Angiogenic potential of skeletal muscle derived extracellular vesicles differs between oxidative and glycolytic muscle. Angiogenic potential of skeletal muscle derived extracellular vesicles differs between oxidative and glycolytic muscle

Skeletal muscle fibers regulate surrounding endothelial cells (EC) via secretion of numerous angiogenic factors, including extracellular vesicles (SkM-EV). Muscle fibers are broadly classified as oxidative (OXI) or glycolytic (GLY) depending on their metabolic characteristics. OXI fibers secrete more pro-angiogenic factors and have greater capillary densities than GLY fibers. OXI muscle secretes more EV than GLY, however it is unknown whether muscle metabolic characteristics regulate EV contents and signaling potential. Extracellular vesicles were isolated from primarily oxidative or glycolytic muscle tissue in mice. MicroRNA (miR) sequencing was done to determine SkM-EV miR contents and cultured endothelial cells were treated with OXI- and GLY-EV to investigate angiogenic signaling potential. There were considerable differences in miR contents between OXI- and GLY-EV and pathway analysis identified that OXI-EV miR were predicted to positively regulate multiple endothelial-specific pathways including nitric oxide (NO) and vascular endothelial growth factor (VEGF) pathways, compared to GLY-EV miRs. OXI-EV improved EC migration (+19%) and tube formation (length: +20%; # of tubes +35%) compared to GLY-EV. These benefits may have been NO pathway mediated, as OXI-EV increased phosphorylation of endothelial nitric oxide synthase (eNOS) and treatment of ECs with the NOS inhibitor L-NAME abolished differences in migration and tube formation between OXI- and GLY-EV. This is the first report to show widespread differences in miR contents between SkM-EV isolated from metabolically different muscle tissue and the first to demonstrate that oxidative muscle tissue secretes EV with greater angiogenic signaling potential than glycolytic muscle tissue. Overall design: 8 total samples were sequenced. Small RNA was isolated from skeletal muscle derived extracellular vesicles oxidative (n=4) and glycolytic (n=4) muscle from mice. Oxidative muscle consisted of the soleus and red portions of the gastrocnemius. Glycolytic muscle consisted of the whole quadriceps.

骨骼肌纤维通过分泌多种血管生成因子（包括骨骼肌来源细胞外囊泡（skeletal muscle derived extracellular vesicles, SkM-EV））调控周围的内皮细胞（endothelial cells, EC）。根据代谢特征，肌纤维可大致分为氧化型（oxidative, OXI）与糖酵解型（glycolytic, GLY）两类。氧化型纤维分泌的促血管生成因子更多，毛细血管密度也高于糖酵解型纤维。不过目前尚不清楚肌肉代谢特征是否会调控细胞外囊泡的内容物与信号活性。
研究从小鼠主要为氧化型或糖酵解型的骨骼肌组织中分离得到细胞外囊泡。通过微小RNA（microRNA, miR）测序分析SkM-EV的miR表达谱，并使用氧化型囊泡（OXI-EV）与糖酵解型囊泡（GLY-EV）处理培养的内皮细胞，以探究其血管生成信号活性。
氧化型与糖酵解型囊泡的miR内容物存在显著差异；通路分析显示，相较于糖酵解型囊泡的miR，氧化型囊泡的miR被预测可正向调控多种内皮特异性通路，包括一氧化氮（nitric oxide, NO）与血管内皮生长因子（vascular endothelial growth factor, VEGF）通路。
相较于糖酵解型囊泡，氧化型囊泡可使内皮细胞迁移能力提升19%、管形成能力增强（管长度增加20%，管数量增加35%）。这类益处可能由一氧化氮通路介导：氧化型囊泡可上调内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）的磷酸化水平，而使用一氧化氮合酶抑制剂L-NAME处理内皮细胞后，氧化型与糖酵解型囊泡间在内皮细胞迁移与管形成能力上的差异便会消失。
本研究首次报道了从不同代谢类型骨骼肌组织中分离的SkM-EV，其miR内容物存在广泛差异；同时首次证明，氧化型骨骼肌组织分泌的细胞外囊泡，其血管生成信号活性高于糖酵解型骨骼肌组织分泌的囊泡。
整体实验设计：共设置8个测序样本。从小鼠氧化型（n=4）与糖酵解型（n=4）骨骼肌来源的细胞外囊泡中提取小RNA。氧化型骨骼肌包括比目鱼肌与腓肠肌的红肌部分；糖酵解型骨骼肌包括整个股四头肌。