Abstract
The senescence of vascular smooth muscle cells (VSMCs) has been implicated as a causal pro-inflammatory mechanism for cardiovascular disease development and progression of atherosclerosis, the instigator of ischemic heart disease. Contemporary limitations related to studying this cellular population and senescence-related therapeutics are caused by a lack of specific markers enabling their detection. Therefore, we aimed to profile a phenotypical and molecular signature of senescent VSMCs to allow reliable identification. To achieve this goal, we have compared non-senescent and senescent VSMCs from two in vitro models of senescence, replicative senescence (RS) and DNA-damage induced senescence (DS), by analyzing the expressions of established senescence markers: cell cycle inhibitors- p16 INK4a, p14 ARF, p21 and p53; pro-inflammatory factors-Interleukin 1β (IL-1β), IL-6 and high mobility group box-1 (HMGB-1); contractile proteins-smooth muscle heavy chain- (MYH11), smoothelin and transgelin (TAGLN), as well as structural features (nuclear morphology and LMNB1 (Lamin B1) expression). The different senescence-inducing modalities resulted in a lack of the proliferative activity. Nucleomegaly was seen in senescent VSMC as compared to freshly isolated VSMC Phenotypically, senescent VSMC appeared with a significantly larger cell size and polygonal, non-spindle-shaped cell morphology. In line with the supposed switch to a pro-inflammatory phenotype known as the senescence associated secretory phenotype (SASP), we found that both RS and DS upregulated IL-1β and released HMGB-1 from the nucleus, while RS also showed IL-6 upregulation. In regard to cell cycle-regulating molecules, we detected modestly increased p16 levels in both RS and DS, but largely inconsistent p21, p14ARF, and p53 expressions in senescent VSMCs. Since these classical markers of senescence showed insufficient deregulation to warrant senescent VSMC detection, we have conducted a non-biased proteomics and in silico analysis of RS VSMC demonstrating altered RNA biology as the central molecular feature of senescence in this cell type. Therefore, key proteins involved with RNA functionality, HMGB-1 release, LMNB-1 downregulation, in junction with nuclear enlargement, can be used as markers of VSMC senescence, enabling the detection of these pathogenic pro-inflammatory cells in future therapeutic studies in ischemic heart disease and atherosclerosis.
Highlights
Inflammation is a hallmark and potent promoter of cardiovascular disease, in addition to other well-defined risk factors that contribute to the multifactorial processes of disease progression (Dzau et al, 2002)
The quantification of these parameters showed that replicatively senescent cells have an increased cell size (Figure 1F) and enlarged nuclei compared to nonsenescent vascular smooth muscle cells (VSMCs) (Figure 1G)
This analysis confirmed that replicative senescence (RS) VSMC nuclei are dominantly large and regular, which is a feature of senescent nuclei, excluding other causes of nuclear enlargement (Figure 1H)
Summary
Inflammation is a hallmark and potent promoter of cardiovascular disease, in addition to other well-defined risk factors that contribute to the multifactorial processes of disease progression (hypercholesterolemia, diabetes, hypertension, and smoking) (Dzau et al, 2002). Elevated inflammatory marker levels, such as interleukin-1β (IL-1β), IL-6, and HMGB-1 are associated with an cardiovascular risk (Ridker, 2016). The CANTOS clinical trial demonstrated that an anti-inflammatory approach through IL-1β blockade improves CV (cardiovascular) outcomes (Ridker et al, 2017). Targeting the source and cause of inflammation, rather than individual cytokines may be a solution for these issues. Cellular senescence has been proposed the key source of inflammation in atherosclerosis and cardiovascular disease (CVD) (Stojanovicet al., 2020). Experimental evidence showed that the SASP includes several pro-inflammatory cytokines (IL-1β, IL-6, HMGB-1), many of which are known cardiovascular risk factors (Ridker, 2014)
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