Delineating the Skeletal Muscle-Microvessel Regeneration Program after Ischemic Injury
Abstract
Understanding the cellular processes involved in skeletal muscle regeneration after damage is critical to advancing translational efforts toward the development of targeted therapeutics. The purpose of this thesis was to ascertain the dynamic interplay between regenerating muscle and regenerating blood vessels after ischemic injury.
First, using a novel systematic investigation of a widely used preclinical mouse model of limb ischemia, I generated a detailed atlas of muscle injury zones and angiogenesis zones in C57BL/6 mice subjected to femoral artery excision. This uncovered previously unrecognized regional variability and an exclusive relationship between angiogenesis and actively regenerating muscle zones. Then, an unbiased, systematic review of 509 manuscripts, criteria-selected from 5147 non-duplicate publications revealed that only 15% of all published studies in the field evaluated data using methods concordant with my findings. These data provide a quality assurance approach that could strengthen the value of this preclinical animal model.
Next, I tested if microvascular mural cells were a source of myogenic progenitor cells upon skeletal muscle ischemia. Using fluorescent lineage tracing techniques, I discovered that a small portion of cells marked by Myosin heavy chain-11 (Myh11) expression possessed the ability to generate new myofibers. The Myh11 lineage cell-derived myofibers were structurally indistinguishable from satellite cell-derived myofibers. Endogenous regulation of this reprogramming process was impaired by Sirtuin 6 (Sirt6) knockout, which provides considerations for future regenerative therapies.
Finally, I further examined if Sirt6 in mural cells influenced the overall regenerative response to hindlimb ischemia. Loss of Sirt6 in mural cells resulted in a necro-fibrotic phenotype that was associated with interstitial and perivascular scarring upon ischemic injury as well as medial fibrosis in larger arteries. I also discovered that the blunted muscle recovery was associated with abnormal regeneration of the microvascular network. Arteriole wrapping by smooth muscle cells was significantly reduced. The capillary density was also reduced along with loss of junctional pericytes and capillary bridges. As well, there was upregulation of p16INK4a, a marker of cell senescence. Together, these findings reveal a role for Sirt6 in mural cell homeostasis during muscle regeneration.
In summary, this thesis provides new insights into the relationships between the microvasculature and functional skeletal muscle regeneration after a severe ischemic insult.