Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions

Abstract

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

Extracellular Vesicles as “Very Important Particles” (VIPs) in Aging

“…Additionally, EVs released by senescent vascular smooth muscle cells (VSMCs) induced the secretion of IL-10, IL-17, TNF-α, and IFN-γ by T cells and monocytes, thereby suggesting that these EVs influence the cytokine milieu modulating the immune cell activity [ 84 ]. Similarly, high levels of inflammatory proteins were found in EVs from diabetic individuals [ 85 ]. An in vitro study reported that human mononuclear cells exposed to cigarette smoke become senescent and release EVs enriched in ICAM-1, IL-8, and MCP-1 [ 86 ].…”

Cell non-autonomous regulation of cerebrovascular aging processes by the somatotropic axis

“…Recent data suggest that GH/IGF-1 may also play a role in regulating cellular senescence in the context of atherosclerosis. Senescent cells accumulate in aging, contributing to cerebrovascular pathologies and atherosclerosis ( 322 – 331 ). Senescent cells can cause tissue dysfunction, and IGF-1 has been shown, in vitro , to suppress the formation of oxidative-stress-induced senescent endothelial cells ( 314 ).…”

“…Multiple types of senescent cells have deleterious effects throughout the timeline of atherosclerosis including endothelial cells, VSMCs, and macrophages/foam cells. These senescent cells contribute to disease progress and plaque rupture by promoting degradation of elastic tissue, thinning of the fibrous cap, increased plaque burden, adoption of a proinflammatory macrophage phenotype, and suppression of a protective migratory phenotype by VSMCs ( 329 – 331 ). Consistent with the previously discussed role of IGF-1 in promoting cap thickening and its importance as a regulator of elastic ECM components, it is not surprising that impaired IGF-1 signaling in VSMCs is important in the context of senescence-induced plaque progression.…”

The Potential of Senescence as a Target for Developing Anticancer Therapy

“…sEVs released by senescent vascular smooth muscle cells (VSMCs) induced the secretion of IL-17, interferon γ (INFγ), and IL-10 by T cells, and tumor necrosis factor α (TNFα) by monocytes. sEVs released by senescent VSMCs promoted M1 macrophage polarization favoring the pro-inflammatory phenotype [ 64 ]. Thus, SASP could modulate immune responses.…”

“…High levels of LCN2 could predict the poor prognosis of patients with breast cancer [ 66 ]. The inactivation of LCN2 enhanced the response to chemotherapeutic drugs in mouse models of breast cancer [ 64 ]. This implies the roles of the SASP in cancer cell proliferation and anticancer drug resistance.…”