Conditioned Medium from Dysfunctional Endothelial Cells Impairs Satellite Cell Expansion and Differentiation

Dysfunction of the endothelial lining of the cardiovascular system is a hallmark symptom of both cardiovascular disease (CVD) and type II diabetes mellitus (T2DM). The dysfunctional endothelial cell phenotype is characterized by an increase in production and release of inflammatory cytokines and reactive oxygen species (ROS) and a decrease in nitric oxide (NO) and growth factor secretion. Evidence has emerged showing a close relationship between endothelial cells and skeletal muscle satellite/progenitor cells. Skeletal muscle cell growth and differentiation are impaired in oxidative environments and many endothelial dysfunction-related diseases. The purpose of the current study is to determine the impact that high glucose induced endothelial cell dysfunction has on the growth and differentiation of skeletal muscle satellite cells (SkMSC). Human Umbilical Vein Endothelial Cells (HUVEC) were grown in normoglycemic, 5 mM glucose (CON) media or hyperglycemic, 30 mM glucose (DYS) media for 48 hours to induce dysfunction, after which the media was collected to be used as conditioned media (CM). Primary human SkMSCs were isolated from the vastus lateralis of eight lean subjects and subsequently treated with CON or DYS CM for 2, 4, or 6 days. Satellite cells not collected at day 6 were directed toward differentiation in CON or DYS CM. SkMSC growth, ROS production and differentiation were analyzed via cell counting, MTT assay, DHE assay, qPCR, immune blotting, and immunocytochemistry. After 6 days of treatment, SkMSC counts and the number of viable cells were decreased in DYS CM treatment. SkMSC cyclin D1 was downregulated and caspase-9 was upregulated in the DYS treatment group. Myogenesis and myotube fusion were impaired in DYS CM treated SkMSC. Abnormal inhibition of p38 phosphorylation and a non-significant increase in NF-κB activity occurred in late stage DYS CM treated satellite cells. This study provides evidence that dysfunctional endothelial cells impair skeletal muscle satellite cell expansion through possible dysregulation of apoptotic and proliferative pathways. CM from DYS ECs also clearly inhibited muscle satellite cell differentiation and myotube fusion via abnormal intracellular signaling. Future studies should focus on expanding current knowledge on the relationship between endothelial cells and muscle satellite cells in endothelial dysfunction-related pathologies