Targeting senescent cells to attenuate cardiovascular disease progression

Cardiovascular disease (CVD) is the most common disease to increase as life expectancy increases. Most high-profile pharmacological treatments for age-related CVD have led to inefficacious results, implying that novel approaches to treating these pathologies are needed. Emerging data have demonstrated that senescent cardiovascular cells, which are characterized by irreversible cell cycle arrest and a distinct senescence-associated secretory phenotype, accumulate in aged or diseased cardiovascular systems, suggesting that they may impair cardiovascular function. This review discusses the evidence implicating senescent cells in cardiovascular ageing, the onset and progression of CVD, and the molecular mechanisms underlying cardiovascular cell senescence. We also review eradication of senescent cardiovascular cells by small-molecule-drug– mediated apoptosis and immune cell-mediated efferocytosis and toxicity as promising and precisely targeted therapeutics for CVD prevention and treatment.


Introduction
Human life expectancy is significantly increasing due to the better quality of water, food, hygiene, housing, and lifestyle, as well as vaccine usage and improved medical care (Foreman et al., 2018). As projected, the percentage of the global population of age ≥ 65 years will increase from 13% in 2010 to 19% in 2030, whereas those age ≥ 85 years will increase from approximately 0.03% in 2010 to approximately 1.4% in 2030 (Kontis et al., 2017). Advanced age has been well recognized as the leading unmodifiable risk factor for chronic fatal diseases (Niccoli and Partridge, 2012), including cardiovascular disease (CVD) (Shakeri et al., 2018), cancer, and neurodegenerative diseases (Baker and Petersen, 2018). Among these, CVD is the most common disease to increase globally as populations continue to age (Partridge et al., 2018). CVD is the leading cause of death in the elderly (Roth et al., 2017). However, the mechanisms underlying development of age-related CVD are largely unknown. Cellular senescence, a state of permanent cell-cycle arrest despite continued viability and metabolic activity, presents in diseased cardiovascular tissues and is strongly associated with cardiovascular ageing (Shakeri et al., 2018). Senescence is different from ageing, which is characterized by progressive functional decline. Senescence generally happens at the cellular level, whereas ageing occurs on the tissue or organ level. Cell senescence drives tissue ageing (McHugh and Gil, 2018) and is also different from cell quiescence characterized by reversible cell cycle arrest. Cell senescence and quiescence have distinct features and roles in the pathophysiology of CVD. Growing evidence indicates that senescent cardiovascular cells tightly trigger or exacerbate the onset and progression of numerous CVDs, including atherosclerosis , arterial stiffening (Schellinger et al., 2019), aortic aneurysms , (re) stenosis, myocardial fibrosis , and heart failure. Here, we discuss the unique features of senescent cardiovascular cells, molecular mechanisms underlying cardiovascular cell senescence, and emerging roles of senescent vascular cells in CVD initiation and progression. We also summarize whether and how senotherapy targeting elimination of senescent cardiovascular cells by senolytics or the immune system could be used to improve cardiovascular function with normal ageing-, disease-, or cancer therapy-induced damage, ideally resulting in healthy longevity Ovadya and Krizhanovsky, 2018; van Deursen, 2019).

Cellular senescence or quiescence and development of CVD
Senescent cardiovascular cells are especially abundant at sites of diseased or impaired cardiovascular systems, and accumulating evidence from human samples and mouse models demonstrates a causal role for senescent cells in the pathogenesis of age-related CVD, including atherosclerosis , abdominal aortic aneurysm (AAA) , arterial stiffness (Roos et al., 2016), hypertension (Boe et al., 2013), and heart failure (Gude et al., 2018). We will review a body of work that, taken together, strongly suggests that cardiovascular cell senescence may have a significant role in the pathogenesis of CVD.

Cardiovascular cell senescence and quiescence
Cardiovascular cell senescence is defined as irreversible and permanent cell cycle arrest while cells remain metabolically active. Vascular cell senescence can be triggered by various detrimental stimuli, including but not limited to, radiation, oxidative stress, shortened telomeres Minamino et al., 2002), DNA damage, mitochondrial dysfunction, abnormal metabolism, and gene mutation. There are two kinds of vascular cell senescence (Bennett et al., 2016;Chi et al., 2019). The first is replicative senescence, irrevocable cell proliferation arrest after multiple cell divisions, which is generally mediated by telomere shortening (Kuilman et al., 2010). The second is stress-induced premature senescence (SIPS), a stable cell cycle arrest in the absence of any detectable telomere loss or dysfunction, which is usually induced by distinct endogenous or exogenous stresses (Kuilman et al., 2010). Cell senescence is a strategy used generally by mitotic cells to prevent dysregulated cell division. Emerging evidence demonstrates that cell senescence also occurs in post-mitotic cells, including cardiomyocytes and mature adipocytes (Sapieha and Mallette, 2018). In general, DNA damage in telomere regions drives post-mitotic cardiomyocyte senescence (Anderson et al., 2019). p53 induction mediates the senescence of post-mitotic adipocytes (Minamino et al., 2009). Upregulation of pro-senescence factor p21 triggers cell senescence in post-mitotic dopaminergic neurons (Riessland et al., 2019). Cardiovascular cell senescence is vital for the maintenance of cardiovascular tissue homeostasis during embryonic development, tissue regeneration, and wound healing (Demaria et al., 2014). However, persistent accumulation of senescent cells in cardiovascular tissues will impair cardiovascular function and has been implicated in the pathogenesis of age-related CVD. In contrast, cardiovascular cell quiescence with reversible cell cycle arrest usually occurs due to a lack of nutrition or growth factors (Blagosklonny, 2011).
2.1.1. Hallmarks of cardiovascular cell senescence-Senescent cardiovascular cells usually differ greatly from non-senescent cardiovascular cells, including proliferating cells and quiescent cells (Table 1). Senescent cardiovascular cells present several morphological and molecular features ( Table 2) that may serve as suitable markers and therapeutic targets for these cells. Senescent cardiovascular cells generally display a characteristic flattened and enlarged morphology (Coleman et al., 2010;Meijles et al., 2017), increased senescence-associated beta-galactosidase (SA β-gal) activity , telomere attrition, and accumulation of cyclin-dependent kinase inhibitor p16 ink4a or p21 (Morgan et al., 2013). The prominent feature of senescent cardiovascular cells is the senescence-associated secretory phenotype (SASP). Senescent vascular cells secrete a variety of pro-inflammatory cytokines (e.g. IL-6, IL-8), growth factors (e.g. vascular endothelial growth factor [VEGF], platelet-derived growth factor AA [PDGF-AA]) (Demaria et al., 2014), chemokines, and matrix metalloproteinases (MMPs). Senescent vascular cells exhibit a SASP that enables them to communicate with other cells, as well as the microenvironment, and to promote the senescence of neighboring cells, tissue regeneration, and embryonic development (Munoz-Espin et al., 2013). A critical feature of senescent cells is that they are more resistant than non-senescent cells to both extrinsic and intrinsic pro-apoptotic stimuli, which may be due to the transcriptional and cap-independent translational upregulation of pro-survival BH2 family proteins (BCL-W, BCL-X L , and BCL-2) (Yosef et al., 2016). Another surrogate marker of vascular cell senescence is the induction of telomere-associated foci (TAF) of DNA damage (Roos et al., 2016). DNA methylation may function as a biomarker for vascular cell senescence and biological ageing (Field et al., 2018).
Notably, one type of cardiovascular cell may have its unique senescent hallmarks with different kinds of senescence. For example, passaged vascular smooth muscle cells (VSMCs) exhibit p16, but not p21, elevation in replicative senescence, whereas p21, but not p16, is expressed in oxidative SIPS . Endothelial cell (EC) SENEX is upregulated in SIPS, but not in replicative senescence (Coleman et al., 2010). Upregulation of fibroblast senescence marker dipeptidyl peptidase 4 (DPP4, also known as CD26) is much stronger in replicative senescence than in ionizing radiation (IR)-induced premature senescence . Middle-aged wild-type lung ECs show elevation of p53 and p21, but not p16, compared with younger counterparts (Meijles et al., 2017). Cyclin D1 2016; Pantsulaia et al., 2016;Regina et al., 2016). Minamino et al. first demonstrated that senescent ECs with strong SA β-gal activity are present in atherosclerotic lesions of human coronary arteries . Atherosclerotic ECs have shortened telomeres compared with the ECs in the normal vessel wall (Ogami et al., 2004). ECs from the aneurysmal region also present a senescent phenotype with shorter telomeres and more severe oxidative DNA damage (Cafueri et al., 2012). Importantly, in a mouse ageing model, EC senescence contributes to heart failure without systolic dysfunction, specific heart failure with preserved ejection fraction (HFpEF), which occurs in approximately 50% of all patients with heart failure . Also, EC senescence mediates thrombosis (complete vena cava occlusion) via elevation of plasminogen activator inhibitor-1 (PAI-1), an established marker and key mediator of cellular senescence (McDonald et al., 2010). EC premature senescence due to sirtuin deacetylase 1 (Sirt1) inhibition (Ota et al., 2007;Zu et al., 2010) may reversibly lead to vascular ageing and age-related decrease in exercise endurance (Das et al., 2018). Senescence of bone ECs (type H ECs with high expression of CD31 and endomucin) may trigger dysfunctional vascular niches for hematopoietic stem cells (Kusumbe et al., 2016), which may accelerate atherosclerosis development in mice (Fuster et al., 2017).

Senescence of vascular smooth muscle cells and CVD-VSMC
senescence is profoundly associated with and contributes to numerous CVDs, including atherosclerosis (Bennett et al., 2016;Gardner et al., 2015;Grootaert et al., 2018), aortic aneurysm (Cafueri et al., 2012), and fibrotic neointima formation . VSMCs from aged thoracic aortas express higher levels of platelet-derived growth factor receptor-alpha (PDGFR-α) and are resistant to apoptosis induced by serum starvation or nitric oxide . VSMCs derived from human atherosclerotic plaques have a lower level of proliferation compared with cells from the regular arterial media, suggesting that plaque VSMCs are prematurely senescent (Bennett et al., 1998). Human plaque VSMCs are characterized by higher p16 and p21 expression, hypophosphorylation of retinoblastoma (RB), stronger SA β-gal activity, and sizeable flattened cell morphology, when compared with normal VSMCs . Matthews et al. reported that senescent VSMCs are present in the fibrous cap of human advanced carotid atherectomies , and VSMCs within the fibrous cap demonstrate remarkable telomere loss compared with medial VSMCs of the same lesion. Furthermore, telomere shortening of intimal VSMCs is tightly linked to increasing severity of atherosclerosis . Angiotensin II (Ang II) has been reported to accelerate the development of atherosclerosis via induction of premature senescence by the p53/p21-dependent pathway in VSMCs, but not bone marrow cells . VSMC senescence due to Sirt1 inactivation increases atherosclerosis (Gorenne et al., 2013). Also, VSMC senescence contributes to plaque vulnerability, leading to myocardial infarction and stroke . VSMC-specific TRF2 overexpression in apolipoprotein E knockout (ApoE −/− ) mice prevents senescence and consequently improves several features of plaque vulnerability .
Medial VSMCs derived from patient AAAs demonstrate accelerated replicative senescence compared to VSMCs from the corresponding adjacent (non-aneurysmal) inferior mesenteric artery of the same patient . Ang II induces VSMC senescence and resultant AAA formation via Sirt1 reduction . Medial VSMC senescence due to NAD + reduction by inhibition of the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) leads to human thoracic aorta (ascending aorta) aneurysm . VSMC senescence in the aorta also increases vascular stiffness . VSMC senescence induced by nicotine (Suner et al., 2004) may drive nicotine-mediated aortic and arterial stiffness . Replicative senescence of VSMCs instigates age-related medial artery calcification that is not concomitant with lipid or cholesterol deposit via runt-related transcription factor-2 (RUNX-2)-mediated osteoblastic transdifferentiation . Ageing exacerbates neointimal formation by wire injury in carotid arteries in mice . However, it is unknown if age-enhanced neointimal formation is due to VSMC senescence.

2.2.3.
Immune cell senescence in CVD-Immune cell senescence (immunosenescence) plays a pivotal role in CVD initiation and progression (Alpert et al., 2019;Yu et al., 2016). Macrophages are the primary type of immune cells that play critical roles in CVD development. Employing CD11b-driving diphtheria toxin (DT) receptor (DTR) transgenic mice, Stoneman et al. showed that monocyte/macrophage content positively contributes to atherosclerotic plaque development, collagen content, and necrotic core formation. However, monocyte reduction has minor effects on the established plaques (Stoneman et al., 2007). Mouse ageing is associated with the accumulation of senescent macrophages that can be induced in young mice by senescent fibroblasts (Hall et al., 2016). Senescent macrophages accumulate in the sub-endothelial space during early atherogenesis . In advanced atherosclerotic plaques, senescent macrophages promote features of plaque instability, including diminished collagen content, elastic fiber fragmentation, and fibrous cap thinning, in descending aorta and brachiocephalic artery, by elevating MMP3 and MMP13 formation. Interestingly, selective removal of these p16positive senescent cells without interfering with the senescence program by genetic or pharmacological strategies reverses atherosclerosis in mice . It was reported that older persons (over the age of 60 years) with the senescent marker of shorter telomeres in leukocyte DNA have a 3.18-fold higher mortality rate from heart disease (Cawthon et al., 2003), implying that senescent immune cells may lead to heart disease. Accelerated telomere shortening also presents in leukocytes of patients with severe coronary artery disease (Samani et al., 2001) and myocardial infarction . Plasmacytoid dendritic cells (pDCs, uniquely produce type I interferon) and regulatory T cells (Tregs) are concomitantly induced and co-localized in mouse atherosclerotic intima (Yun et al., 2016). Although the accumulation of intimal DCs increases in aged mice with accelerated atherogenesis (Liu et al., 2008), the causal function of senescent DCs and T cells in CVD development remains an unmet challenge. Recently, it was reported that human carotid artery plaques contain immune cells, including CD4 + or CD8 + T cells, natural killer (NK) cells, and macrophages . However, it is totally unknown whether the patient's plaque immune components are senescent and the role of senescent immune components in human atherogenesis.

Senescent myofibroblasts and fibroblasts in CVD-Senescence
of cardiac myofibroblasts is increased in perivascular fibrotic areas after transverse aortic constriction (TAC) compared with the sham-treated heart. Inhibition of premature senescence by genetic deletion of both p53 and p16 leads to enhanced fibrosis and cardiac dysfunction after TAC compared with the wild-type control heart. In contrast, induction of premature senescence by cardiac-specific adeno-associated virus serotype 9 (AAV9) (Suckau et al., 2009) gene transfer-mediated expression of cysteine-rich angiogenic inducer 61 (CYR61) (Jun and Lau, 2010) results in an approximately 50% reduction of perivascular fibrosis and improved cardiac function after TAC . These data imply that premature senescence of myofibroblasts functions as an essential anti-fibrotic mechanism and is a promising therapeutic target for myocardial fibrosis (Condorelli et al., 2016). The role and regulation of senescent fibroblasts and myofibroblasts in the development of CVD, including AAA, cardiac fibrosis, and arterial stiffness, warrant further investigation.

Senescence of vascular stem/progenitor cells and CVD-Ageing is
frequently associated with dysfunction of stem or progenitor cells. Although cellular senescence of progenitor cells (PCs) contributes to multiple diseases (Nicaise et al., 2019), senescence of cardiovascular PCs in CVD progression has been less investigated. Circulating endothelial progenitor cells (EPCs) from human subjects at high risk for cardiovascular events or older subjects have higher percentages of in vitro senescence  or functional impairment (e.g. decreased migration and proliferation) (Heiss et al., 2005), which is correlated with vascular or EC dysfunction, a key trigger of atherogenesis. Depletion of growth differentiation factor 11 (GDF11) or telomerase reverse transcriptase (TERT) causes senescence of young VEGFR2 + /CD133 + EPCs, leading to impaired vascular function and angiogenesis in vitro and in vivo .
However, it is unknown whether EPC senescence contributes to the onset and progression of CVD.
Although the endogenous cardiomyocyte renewal capacity of adult cardiac stem/progenitor cells (CSCs/CPCs) is still a matter of debate (van Berlo et al., 2014;Vicinanza et al., 2018), they exert a beneficial effect on cardiac function in animal models of cardiac ischemic injury (Vagnozzi et al., 2020). Age affects the senescence of human CSCs from older patients Nakamura et al., 2016), and it also enhances mouse CSC senescence (Torella et al., 2004). Indeed, c-kit + cardiac CPCs from aged (24 months) C57BL/6 mice have increased senescent phenotype, decreased stemness, and impaired ability to upregulate paracrine factors for angiogenesis (Castaldi et al., 2017). Overall, CSC senescence mediates cardiac ageing and heart failure (Cianflone et al., 2019;Torella et al., 2004). Interestingly, elimination of senescent CPCs using dasatinib + quercetin (D + Q) senolytics attenuates the SASP and its effect on promoting senescence of healthy nonsenescent CPCs in vitro. Moreover, systemic ablation of senescent cells in aged mice in vivo using senolytics (D + Q) leads to resident CPC activation and enhanced heart regenerative capacity . Ageing induces senescence of cardiac mesenchymal stem cells (MSCs) associated with decreased CD90 expression, resulting in impaired EC differentiation potentials and enhanced SASP (Martini et al., 2019), which may contribute to cardiac disease. Additionally, CVD risk factors, such as type 2 diabetes, depletes circulating pro-vascular PCs characterized by high aldehyde dehydrogenase activity and CD34 + (Terenzi et al., 2019). Importantly, in patients with their first acute myocardial infarction, tight glycemic control reduces senescent myocyte precursor cells, thus increasing the regenerative potential of the ischemic myocardium (Marfella et al., 2012).

Molecular mechanisms of cardiovascular cell senescence
There are multiple mechanisms involved in cardiovascular cell senescence. Here, the review summarizes several key underlying molecular mechanisms.

Progeria and vascular cellular senescence in cardiovascular ageing and diseases
The homeostasis of the cell nucleus is profoundly modified during cellular senescence. Defects of the nuclear lamina have been associated with several different diseases of accelerated ageing, including Hutchinson-Gilford progeria syndrome (HGPS) (Gonzalo et al., 2017;Gordon et al., 2014), mandibuloacral dysplasia (Novelli et al., 2002), and atypical Werner syndrome (Bonne and Levy, 2003). HGPS is an ultra-rare, early-onset, and severe genetic disease of premature ageing caused by a point mutation (C1824 T) in Lmna (G608 G) or Zmpste24 that disrupts nuclear lamin A processing, leading to the formation of mutated (truncated and farnesylated) prelamin A, generally referred to as progerin (50 amino acids deleted from the tail of prelamin A) (Kim et al., 2018a;Lee et al., 2016). Prelamin A elevation is linked to oxidative stress-mediated reduction of the lamin A-processing enzyme Zmpste24/FACE1 ( Fig. 1)  . HGPS patients exhibit severe premature arteriosclerosis characterized by VSMC calcification and attrition, as well as prominent adventitial fibrosis, and die in their early teens (younger than 15 years), mainly due to myocardial infarction or stroke (Olive et al., 2010).
Prelamin A accumulation in multiple cardiovascular cells contributes to their senescence. For example, senescent VSMCs rapidly accumulate prelamin A and present defective nuclear morphology in vitro, both of which are reversible by treatment with farnesylation inhibitors and statins ( Fig. 1)  . In human arteries, prelamin A does not accumulate in young and healthy vessels but is prevalent in medial VSMCs from aged individuals or in atherosclerotic lesions, where it often colocalizes with senescent and degenerative VSMCs. Knockdown of FACE1 recapitulates the prelamin A-induced defects of nuclear morphology in aged VSMCs, whereas prelamin A overexpression promotes VSMC senescence through disrupting mitosis and inducing DNA damage in VSMCs, leading to premature senescence . Selective overexpression of progerin in VSMCs, but not macrophages, leads to VSMC loss and promotes LDL retention in the aorta and the resultant atherogenesis and death in a mouse model of HGPS (Hamczyk et al., 2018). Disruption of the linker of the nucleoskeleton and cytoskeleton (LINC) complex in VSMCs ameliorates progerin-induced VSMC apoptosis and limits the accompanying adventitial fibrosis (Kim et al., 2018a). Furthermore, VSMC-derived progerin accelerates atherogenesis via inducing endoplasmic reticulum (ER) stress in the aorta (Hamczyk et al., 2019). Mice with progerin overexpression in ECs (progerin ecTg ) develop perivascular and cardiac fibrosis, cardiac hypertrophy (Fig. 1), and premature death without VSMC depletion (Osmanagic-Myers et al., 2019). Also, progerin expression is increased in human hearts with dilated cardiomyopathy and is strongly associated with left ventricular remodeling and myocardial ageing (Messner et al., 2018). Left ventricular diastolic dysfunction is the most prevalent echocardiographic abnormality in HGPS patients, and its prevalence increases with age (Prakash et al., 2018). Recently, Beyret and colleagues employed a single-dose systemic administration of AAV9-delivered CRISPR-Cas9 components with lamin A/progerin reduction via facial vein injection to repress HGPS in a mouse model (Beyret et al., 2019). At the same time, another group using intraperitoneal injection of AAV9-mediated CRISPR-Cas9 to ameliorate HGPS in Lmna G609G/G609G mice (Santiago-Fernandez et al., 2019). All the results indicate that prelamin A accumulation in different cardiovascular cells due to impaired lamin A processing is a novel biomarker of cardiovascular ageing and contributes to CVD development ( Fig. 1) and therefore represents a novel therapeutic target to ameliorate the effects of age-induced cardiovascular dysfunction.

Impaired autophagy leads to cardiovascular cell senescence
Autophagy is a "housekeeping" cellular process recognized as a mechanism for cell survival when cells encounter stress, including nutrient deprivation or hypoxia, in which cells degrade their dysfunctional proteins, macromolecules, or sub-organelles in lysosomes and recycle them to produce the required raw materials for biosynthesis or energy generation (Anding and Baehrecke, 2017; Grootaert et al., 2018). In general, autophagy appears to be constitutively active in the cardiovascular system, but its activity decreases with age (Kroemer, 2015;Shirakabe et al., 2016). Importantly, inhibited general autophagy or special autophagy of mitochondria (mitophagy) leads to or accelerates cardiovascular ageing (Abdellatif et al., 2018). Dysfunctional autophagy in ECs, VSMCs, and macrophages, plays a detrimental role in atherogenesis (Fig. 2). Growing evidence implies that decreased autophagy results in cardiovascular cell senescence (Sasaki et al., 2017). For instance, VSMC-specific deficiency of the essential autophagy factor autophagy-related 7 (ATG7) causes accumulation of SQSTM1/p62 and accelerates SIPS. ATG7 deletion in VSMCs of ApoE −/− mice promotes ligation-induced neointima formation and Western diet-induced atherogenesis in mice (Grootaert et al., 2015). Interestingly, moderate activation of autophagy by rapamycin has been shown to repress VSMC replicative senescence (Tan et al., 2016) and stabilize progressed atherosclerotic plaques (Luo et al., 2017). Inhibition of autophagic adaptor p62-mediated selective autophagy stabilizes and increases GATA4 protein, which initiates and maintains the SASP, thus triggering senescence of fibroblasts (Kang et al., 2015).

Mitochondrial dysfunction causes cardiovascular cell senescence
Mitochondrial dysfunction usually drives cellular senescence (Chapman et al., 2019;Wiley et al., 2016), which is characterized by lower NAD + /NADH ratios (Mouchiroud et al., 2013;Watson et al., 2017;Wiley et al., 2016), excluding RAS oncogene-induced fibroblast senescence (Nacarelli et al., 2019). In general, mitochondrial fission reduction-caused inhibition of mitophagy contributes to senescence in multiple cell types by mitochondrial dysfunction (Fig. 3). For example, mouse heart with mitochondrial imbalance between fission (fragmentation) and fusion develops mitochondrial senescence and heart failure due to the impaired mitophagy (Song et al., 2017). Furthermore, increased mitochondrial fission associated with elevation of mitochondrial reactive oxygen species (ROS), but not ER stress, triggers EC senescence and dysfunction, including impaired EC-dependent vasorelaxation and angiogenesis (Kim et al., 2018b). Kim and colleagues recently identified protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein dynamin-related GTPase1 (Drp1) at Cys 644 . Diabetic reduction of PDIA1 induces Drp1 sulfenylation (oxidation) at Cys 644 , promoting Drp1 GTPase activity, which leads to mitochondrial fission contributing to EC senescence (Kim et al., 2018b). On the other hand, ageing also leads to mitochondrial dysfunction. For example, ageing elevates RNA-binding protein Pumilio2 (PUM2) in mouse muscle, which translationally downregulates mitochondrial fission factor (MFF, an outer mitochondrial membrane protein) and thereby inhibits mitochondrial fission and mitophagy, resulting in mitochondrial dysfunction (D'Amico et al., 2019). Interestingly, NAD + replenishment restores defective mitophagy and mitochondrial function in fibroblasts and consequently restrains the accelerated ageing in Caenorhabditis elegans and Drosophila melanogaster models of Werner syndrome (Fang et al., 2019), a human premature ageing disease. It is unknown whether clearance of dysfunctional fragmented mitochondria by guanine derivative-targeted cargo-mediated mitophagy (Takahashi et al., 2019) attenuates cardiovascular cell senescence.
Mitochondrial dysfunction may induce cell senescence through the following mechanisms: 1) instigation of oxidative stress, triggering activation of DNA damage response or telomere damage in cardiomyocytes (Anderson et al., 2019;Chapman et al., 2019); 2) leakage of mitochondrial DNA into the cytoplasm of tubular cells (Chung et al., 2019;Maekawa et al., 2019) or triggering of cytoplasmic chromatin fragmentation in fibroblasts (Vizioli et al., 2020) and consequently driving activation of the cGAS-STING (stimulator of interferon genes) pathway to mediate SASP and senescence; and 3) AMPK-p53 activation-mediated mitochondrial dysfunction-associated senescence with distinct SASP profiles in fibroblasts (Wiley et al., 2016). Mitochondrial DNA polymerase (PolG)-mutated (POLG D257A ) mice showing mitochondrial dysfunction with lower NAD + /NADH ratios in inguinal adipose tissue demonstrate more senescent cells in adipose tissue and skin compared to that of agematched wild-type mice (Wiley et al., 2016). Moreover, overexpression of mitochondriatargeted catalase partially reverses cell senescence in heart and age-related cardiomyopathy in POLG D257A mice in vivo (Dai et al., 2010).

cGAS-STING signaling in cardiovascular cell senescence and disease
Although DNA damage responses have been tightly linked to cardiovascular cell senescence Matthews et al., 2006), the underlying mechanism remains incompletely understood. Damaged or stressed cells usually have increased chromatin fragmentation and cytosolic DNA, which binds and activates cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS) (Ablasser and Chen, 2019). The activation of cGAS, in turn, increases the second messenger molecule 2′3′ cyclic GMP-AMP (cGAMP), which binds and activates the ER protein STING (Motwani et al., 2019), which triggers the production of SASP factors (including IL-6 and TNF-α) and paracrine senescence (Gluck et al., 2017). Numerous stimuli (including oxidative stress) of cellular senescence engage the cGAS-STING pathway in fibroblasts in vitro (Gluck et al., 2017). In pre-senescent hepatic stellate cells and human diploid fibroblasts, transcriptional downregulation of E2F-mediated cytoplasmic DNases (DNase2 and DNA 3' repair exonuclease 1 [TREX1]) results in cytoplasmic accumulation of nuclear DNA, which provokes aberrant activation of cGAS-STING signaling and resultant SASP and cellular senescence . The cGAS-STING pathway mediates irradiation-and NRas V12 oncogene-induced senescence and SASP in mice in vivo (Gluck et al., 2017).
Interestingly, cGAS activity can be post-translationally regulated. Dai et al. reported that aspirin-induced cGAS acetylation at one of three lysine residues (K384, K394, or K414) robustly suppresses cGAS activity and self DNA-induced autoimmunity in a mouse model of Aicardi-Goutières syndrome (AGS) (Dai et al., 2019). Whether senescence stimuli lead to deacetylation of cGAS in the cardiovascular system remains undetermined. It has been reported that cGAS-STING signaling from ischemic cell death results in a fatal response to myocardial infarction (MI). Inhibition of the cGAS-STING-IRF3-type I interferon axis blocks pathological myocardial remodeling, maintains cardiac function, and improves post-MI cardiac repair and survival in mice (Fig. 4) King et al., 2017). These studies suggest a novel molecular mechanism for cellular senescence and suggest that modulation of cGAS activity may be a new strategy to treat senescence-associated cardiovascular disease. Cytosolic DNA from dysfunctional mitochondria and nuclei of senescent cardiovascular cells would activate cGAS-STING signaling. Whether and how cGAS-STING signaling plays causative roles in cardiovascular cell senescence warrants further exploration. It remains to be determined whether the regulation of cGAS or STING is beneficial in CVD prevention and therapy.

Other mechanisms
There are other mechanisms underlying cardiovascular cell senescence. Epigenetic events, including DNA methylation, regulate cell senescence (known as an epigenetic clock) (Cheng et al., 2017;Ermolaeva et al., 2018). For example, hypermethylation of DNA cytosinepreceding-guanosine (CpG) islands in the NAMPT promoter is present within both dilated thoracic aortas and VSMCs, is inversely associated with NAMPT mRNA level, leading to NAD + reduction and consequent VSMC premature senescence . Recently, a high-throughput screen of a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins showed that histone acetyltransferase p300 positively controls replicative senescence of IMR-90 lung fibroblasts via inducing a dynamic hyperacetylated chromatin state .
Noncoding RNAs (ncRNAs) also play crucial roles in cell senescence. Notably, long ncRNAs (lncRNAs; > 200 nt in length) have recently been demonstrated to play critical roles in ageing and age-related diseases (Kour and Rath, 2016;Zhang et al., 2018). Abdelmohsen et al. used RNA sequencing and reported that lncRNA MALAT1 (metastasisassociated lung adenocarcinoma transcript 1) is decreased in senescent fibroblasts (Abdelmohsen et al., 2013). lncRNA MALAT1 may be reduced in senescent ECs as proliferating human ECs have higher levels of lncRNA MALAT1 (Michalik et al., 2014). lncRNA Meg3 (maternally expressed gene 3) is upregulated in senescent human umbilical vein endothelial cells (HUVECs). Meg3 reduction in HUVECs blocks age-induced inhibition of sprouting angiogenesis in vitro. Meg3 silencing restores blood flow impaired in an aged mouse ischemic hind limb in vivo (Boon et al., 2016). Recently, it was reported that oncogene HRas-induced senescent fibroblasts had increased lncRNA-OIS1, which transcriptionally upregulates DPP4 protein . lncRNA-OIS1 may also be elevated in senescent ECs because senescent ECs have higher DPP4 levels . However, the functions and regulation of lncRNAs implicated in cardiovascular senescence are largely unknown.

Clearance of senescent cardiovascular cells alleviates CVD
Compelling data indicate that senescent cardiovascular cells lead to and accelerate CVD onset and development; thus, senescent cells are an emerging target for age-related disease, including CVD . Targeting senescent cardiovascular cells is a potential strategy to prevent or cure CVDs. For example, inhibiting vascular cell senescence by βhydroxybutyrate , which is elevated by fasting and calorie restriction, may be beneficial for prevention of CVD having diverse risk factors (Chakraborty et al., 2018). Rapamycin (Flynn et al., 2013;Singh et al., 2016) or metformin (Barzilai et al., 2016;Yin et al., 2011), acting on the senescent cell property of SASP, also attenuates or reverses CVD development. Interestingly, therapeutic removal of senescent cells is emerging as a promising and innovative strategy to delay cardiovascular ageing or disease progression. Currently, several approaches are being used for the elimination of senescent cardiovascular cells in in vitro and in vivo models.

Induction of apoptosis in senescent cardiovascular cells by small-molecule drugs
Because senescent cells have a pivotal feature, resistance to apoptosis due to elevation of pro-survival molecules, the B cell lymphoma 2 (BCL-2) family proteins (BCL-2, BCL-W, and BCL-X L ) (Singh et al., 2019), the development of novel small-molecule inhibitors of these proteins, known as BH3 mimetics, has been used to selectively induce apoptosis of senescent cells (Yosef et al., 2016), preparing for elimination of apoptotic cells by phagocytosis. Senotherapeutic agents are used to target features of cellular senescence (Table 3). For example, senolytics are used to target anti-apoptotic signaling molecules and induce cell death of senescent vascular cells Zhu et al., 2016). Elegant experiments by Childs and colleagues demonstrated that clearance of senescent cells by ABT-263 (navitoclax) dramatically inhibits atherogenesis onset in the aortic arch of high-fat diet (HFD)-fed Ldlr −/− mice . Treatment of aged (2-year-old) mice with the senolytic drug ABT-263 eliminates senescent cardiomyocytes and consequently reduces fibrosis and cardiomyocyte hypertrophy (Anderson et al., 2019). Importantly, clearance of senescent cells by ABT-263 attenuates myocardial remodeling and improves diastolic function, as well as overall survival in aged mice following myocardial infarction mimicked by ligation of the left anterior descending coronary artery (Walaszczyk et al., 2019). BH3 mimetics ABT-737 and ABT-199 targeting BCL-2 specifically eliminate senescent pancreatic beta cells without effect on the abundance of the immune cell (lymphoid or myeloid) types in a non-obese diabetic mouse model and prevent type 1 diabetes (Thompson et al., 2019).
As senescent cells share common SASP and apoptosis-resistance features with cancer cells, dasatinib (D), which is used in the cancer treatment, may have a role in clearing senescent Song et al. Page 12 Ageing Res Rev. Author manuscript; available in PMC 2021 July 01. cells. Zhu et al. demonstrated that oral gavage administration of single-dose dasatinib + quercetin (D + Q) dramatically decreases senescent cell number and improves cardiac function of 24-month-old mice as shown by improved left ventricular ejection fraction and fractional shortening (Zhu et al., 2015). A single D + Q treatment significantly improves vascular endothelial function and vascular smooth muscle sensitivity to nitroprusside. However, senescent cell elimination does not change smooth muscle contractile function (Zhu et al., 2015). Intermittent treatment with D + Q by oral gavage reduces the number of TAF-positive senescent VSMCs in the aorta media of aged (24-month old) and atherosclerotic ApoE −/− mice (fed a western diet for two months), but not in established intimal atherosclerotic plaques. Treatment with D + Q also improves vasomotor function in aged mice, as well as reduced aortic calcification in ApoE −/− mice. However, D + Q treatment does not affect intimal plaque size (Roos et al., 2016). Additionally, clearance of senescent glial cells from HFD-fed or leptin receptor-deficient obese mice by D + Q restores neurogenesis and alleviates neuropsychiatric disorders, including anxiety and depression (Ogrodnik et al., 2019). D + Q senolytic treatment selectively clears amyloid beta (Aβ)triggered senescent oligodendrocyte progenitor cells (OPCs) characterized by upregulation of p21, p16, and SA β-gal activity, and decreases Aβ plaque load and subsequent cognitive improvement in Alzheimer's disease mice . In clinical trial, D + Q treatment (D, 100 mg/day plus Q, 1250 mg/day, 3 times per week for three weeks) improves physical function of patients with idiopathic pulmonary fibrosis (Justice et al., 2019). Another D + Q phase 2 pilot study (oral D 100 mg and Q 1000 mg for three days) on subjects with diabetic kidney disease decreases adipose tissue senescence and circulating key SASP factors (Hickson et al., 2019). It is noteworthy that dasatinib treatment increases susceptibility to experimental pulmonary hypertension development in rats (Guignabert et al., 2016).
More approaches have been used to induce apoptosis of senescent cells. Compared with healthy cells, senescent cells upregulate transcription factor forkhead box protein O4 (FoxO4), which interacts with p53. FoxO4-DRI peptide, designed to interfere with the interaction of FoxO4 and p53, thus directs p53 from the nucleus to mitochondria for apoptosis induction. Selective downregulation of FoxO4 by inhibitory RNA triggers apoptosis in senescent, but not healthy, cells via release and activation of p53 .
Intriguingly, senolytic drugs seem to exert their effects in a cell type-specific manner. For example, dasatinib is more effective in selectively killing senescent human pre-adipocytes than HUVECs, whereas quercetin (polyphenol, PI3K inhibitor) is more effective in killing senescent HUVECs and mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) than senescent adipocytes (Zhu et al., 2015). ABT-263, targeting the anti-apoptotic BCL-2 family, selectively increases apoptosis and decreases cell viability of senescent but not proliferating HUVECs, while does not affectprimary human preadipocytes . D + Q does not affect the viability of proliferating or quiescent cells. The HSP90 inhibitor Ganetespib exhibits senolytic activity in IR-induced senescent HUVECs, but not in pre-adipocytes (Fuhrmann-Stroissnigg et al., 2017).

Immune clearance of senescent or apoptotic cells
Accumulating data indicate that immune surveillance of senescent cells is mediated by immune cells, such as macrophages, natural killer (NK) cells, neutrophils, and cytotoxic T cells in tumors (Burton and Krizhanovsky, 2014;Kang et al., 2011;Xue et al., 2007) and liver cirrhosis (Krizhanovsky et al., 2008). Different senescent cells generate unique ligands that attract different immune cells. For example, senescence-related hepatic stellate cells elevate cell surface MICA and ULBP2, ligands of activating receptor NKG2D, on NK cells (Krizhanovsky et al., 2008). Senescent cells may express specific surface antigens, such as major histocompatibility complex class II (MHCII) molecules that will be recognized by distinct cells (such as CD4 + T) of the immune system and subsequently killed (Kang et al., 2011). At present, senescence immunotherapy is an emerging research field (Burton and Stolzing, 2018;Hoenicke and Zender, 2012;Krizhanovsky et al., 2008;Sagiv et al., 2013). Senescence immunotherapy strategies are also a promising alternative to senolytics for removing senescent cardiovascular cells in CVD prevention and therapy (Fig. 5).

Macrophages engulf apoptotic or senescent cells-It was reported that macrophages engulf senescent cells in cancer.
Kang and colleagues presented that CD4 + T cells need monocytes or-macrophages, but not NK cells, to clear pre-malignant senescent hepatocytes and subsequently restrain liver cancer development (Kang et al., 2011). Interestingly, p53 restoration induces liver tumor cell senescence with upregulated p16 and SA β-gal activity, but not apoptosis, in mice in vivo. The senescent tumor cells attract innate immune cells, including macrophages, neutrophils, and NK cells, resulting in clearance of senescent tumor cells and resultant tumor regression (Xue et al., 2007). Whether macrophages remove senescent cardiovascular cells in aged or diseased cardiovascular systems remains to be elucidated.
It is well known that macrophages can clear apoptotic cells in a process known as efferocytosis, which prevents apoptotic cells from becoming necrotic or acquiring proinflammatory activity (Henson, 2017;Roberts et al., 2017). Impaired macrophage efferocytosis would enhance atherosclerotic lesion development (Kojima et al., 2017;Proto et al., 2018;Schrijvers et al., 2005) and vulnerable plaque formation (Seneviratne et al., 2017;Thorp et al., 2008;Yurdagul et al., 2017). For example, transcription factor interferon regulatory factor (IRF)-5 enhances fragile plaque formation through maintenance of proinflammatory CD11c + macrophages within atherosclerotic lesions and by stimulating the expansion of the necrotic core by impairing macrophage efferocytosis mediated by downregulated integrin-β3 and its ligand, milk fat globule-epidermal growth factor 8 ( Fig. 6) (Seneviratne et al., 2017) Both the macrophage itself and the features of apoptotic or senescent cells regulate macrophage efferocytosis capability. Tissue-resident macrophages silently eradicating apoptotic cells with limited recognition of nucleic acids within the apoptotic cells are characterized by a lack of Toll-like receptor 9 (TLR9) expression (Roberts et al., 2017). Recently, Yang et al. reported that C-type lectin receptor LSECtin (Clec4g) in colon macrophages is needed for macrophage engulfment and elimination of apoptotic cells . It is noteworthy that Treg cells secrete interleukin-13 (IL-13), thus stimulating IL-10 production in macrophages. The upregulated IL-10 signaling elevates macrophage Vav1 (a guanine nucleotide exchange factor), which activates GTPase Rac1 to promote apoptotic cell engulfment by macrophages (Proto et al., 2018). Continued clearance of multiple apoptotic cells by macrophages requires Drp1-mediated macrophage mitochondrial fission, which is initiated by the first uptake of apoptotic cells . Drp1deficient macrophages show defective efferocytosis and subsequently increased plaque necrosis in western diet-fed Ldlr1 −/− mice . On the other hand, apoptotic cell fate also affects macrophage efferocytosis. For example, apoptotic cells expressing cellsurface protein CD47, a "don't eat me" signal, impair macrophage efferocytosis. Antibodies against CD47 markedly recover efferocytosis without cellular apoptosis alternation, as well as reduce atherosclerosis in both aortic sinus and en face aorta (Kojima et al., 2016).
Moreover, the anti-CD47 antibody ameliorates AAA formation in an ApoE −/− /AngII model and a porcine pancreatic elastase model (Kojima et al., 2018). Cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient apoptotic cells are resistant to efferocytosis leading to accelerated atherogenesis due to the reduction of calreticulin, a principal phagocyte receptor ligand (Gardai et al., 2005). Supplementation with exogeneous calreticulin normalizes the engulfment of CDKN2B-deficient apoptotic cells (Kojima et al., 2014). Thus, it is critical for us to know the molecular mechanisms regulating the phagocytic ability and senescent cell clearance by macrophages in CVD progression and therapy.

NK cells eradicate senescent cells-
The human NK cell line YT selectively targets etoposide-induced senescent and activated hepatic stellate cells, but not proliferating cells, in vitro. Also, YT cells preferentially attack senescent IMR-90 cells, which then undergo apoptosis and detach from the surface of the culture dish (Krizhanovsky et al., 2008). This selectivity is because expression of NKG2D ligands MICA and ULBP2 is selectively upregulated in senescent IMR-90 fibroblasts, but not in growing or quiescent cells (Sagiv et al., 2016). Furthermore, NK cell activation with polyinosinic-polycytidylic acid (Radaeva et al., 2006) decreases senescent cell number in the liver in vivo resulting in the resolution of liver fibrosis (Krizhanovsky et al., 2008). NKG2D receptor deletion enhances the accumulation of senescent stellate cells leading to increased liver fibrosis in mice (Sagiv et al., 2016). Chemotherapeutic agents, including doxorubicin, melphalan, and bortezomib, increase both DNAM-1 (DNAX accessory molecule-1; CD 226) ligand PVR (poliovirus receptor; CD155) and NKG2D ligands (MICA and MICB) on multiple myeloma cells exhibiting a senescent phenotype. These ligands promote NK cell susceptibility . Interestingly, PVR and Nectin-2 are expressed at cell junctions on primary vascular ECs. Moreover, the specific binding of DNAM-1-Fc molecule was detected at endothelial junctions. This binding is almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but is not modified by -mAbs anting Nectin-2, which demonstrates that PVR is the major DNAM-1 ligand on ECs. Both anti-DNAM-1 and anti-PVR mAbs strongly block the transmigration of monocytes through the endothelium (Reymond et al., 2004). Moreover, granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK cell-mediated killing of senescent cells. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent (Sagiv et al., 2013). Unfortunately, the roles of NK cell-mediated depletion of senescent cardiovascular cells in CVD progression remain unknown.
Senescent human diploid fibroblasts selectively and robustly elevate expression of DPP4 on the cell surface, but not in the cytosol, compared with proliferating fibroblasts . Anti-DPP4 antibodies have been used to recognize the specific antigen DPP4 on the cell surface of senescent cells and guide NK cells to selectively destroy the antibody-labeled senescent cells in vitro (Fig. 5). Because senescent HUVECs and HAECs also express higher levels of DPP4 mRNA , whether we can use a DPP4-based mechanism to eradicate senescent cardiovascular cells needs further exploration. Whether senescent cardiovascular cells generate specific surface ligands recognized by NK cell receptors, such as NKG2D and DNAM-1, is another exciting research arena.

Dendritic cells and senescent or apoptotic vascular cells-Dendritic cells (DCs)
, one kind of professional phagocytic cells, can also recognize and remove apoptotic cells (Albert et al., 1998). For example, DCs exclusively traffic mouse apoptotic intestinal epithelial cells (IECs) to mesenteric lymph nodes, which serve as crucial determinants for the induction of tolerogenic regulatory CD4 + T-cell differentiation and activation (Cummings et al., 2016). DC accumulation in aorta intima of aged wild-type mice, but not of young mice, is associated with increased atherosclerosis (Liu et al., 2008). CD11b + DCs with impaired autophagy as a result of ATG16l1 deficiency expand aortic CD4 + Treg cells and inhibit atherosclerosis in Ldlr −/− mice (Clement et al., 2019). Chemokine (C-C motif) receptor 9 (CCR9) + pDCs expressing indoleamine 2,3-dioxygenase 1 (IDO1) in aorta locally induce aortic Treg cells, which produce IL-10 and subsequently prevent atherogenesis (Yun et al., 2016). However, it is largely unknown whether and how DCs eliminate apoptotic or senescent cells in cardiovascular systems.

Chimeric antigen receptor T cells eliminate senescent cardiovascular cells-
Redirecting cytotoxic T cells to recognize the particular antigens on cancer cells using either a modified T-cell receptor or a chimeric antigen receptor (CAR) has been successfully used for certain cancer therapies (June et al., 2018). Fibroblast activation protein (FAP), a cell-surface glycoprotein (Scanlan et al., 1994), is selectively and highly expressed in activated cardiac fibroblasts, but not cardiomyocytes (Aghajanian et al., 2019). High FAP expression contributes to cardiac fibrosis and resultant myocardial disease.
Recently, adoptive transfer of engineered antigen-specific CD8 + T cells specifically targeting FAP dramatically ablated cardiac fibrosis and restored both systolic and diastolic cardiac function in Ang II-and phenylephrine-exposed mice (Aghajanian et al., 2019). Because senescent cells produce specific cell-surface antigens, such as band 3 (Kay, 1993) and an oxidized form of membrane-bound vimentin (Frescas et al., 2017), developing particular CAR T cells to selectively deplete senescent cardiovascular cells is a promising strategy.

Conclusions and perspectives
Homeostasis of senescent cardiovascular cells is required for a healthy cardiovascular system. Multiple complex molecular pathways regulate cardiovascular cell senescence in vitro and in vivo. Emerging evidence suggests that permanent accumulation of senescent  (Gorgoulis et al., 2019). It is still challenging to spatiotemporally identify and quantify individual senescent cardiovascular cells in vivo in a noninvasive manner (Biran et al., 2017). All of these circumstances have prevented the development of effective treatments for CVD. Development of novel therapeutic approaches to target senescent cardiovascular cells and reduce significant clinical consequences such as MI or stroke, will depend on a rigorous understanding of the senescence biology of each of the major cell types that contribute to the pathogenesis of CVD. So far, only D + Q has been assessed in the clinical setting, and none of the current clinical trials is testing whether senolytic agents can prevent cardiovascular disorders. A more in-depth understanding of molecular mechanisms underlying activation of the immune response, as well as special recognition and targeting of a senescent cardiovascular cell, is warranted. Taken together, to target the senescent cardiovascular cells accurately, effectively, and safely, it is essential to do the following research: 1) identify the unique spatiotemporal biomarkers (particularly the cell surface markers) and targets for senescence of different cardiovascular cells in vivo; 2) investigate the mechanism underlying cardiovascular cell senescence and its function in CVD onset and progression; 3) validate the efficiency and potential side effects of known senolytics in animal models and the cardiovascular clinic; 4) explore novel senolytic agents or local delivery methods that can act on specific senescent cardiovascular cells or tissues and optimize the dosage, mode of administration, and combinations for the treatment of various CVDs; and 5) develop a novel strategy for clearance of senescent cardiovascular cells by immunosurveillance. Prelamin A accumulation leads to vascular cell senescence and multiple cardiovascular diseases. ⊥, inhibits. Refer to the text for the expanded form of abbreviations.    Table 3 Major compounds for eliminating senescent cells