapidly advance our understanding of miR-93 in both experimental and human PAD.