College - Author 1

College of Science and Mathematics

Department - Author 1

Biological Sciences Department

Degree Name - Author 1

BS in Biological Sciences

Date

3-2026

Primary Advisor

Trevor Cardinal, College of Engineering, Biomedical Engineering Department

Abstract/Summary

Peripheral Artery Disease (PAD) is characterized by the buildup of atherosclerotic plaque in the lower extremities that occludes blood flow, and affects over 200 million people worldwide. Without effective treatment, PAD can lead to claudication, ischemia, ulcers, and gangrene. PAD causes more amputations in the United States than any other condition, and for severe cases that require revascularization, procedures benefit fewer than 50% of patients. This demonstrates a clear need for alternative therapies. Potential therapies for PAD target expansion of collateral blood vessels around the area of occlusion – a process called arteriogenesis to compensate for loss of blood flow to extremities. Researchers have tested multiple cell therapy candidates for increasing arteriogenesis, but none has improved patient outcomes in large, randomized clinical trials. An alternative cell therapy candidate for enhancing arteriogenesis is the skeletal muscle progenitor cell, or myoblast. Myogenic cells such as myoblasts are essential in muscle repair and represent a promising therapeutic candidate due to their proximity to collateral vessels, as well as their ability to communicate with macrophages and secrete proangiogenic factors. Sharing a niche with these collateral vessels, myoblasts may have more effective signaling ability to other local cell types that are crucial for arteriogenesis. Previous experiments demonstrated that myoblasts stimulate arteriogenesis in vivo in mice with diet-induced obesity (DIO). However, the cellular mechanisms underlying this enhancement remain unclear. Macrophages are the main orchestrators of arteriogenesis, initially recruited to the collateral as the classically activated M1 polarization, responsible for secreting proinflammatory cytokines and regulating immune cell infiltration. There is then a ‘phenotypic switch’ to the alternatively activated M2 polarization, responsible for collateral expansion through paracrine signaling and matrix degradation. Interestingly, macrophages have an analogous role after muscle damage, and during muscle regeneration. In vivo studies in muscle regeneration suggest that myoblasts recruit and polarize macrophages to the site of injury, suggesting they may be able to have a similar role in collateral arteriogenesis. Indeed, when cocultured with mouse myoblasts, M0 (naïve) and M1 (proinflammatory) macrophages repolarize toward an M2 (proregenerative)-like phenotype. To further this line of investigation, and to produce more translational data, the next logical step is to determine if human myoblasts have a similar effect. In this work, we cultured, plated, and polarized macrophages (RAW 264.7) into M0, M1 (LPS-treated), or M2 (IL-4- treated) phenotypes prior to coculture. Primary human myoblasts were expanded separately and their conditioned media was collected. A transwell coculture system was used to expose macrophages to myoblast-derived signals without direct cell contact, with monoculture controls maintained in parallel. Macrophage polarization was assessed over 72 hours using phase-contrast imaging and quantified by cellular axial ratio. Overall, coculture with myoblasts increased the axial ratio of macrophages compared to monoculture in M0, M1, and M2 polarizations at Day 2, indicating greater M2 polarization over time compared to monoculture. Future work will focus on increasing the translational relevance of this model by implementing an all-human coculture system using primary human macrophages and myoblasts. In addition, we will also investigate fractionation of myoblast-conditioned media to identify the specific soluble factors responsible for driving macrophage polarization.

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