Non-coding RNAs and chronic limb threatening ischemia - Peripheral artery disease (PAD), affecting over 200 million people globally, often advances to chronic limb- threatening ischemia (CLTI), characterized by chronic ischemic rest pain with high risk for amputation and heightened mortality. CLTI is the leading cause of amputations in adults, but there are no medical therapies available that generate a long-term clinical benefit. Recent clinical findings suggest that in CLTI, vascular growth factor overexpression acting on cells in ischemic skeletal muscle result in the formation of abnormally enlarged capillaries with decreased blood transit time and capillary detachment from skeletal muscle fibers. We hypothesize that future therapeutics in CLTI must address microvascular abnormalities to form stable, functional capillaries to improve perfusion to the ischemic limb. Non-coding RNAs (both microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)) are an emerging class of regulators of epigenetic modifiers, RNA, or protein-coding genes that has garnered attention for impacting diverse biological processes relevant to ischemic injury and for their therapeutic potential. However, the identity and roles of specific ncRNAs involved in CLTI are not well defined. Our published and preliminary studies reveal how ncRNAs can control stage-specific aspects of angiogenic growth, migration, sprouting, and permeability in CLTI. Furthermore, we have elucidated ncRNA functions in vivo and translated these findings into novel therapeutic approaches, including the demonstration that local delivery of ncRNAs potently regulate the angiogenic response in experimental limb ischemia in mice without toxicity. Looking forward, we hypothesize the existence of driver ncRNAs and their targets in early and advanced phases of disease and seek to uncover their expression, function, mechanism, and interactomes. Using a robust ncRNA platform established in the lab, we will bring together cross-disciplinary expertise to address focused questions that explore new molecular and cellular targets and abnormal endothelial cell (EC)-cell interactions that underlie defects in microvascular perfusion and function in CLTI. For this program, promising new leads have been discovered related to targets involved in: (1) EC permeability and angiogenesis; and (2) vascular smooth muscle cell-EC crosstalk as critical drivers in the microvascular response to CLTI. Single cell and spatial transcriptomics combined with other -omics-based technologies in specimens from patients with CLTI will illuminate driver cell types and targets underlying impaired microvascular perfusion. Novel bioengineered delivery platforms (e.g. colloidal hydrogels incorporating ncRNAs) will be utilized for sustained delivery of promising targets. The outstanding qualifications of our multi-disciplinary team in the ncRNA field, molecular imaging, vascular biology, nanomedicine, bioinformatics, and CLTI coupled with a strong training program uniquely position us to establish an unprecedented molecular view of ncRNAs in the development of CLTI that can inform new paradigms of ncRNA-based therapeutics for this and other ischemic disease states.
