Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes, Fibroblasts and Endothelial Cells In Vitro

Abstract

Numerous studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes may contribute to improved cardiac function. However, it has not yet been demonstrated by a genetic approach that exosome release contributes to the therapeutic effect of transplanted CDCs. By employing a lentiviral knockdown (KD) strategy against neutral spingomyelinase 2 (nSMase2), a crucial gene in exosome secretion, we have defined the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblast, endothelial cell and primary cardiomyocyte proliferation, cell death, migration and angiogenesis using a series of in vitro coculture assays. We found that secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 lentiviral KD and shRNAi expression was stable and constitutive. hCDC exosome release contributed to the angiogenic and pro-migratory effects of hCDCs on HUVECs, decreased proliferation of fibroblasts, and decreased apoptosis of cardiomyocytes. These in vitro reactions support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Importantly, we have established a novel tool to test constitutive inhibition of exosome secretion in stem cell populations in animal models of cardiac disease.

Beneficial effects of exosomes secreted by cardiac-derived progenitor cells and other cell types in myocardial ischemia

“…Similar results were reported after intramyocardial injection of human CDC exosomes in immunodeficient mice (30). Pharmacological blockade of exosome secretion by GW4869, which inhibits neutral spingomyelinase 2 [nSMase2; a crucial enzyme in exosome secretion (32)], nullified CDC bioactivity. Exosomes from both CPCs and CDCs were highly enriched with miR-146a, as compared with fibroblast exosomes (5,30).…”

“…Using lentiviral knockdown of nSMase2, Lang et al (32) demonstrated that CDC exosomes contributed to the proangiogenic and promigratory activities of human CDCs in HUVECs while also reducing both apoptosis of cardiomyocytes and proliferation of fibroblasts in vitro. Chen et al (33) reported that mouse CPC exosomes inhibited cardiomyocyte apoptosis in mouse hearts after IR in vivo.…”

The clinical impact of exosomes in cardiovascular disorders: From basic science to clinical application

“…hCDCs have markers including CD90 and CD105 (mesenchymal markers), CD63 and HSP90 (exosomal markers), GATA4 and Nkx2.5 (markers of early cardiac development), and could be internalized by endothelial cells, cardiac fibroblasts, and myocytes. hCDCs exosomes could increase endothelial migration, tube formation, and branch point complexity, while reducing the human cardiac fibroblast proliferation (Lang, Young, Ashraf, & Canty, ). Secreted exosomes by cardiomyocytes in ischemic condition could increase mouse cardiac endothelial cell proliferation, endothelial cells migration, sprouting, and capillary‐like structures.…”

Exosomes as agents of change in the cardiovascular system

“…Conditioned media from CDCs was enriched with RNA, the majority of which was contained within exosomes [88]. Co-culture of human CDCs with HUVECS was found to have proangiogenic effects that were not observed when exosome production was disrupted by nSmase2 knockdown in CDCs [89]. Injection of CDC derived exosomes in an acute MI mouse model mimicked the same benefits seen with injection of CDCs themselves [88]; the exosome treated hearts showed decreased scar mass and increased viable mass, as well as lower levels of pro-inflammatory cytokines, which may be a result of previously reported reduction in inflammatory macrophages by CDCs [90].…”