Jinli Wang

Mesenchymal stem cell (MSC)-derived exosomes mediate tissue regeneration in a variety of diseases including ischemic heart injury, liver fibrosis, and cerebrovascular disease. Despite an increasing number of studies reporting the therapeutic effects of MSC exosomes, the underlying molecular mechanisms and their miRNA complement are poorly characterized. Here we microRNA (miRNA)-profiled MSC exosomes and conducted a network analysis to identify the dominant biological processes and pathways modulated by exosomal miRNAs. At a system level, miRNA-targeted genes were enriched for (cardio)vascular and angiogenesis processes in line with observed cardiovascular regenerative effects. Targeted pathways were related to Wnt signaling, pro-fibrotic signaling via TGF-β and PDGF, proliferation, and apoptosis. When tested, MSC exosomes reduced collagen production by cardiac fibroblasts, protected cardiomyocytes from apoptosis, and increased angiogenesis in HUVECs. The intrinsic beneficial effects were further improved by virus-free enrichment of MSC exosomes with network-informed regenerative miRNAs capable of promoting angiogenesis and cardiomyocyte proliferation. The data presented here help define the miRNA landscape of MSC exosomes, establish their biological functions through network analyses at a system level, and provide a platform for modulating the overall phenotypic effects of exosomes.

A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs.

The regulation of AMPK in the ischemic heart remains incompletely understood. Recent evidence implicates the role of Sestrin2 in the AMPK signaling pathway, and it is hypothesized that Sestrin2 plays an influential role during myocardial ischemia to promote AMPK activation. Sestrin2 protein was found to be expressed in adult cardiomyocytes and accumulated in the heart during ischemic conditions. Sestrin2 knockout (KO) mice were used to determine the importance of Sestrin2 during ischemia and reperfusion (I/R) injury. When wild-type (WT) and Sestrin2 KO mice were subjected to in vivo I/R, myocardial infarct size was significantly greater in Sestrin2 KO compared with WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated postischemic contractile function in Sestrin2 KO hearts compared with WT. Ischemic AMPK activation was found to be impaired in the Sestrin2 KO hearts. Immunoprecipitation of Sestrin2 demonstrated an association with AMPK. Moreover, liver kinase B1 (LKB1), a major AMPK upstream kinase, was associated with the Sestrin2-AMPK complex in a time-dependent manner during ischemia, whereas this interaction was nearly abolished in Sestrin2 KO hearts. Thus, Sestrin2 plays an important role in cardioprotection against I/R injury, serving as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults.