Senescence-induced inflammation: an important player and key therapeutic target in atherosclerosis

Abstract

Inflammation is a hallmark and potent driver of pathological vascular remodelling in atherosclerosis. However, current anti-inflammatory therapeutic strategies have shown mixed results. As an alternative perspective on the conundrum of chronic inflammation emerging evidence points towards a small subset of senescent cells as a critical player and central node driving atherosclerosis. Senescent cells belonging to various cell types are a dominant and chronic source of a large array of pro-inflammatory cytokines and various additional plaque destabilizing factors, being involved with various aspects of atherosclerosis pathogenesis. Antagonizing these key agitators of local chronic inflammation and plaque instability may provide a causative and multi-purpose therapeutic strategy to treat atherosclerosis. Anti-senescence treatment options with translational potential are currently in development. However, several questions and challenges remain to be addressed before these novel treatment approaches may enter the clinical setting.

Introduction

Inflammation is a critical driver of atherosclerosis and an independent risk factor for myocardial infarction and cardiovascular death.¹ The Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) showed that targeting inflammation can provide significant, clinically relevant cardiovascular benefit.² In another attempt to target inflammation, the Cardiovascular Inflammation Reduction Trial (CIRT)³ yielded negative, but highly educative results. These trials highlighted the potential, but also the limitations of current anti-inflammatory approaches, including immune suppression and the associated risk of sepsis.⁴ Furthermore, targeting individual cytokines of the pro-inflammatory cascade may not fully address the effects of various other pro-inflammatory elements.⁵ Strategies to target upstream effectors in the inflammatory signalling cascade may provide a solution for these issues.⁵

A strong suspect for the central upstream source of inflammatory factors is ageing, being the strongest independent risk factor for cardiovascular disease (CVD).⁶ On the cellular level, ageing is driven by the accumulation of senescent cells.⁷ Cellular senescence is defined as an irreversible cell cycle arrest (mechanism outlined in Figure 1)⁸ and is accompanied by a pro-inflammatory phenotype. The phenotype is referred to as the senescence-associated secretory phenotype (SASP), producing pro-inflammatory cytokines [interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-18, CCL-2, tumour necrosis factor α (TNF-α)], metalloproteinases (MMP-1, -2, -3, -7, -8, -9, -10, -12, -13, and -14), and other factors.^9,10 Emerging experimental evidence indicates that, in contrast to an acute inflammatory response, the SASP is a protracted and chronic source of inflammatory and other plaque-destabilizing factors,^9,10 gradually contributing to atherosclerosis pathogenesis.

Figure 1.

Cell cycle arrest mechanisms in senescence. CDK, cyclin-dependent kinase; hypo-p-Rb, hypo-phosphorylated retinoblastoma protein. G1, S, G2, and M are cell cycle phases; p16, p53, and p21 are cell cycle inhibitors.

Intriguingly, cellular senescence and atherosclerosis share multiple causative stimuli^1,11,12: oxidized and electronegative low-density lipoprotein (LDL),^13,14 inflammation,¹⁵ reactive oxygen species,⁸ cigarette smoke,¹⁶ hypertension,¹⁷ hyperglycaemia,¹⁸ cytomegalovirus (CMV) infection,¹⁹ telomere attrition,²⁰ mitochondrial failure,²¹ dysfunctional autophagy,²² and genomic damage.⁸ Senescent cells are more than simple bystanders, as multiple molecules involved in the SASP act as promoters of atherosclerosis and are biomarkers of CVD risk.²³ Furthermore, effects of experimental overexpression of many molecules drives both atherosclerosis and senescence. On the other hand, antagonizing cellular senescence and ageing processes has anti-atherosclerotic effects. These overlapping features of senescence and atherosclerosis are shown in Table 1. For a better understanding of the cell-specific roles of these molecules refer to Figure 2, which explains expression patterns on a cellular level of senescence and their roles in atherogenesis.

Table 1.

Clinical and experimental expression of molecules in atherosclerosis and senescence

Molecules	Effect on CVD risk	Over-expression effect on atherosclerosis	Over-expression effect on senescence	Down-regulation effect on atherosclerosis	Down-regulation effect on senescence	References
Ang II						24–26
AMPK						27–29
CCL2						30–32
FOXO3						33–35
GATA4						36–38
HMGB1						39–41
HSP90						42–44
IL1α/β						1 , 30 , 45
IL6						1 , 30 , 45
IL18						46–48
MMP9						45 , 48–50
MMP12						51–53
mTOR						27 , 54
NF-κB						55 , 56
NLRP3						57–60
NR3C2						25 , 61
OPG						62–65
PI3K						27 , 54 , 66 , 67
p66Shc						68–71
SIRT1						27 , 72–74
STAT3						75 , 76
TRAF6						77–79
TRF2						80

, promoting effect; , inhibitory effect; , unknown/unclear evidence; Ang II, angiotensin II; AMPK, AMP-activated protein kinase; CVD, cardiovascular risk; FOXO, forkhead box class O; HMGB1, high-mobility group box 1; IL, interleukin; MMP, matrix metalloproteinase; mTOR, mechanistic target of rapamycin; NF-κB, nuclear factor kappa B; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; NR3C-2, mineralocorticoid receptor; PI3K, phosphoinositide 3-kinase; OPG, osteoprotegerin; SIRT, sirtuin; STAT3, signal transducer and activator of transcription 3; TRAF6, tumour necrosis factor receptor-associated factor 6; TRF2, telomeric repeat-binding factor 2.

Figure 2.

Roles of different senescent cell types in atherosclerosis, represented in blue. CRP, C-reactive protein; CVD, cardiovascular disease; EC, endothelial cell; ET-1, endothelin-1; ICAM, intercellular adhesion molecule; IL, interleukin; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NO, nitric oxide; NF-κB, nuclear factor κB; TNF-α, tumour necrosis factor α; VCAM, vascular cell adhesion molecule; VSMC, vascular smooth muscle cell.

These observations suggest that senescent cells are an important source of local, chronic, low-grade inflammation and plaque destabilization, and thus, a promising upstream therapeutic target. Targeting of senescent cells and ageing processes therefore holds unexplored potential to ameliorate atherosclerosis as a causal approach to decrease inflammation and provide plaque stabilization, possibly without overt immunosuppression.

In this review, we present the current experimental knowledge close ties of senescence with many aspects of atherosclerosis pathophysiology. Further on, we present clinical evidence connecting cellular senescence and atherosclerosis. We elaborate on how targeting senescence can be utilized to develop novel therapeutic strategies for atherosclerosis. Finally, we discuss the challenges and unmet needs that remain to be addressed before anti-senescence therapies can be introduced in the clinical setting.

Evidence on the cellular level of the contribution of senescence to atherosclerosis

Cellular senescence evolves with ageing and in sites of disease⁷ in response to sub-lethal exposure to various stimuli,⁸¹ which are outlined in the Introduction section. Senescent cells, within their physiological roles, facilitate tissue repair in response to acute damage,⁸² but are promptly cleared by the innate immune system after injury resolution.⁸³ However, some senescent cells remain and gradually accumulate in tissues.⁸⁴ These persisting senescent cells have been recently tied to multiple chronic diseases other than atherosclerosis, including diabetes mellitus,⁸⁵ obesity,⁸⁵ Alzheimer’s disease,⁸⁶ heart failure,^10,87 idiopathic pulmonary fibrosis,^88,89 chronic obstructive pulmonary disease,⁹⁰ chronic kidney disease,⁹¹ cancer,⁹² and osteoarthritis.⁹³ It is thus not surprising that senescent cell burden is a good predictor of animal lifespan.^94,95

Cellular senescence is observed in early stages of atherosclerosis,⁹ affecting different cell types in the arterial wall that have distinct roles in the pathogenesis of this disease: endothelial cells (ECs),^96–98 vascular smooth muscle cells (VSMCs),^45,99–101 macrophages, foam cells,^9,102 monocytes,¹⁰³ fibroblasts,^104–106 and T cells.^107,108

Originating from these different cell types, senescent cells directly facilitate multiple key pathophysiological processes in atherosclerosis, which are displayed in Figure 2 (necrotic core enlargement, extracellular matrix degeneration and cap thinning, erosion, calcification, intra-plaque angiogenesis).¹⁰⁹Take home figure details the complex mechanisms leading to plaque destabilizing outcomes. Take home figure also exemplifies that some features of cellular senescence are shared by different senescent cell types, while some are cell population specific.

Take home figure.

Senescent cell contributions to the mechanisms of atherosclerotic plaque rupture.

All senescent cells contribute to plaque inflammation. Consistent SASP elements in the majority of senescent cells are IL-1α, IL-1β, IL-6, IL-8, IL-18, and TNF-α,^15,105,110 all of which are clinically validated CVD risk factors.^1,5 These inflammatory cytokines promote senescence locally in a paracrine manner³⁰ and perhaps, at a systemic level.¹¹¹ Other features depend on the cell type.

Endothelial cell senescence directly compromised the endothelial barrier through disruption of cellular proliferation, permeability, and motility,^112–114 possibly contributing to endothelial erosion and intra-plaque haemorrhage. As seen in a retinopathy model, the overall effect of the SASP leads to pathological neo-angiogenesis.¹¹⁵

Vascular smooth muscle cell senescence was associated with necrotic core enlargement⁸⁰ and plaque calcification⁶² in human atherosclerosis. Senescent VSMCs acquire an osteoblastic secretory phenotype and activate several osteogenic pathways [e.g. Runt-related transcription factor 2 (RUNX-2), bone morphogenetic protein 2 (BMP-2), alkaline phosphatase (ALP), osteopontin (OPN), and osteoprotegerin (OPG)], thus contributing to plaque calcification.⁶² OPG, a soluble factor and key element of the SASP,⁶³ is a CVD risk factor.⁶⁴ Plaque destabilization through cap thinning is promoted by various MMPs,⁴⁹ such as MMP-1, -2, -3, -7, -8, -9, -10, -12, -13, and -14,^45,49,51 which are secreted as part of the SASP of senescent VSMCs, monocytes, macrophages, and foam cells.

Furthermore, CD4+, CD28-null, CD45 re-expressing senescent T-cells (also known as CD4+ T-EMRA cells) were found to be highly active subpopulations, expressing increased amounts of TNF-α, INF-γ, CXCR-3 (C-X-C chemokine receptor), CCR-6, CCR-7 (C-C motif chemokine receptor 6 and 7), and the anti-apoptotic Bcl-2 family proteins.^19,116 In addition to the pro-inflammatory phenotype, T-EMRA cells showed atypical cytotoxic activity towards the plaque endothelium,¹¹⁷ likely contributing to plaque erosion.

Together, these findings indicate that different types of senescent cells in the vasculature exhibit features that are considered highly unfavourable in the setting of atherosclerosis.

Clinical evidence linking ageing and senescence processes to atherosclerosis

Ageing is well-documented as a risk factor for CVD¹¹⁸ and age is considered in various cardiovascular risk scoring systems (Framingham, Reynolds, PROCAM, ESC, and Diamond Forrester). In addition, ageing-associated morphological and haemodynamical arterial disturbances are known.^119,120 The majority of individuals older than 70 years of age (57.1% male and 68.7% female) have atherosclerotic lesions.¹²¹ The trend progressively worsens, as 87.4% of male and 88.9% of female patients older than 85 were affected by subclinical or clinical forms of vascular disease.¹²² However, ageing has a strong residual effect that cannot be simply explained by longer exposure to classical risk factors. Only 11.9% and 40.3% of the effect of age can be attributed to longer exposure to risk factors in men and women, respectively.¹²³

Cellular senescence may account for a part of this void, acting a hub between various cardiovascular risk factors. As depicted in Figure 3, cellular senescence can be a consequence of two processes which may run in parallel and are intertwined. The first is replicative exhaustion resulting from chronological age or intense proliferation (telomere dependent)^20,124,125 and the latter is exposure to cardiovascular risk factors (stress-induced).^13–18 Therefore, cellular senescence burden is a phenomenon only partly correlated with age from birth; it is rather a consequence of a combined effect of chronological ageing and risk factor exposure. Being a shared consequence of the effect of all of these various factors, senescence is an important upstream effector that promotes atherogenesis.

Figure 3.

Putative model of the role of cellular senescence in inflammation-associated cardiovascular disease risk.

In clinical practice, the capability of cellular senescence to drive atherosclerosis independently of chronological ageing and classical risk factor exposure is seen in drastic examples of human progeria syndromes, such as Hutchinson–Gilford progeria (HGS), Werner syndrome, and other laminopathies. Patients with these syndromes are characterized by a strong accumulation of senescent cells early in life and suffer from strikingly increased CVD risk due to a large atherosclerotic plaque burden.^101,126,127 Patients with HGS suffer from myocardial infarction or stroke in the absence of classical risk factors at the mean age of 13 years.¹²⁸

Furthermore, cancer therapies that induce cellular senescence, such as cisplatin, doxorubicin and radiation,¹²⁹ accelerate atherosclerosis.¹³⁰

Additional clinical evidence of the involvement of cellular senescence in the atherosclerotic vessel wall in the general population comes from post-mortem histological analysis,^96,131–134 that showed that senescent EC and VSMC accumulate substantially more in atherosclerotic than in physiologically aged healthy arteries.^96,131–134 Expression of senescence marker p16 INK4a in the diseased human coronary arteries positively coincided with unstable plaques and correlated with intra-plaque TNFα levels.¹³⁵ Furthermore, coronary vessels from ischaemic heart disease patients showed significant endothelial senescent cell burden, while the mostly plaque-free internal mammary arteries from the same donors had no evidence of senescence.⁹⁶ In human carotid artery atherosclerosis, senescent VSMCs were associated with phenotypical features of plaque instability^{80,134,136,137} and accounted for 18% of all plaque cells.¹³⁴ Senescent T-EMRA cells were also found inside unstable plaques.¹¹⁷ Additional studies with detailed quantification of senescent cells, plaque features, and correlation to clinical data are necessary to strengthen the causal link between arterial wall cellular senescence and atherosclerosis.

As specific and safe imaging methods to evaluate senescent cell burden in the arteries of patients still do not exist, tracking senescent cell populations in the blood appears to be the best option to evaluate the contribution of cellular senescence to CVD risk.

Senescence of blood–borne immune cells has been tied to CVD risk. Bulk analysis of leucocyte populations showed that short telomeres predict atherosclerosis development and CVD.^124,125 The ageing pro-oxidative marker p66Shc⁶⁸ is overexpressed in leucocytes from patients with acute coronary syndromes, but not stable coronary artery disease.⁶⁹ As these results from bulk leucocytes may be affected by telomere length and expression profiles of short-lived myeloid cells, the stable lymphoid populations attracted significant attention, specifically senescent T-EMRA cells. Accumulation of T-EMRA was associated with increased acute mortality and re-occurrence of MI, particularly among diabetic patients.¹⁹ Increased numbers of these cells were found in hypertension and rheumatoid arthritis, predicting worsened CV outcomes within these high-risk conditions.¹⁹ Both CD4+ and CD8+ T-EMRA cells are strong independent predictors of cardiovascular mortality in the elderly.¹⁰⁷ Cytomegalovirus infection activated senescence mostly in CD4+ EMRA cells,^19,107 providing an interesting connection between CMV infection, CD4+ EMRA senescence, and atherosclerosis.

In line herewith, the role of age-dependent accumulation of pro-inflammatory lymphocytes with somatic mutations (clonal haematopoiesis of indeterminate potential), mainly in TET2 and DNMT3a, is emerging.¹³⁸ Senescence is associated wide perturbations in DNA methylation.¹³⁹ Given its critical functions in this process, it is possible that DMNT3a plays a role in the immuno-senescence programme. Silencing of DMNT3a reportedly activated senescence in cancer and skin experimental models.^140,141 However, it is unclear whether these mutations are involved in haematopoietic cellular senescence or SASP activation.

In summary, chronic senescent cells accumulate over time as a result of repeated tissue damage. Through the SASP, cellular senescence exerts many pro-atherogenic effects and it possibly is a key aetiologic driver of aberrant vascular remodelling, forming a perpetual loop that chronically amplifies the effects of risk factor exposure (Figure 3). Therefore, senescence appears to be a therapeutic target worth exploring for the prevention or treatment of atherosclerosis.

Treatment options for targeting senescence

Multiple clinically approved treatment strategies for CVD have an anti-senescent effect upon closer analysis. Aldosterone and Angiotensin II drove VSMC senescence independently and synergistically, this being prevented by mineralocorticoid receptor and angiotensin II receptor blockers.^24,25 Pioglitazone ameliorated endothelial cell senescence by telomerase activation.¹⁴² Statins were shown to delay endothelial and T-cell senescence, through p38-mediated SASP inhibition, telomerase and cell cycle regulation.¹⁴³ Metformin has anti-senescent and anti-atherosclerotic effects through SIRT-1 agonism.²⁷ Rivaroxaban attenuates VSMC senescence, by inhibiting the signalling cascade between Factor Xa and Insulin-like growth factor-binding protein 5.¹³⁷ The cardiovascular benefits of exercise in adulthood can be explained by a decrease of immuno-senescence.¹⁴⁴

In a direct senescence-specific approach, several inflammatory and energy-sensing pathways and complex epigenetic modulators provide valid entry points to prevent, stabilize, or reverse atherosclerosis. Given the status quo of the clinical application of genome-based therapies in the CVD field, pharmacological approaches are discussed in detail below because of their immediate translational potential. Figure 4 provides an overview of the known molecular therapeutic targets in senescence and divides them into six most promising therapeutic strategies: direct anti-inflammatory, anti-SASP, energy sensing, epigenetic modulation, senolytics, and reprogramming. These approaches and the complex relationships between various molecular targets are explained in further detail below.

Figure 4.

Anti-senescence strategies. Akt, protein kinase B; AMPK, AMP-activated protein kinase; cGAS, cyclic GMP-AMP synthase; DPP4, dipeptidyl peptidase 4; FOXO, Forkhead box class O; HMGB-1, high-mobility group box 1; IL, interleukin; MAPK, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; NF-κB, nuclear factor kappa B; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; ox-vimentin, oxidized vimentin; PI3K, phosphoinositide 3-kinase; SASP, senescence-associated secretory phenotype; SIRT, sirtuin; STING, stimulator of interferon genes; TBK, serine/threonine-protein kinase TBK1; TNFα, tumour necrosis factor α.

Targeting intracellular signalling molecules at the crossroads of inflammation and senescence

The idea of antagonizing pro-inflammatory molecules upstream of the IL-1-IL-6/TNFα-CRP cascade or other immune regulators¹⁴⁵ is viable, not only for a direct anti-inflammatory effect but also to ameliorate senescence. Inhibition of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome (NLRP3) suppressed T-cell senescence⁵⁷ and atherosclerosis.⁵⁸ Even more upstream, inhibition of nuclear factor κB (NF-κB) abrogated p53-mediated senescence.⁵⁵ However, while inhibiting NF-κB in ECs was beneficial in atherosclerosis, in macrophages, this had a pro-atherogenic effect.⁵⁶ Immunosuppression and the inhibition of cellular debris traffic are strong reasons not to target these and other major inflammatory pathways.

A related concept is the inhibition of the SASP. Recent data suggest DNA leakage from the senescent nucleus into the cytoplasm and consequent activation of the GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) anti-viral pathway as a mechanism of SASP onset.^146,147 This pathway is an attractive target to antagonize the SASP, but may come at the cost of lowering anti-viral defences. Alternatively, it is possible to modulate individual components of the SASP by inhibiting Janus kinase (JAK) 1/2¹⁴⁸ and JAK2/signal transducer and activator of transcription 3 (STAT3).¹⁴⁹ TNF receptor-associated factor 6 (TRAF6) inhibition in macrophages is another anti-inflammatory strategy to counter atherosclerosis development.⁷⁷ The ATM-TRAF6-TAK1 axis is a driver of the SASP,⁷⁸ and an Serine-protein kinase ATM inhibitor was found to have an anti-senescence effect.⁷⁹ p38 Mitogen-activated protein kinase (MAPK) inhibition has been suggested as an anti-SASP strategy.¹⁵⁰ Independently of the senescence context, blockade of this pathway failed to improve cardiovascular outcomes 12 h to 2 weeks post-myocardial infarction.¹⁵¹ However, anti-SASP strategies may not be effective in acute or subacute disease (see Future directions section).

Energy-sensing pathways, like those regulated by mammalian target of rapamycin (mTOR), sirtuin (SIRT)-1, Forkhead box class O 3 (FOXO3), and AMP-activated protein kinase (AMPK), have been shown to be largely responsible for the anti-ageing benefits of caloric restriction.²⁷ Inhibition of mTOR complex 1 by rapamycin prolonged the healthy lifespan of mice and exerted anti-senescence and anti-atherosclerotic effects.^152,153 However, mTOR inhibition can cause muscular atrophy.¹⁵⁴ The SIRT-1 agonist resveratrol ameliorated ageing pathology, in part by activating FOXO3.^33,155 Clinical application of resveratrol and related SIRT agonists is challenging due to stability and bioavailability issues.¹⁵⁶ Direct FOXO3 agonism is pharmacologically possible.³³ Importantly, more than 12.5-fold overexpression of FOXO3 has been shown to be harmful, possibly because of nicotinamide adenine dinucleotide (NAD) depletion¹⁵⁵; thus, a careful dose-finding study is imperative. Recently, fine-tuning of FOXO3 levels has been achieved via a miRNA-132 antagonistic approach,¹⁵⁷ that is currently clinically tested (NCT04045405). NADP boosters (nicotinamide mononucleotide,¹⁵⁸ nicotinamide riboside,¹⁵⁹ P7C3,¹⁶⁰ apigenin¹⁶¹) can be used resolve the NAD depletion issue and are able to prevent senescence in ECs¹⁵⁸ and VSMCs.¹⁵⁹ The small scale Metformin in Longevity Study (MILES, NCT02432287) trial studied AMPK activation for anti-ageing purposes and the results are currently analysed. A larger scale study with a similar design is planned [Targeting Aging with Metformin (TAME) trial].

Epigenetic manipulation through long non-coding RNAs, miRNA modulators, and other oligonucleotides has yielded promising outcomes in experimental settings.^162–165 Some of these molecules are currently clinically evaluated in CVD.¹⁶⁶ Epigenetic modification through histone modification via bromodomain inhibitors could suppress atherosclerosis,¹⁶⁷ senescence,¹⁶⁸ and the SASP,¹⁶⁹ indicating high potency for such interventions. The non-histone DNA-binding protein high mobility group protein B1 (HMGB1) is a major pro-inflammatory SASP factor, when externalized from the nucleus into the extracellular environment.³⁹ Soluble HMGB1 promoted atherosclerosis^40,41 and CVD risk.¹⁷⁰ It remains to be seen if HMGB1 nuclear retention ameliorates the epigenetic landscape of senescent cells and provides benefits in atherosclerosis. Another promising strategy is the epigenetic blockade of p66Shc, since pharmacological inhibitors are not currently available. p66Shc is a promoter of oxidative stress⁷⁰ and is highly expressed in senescent cells⁶⁸ and atherosclerosis.¹⁷¹ Inhibiting p66Shc its key regulator MiR-34a is promising,¹⁷² as p66Shc knock-out led to senescence prevention,⁶⁸ 30% animal lifespan extension,¹⁷³ and strong inhibition of atherogenesis.^70,71

Targeting senescent cells as the root of inflammation: senolytic approaches or cellular reprogramming

Senolytic approaches

Safe removal of chronic senescent cells is an attractive approach to blocking ageing pathology and atherosclerosis. It is possible to specifically delete senescent cells through activation of apoptosis by compounds that selectively target senescent cells, called senolytics. INK-ATTACK transgenic mice contain an inducible suicide gene in the CDKNA2 locus, which encodes p16 INK4a, a key molecule in senescent cells. This elegant transgenic model has been used to selectively eliminate senescent cells in vivo, leading to reversal of age-related characteristics without noticeable side effects.^94,95 The positive effect of senescent cell removal in atherosclerosis in LDL receptor-knockout mice has been replicated in INK-ATTACK, p16-3MR, and INK-nitroreductase mouse models using the senolytic navitoclax (ABT-263).⁹ Senescent cell elimination diminished plaque size, inflammation, and instability features.⁹ These data provide a rational basis for the use of senolytics in the treatment of atherosclerosis.

Recently, multiple senolytic compounds have been developed and have been tested for the treatment of CVD (Figure 4).^42,174–185 Navitoclax, a Bcl-2 inhibitor, has beneficial effects in atherosclerosis,⁹ and may be a good candidate for the targeting of T-EMRA cells, which overexpress Bcl-2 family proteins.¹¹⁶ Combination therapy with dasatinib and quercetin (D+Q) removed senescent cells from the tunica media, but not the intima, improving vascular function parameters, but not arterial compliance and plaque burden.³³ Senolytic drugs were known to be more effective against particular senescent cell types in vitro, and less effective against others, for unclear reasons.^42,174–185 It is otherwise unknown how the cell type specificity of senolytics affects cells of the arterial wall, but it may explain why D+Q removed senescent cells only within the tunica media. Further studies are needed to corroborate the senolytic effect within plaque.

Another option for using senolytics may with the goal of improving outcomes of autologous transplantation therapies of bone marrow-derived angiogenic cells in ischaemic heart failure. In ischaemic heart failure patients, these cells are severely impaired^186,187 and display markers of senescence,^188,189 disabling their use for patient-derived cellular therapy. Senolytics may be used to remove senescent cells, either in vivo or ex vivo, leading to a rejuvenated population and more effective therapeutic outcomes.^186,189

In the context of drug repurposing, some senolytic compounds are already clinically approved (dasatinib) or in clinical trials (navitoclax, HSP90 inhibitors) for indications in oncology. The results of the first human trial of dasatinib and quercetin in idiopathic pulmonary fibrosis (IPF) have been recently published.⁸⁸ This small scale single-arm study reported improvements of 6-min walk distance, 4 m gait speed, chair-stands, and Short Physical Performance Battery (SPPB) score, showing the promise of this therapy for IPF. An ongoing trial is evaluating dasatinib and quercetin for their senolytic ability in chronic kidney disease (NCT02848131).

Currently available senolytics drugs are limited by adverse effects. When used for cancer treatment, these drugs often exhibit adverse effects such as nausea, vomiting diarrhoea, and skin rashes; notably, Bcl-xl inhibitors induce severe thrombocytopenia.¹⁹⁰ To avoid these effects, localized delivery via percutaneous intervention may be required to target senescence in a CVD setting. This could be combined with senescent cell-specific delivery methods, such as the galactose-encapsulation of drugs that reduced thrombocytopenia after navitoclax application in an experimental setting.¹⁹¹ Alternatively, the drugs could be applied periodically, with long off-drug periods.¹⁷⁵

Cellular reprogramming

The induction or reprogramming of mesenchymal cells into a pluripotent state is possible via octamer-binding transcription factor (Oct)3/4, Sox2, c-Myc, and Krüppel-like factor (KLF4).¹⁹² Cells can potentially be reverted to a non-senescent state, reversing their cellular biological clock. Short-term cyclic expression of Oct4, Sox2, KLF4, and c-Myc in LAKI 4F mice suppressed the ageing phenotype, prevented VSMC degeneration, and reversed bradycardia without observed tumorigenesis.¹⁹³ These results suggest an intriguing possibility that low-dose short-term reprogramming may be beneficial for atherosclerosis. A drug cocktail of HDACs, GSK-3β, and TGF-β kinase enabled cell reprogramming in vitro, thus improving liver regeneration and function after acute injuries in mice.¹⁹⁴ The authors attributed this effect to the up-regulation of pluripotency genes. Extensive reprogramming may be tumourigenic, as it leads to up-regulation of strong oncogenes, such as c-Myc.¹⁹⁵ Extremely precise and reliable control of this anti-senescence strategy is an absolute must to avoid malignant transformation. Of note, senescent cells from elderly people could be reverted to a pluripotent state only by the additional over-expression of NANOG and LIN2 in addition to Oct3/4, Sox2, c-Myc, and KLF4.¹⁹⁶

Another intriguing strategy is direct reprogramming or trans-differentiation, which enables a phenotypic change from one differentiated cell type into another without achieving pluripotency.¹⁹⁷ Hypothetically, senescent secretory VSMCs could thereby regain a contractile phenotype.

Future directions towards clinical application of anti-senescence strategies

Research regarding ageing mechanisms and cellular senescence is confronted with pre-clinical and clinical challenges that need to be addressed in order to pinpoint the role of cellular senescence in atherosclerosis and define optimal therapeutic strategies. Firstly, in pre-clinical research, the detection of senescent cells is hampered by the lack of specific markers. In vitro, the gold standard senescence-associated β-galactosidase staining and p16 INK4a expression detection can yield false-positive results in macrophages.¹⁹⁸In vivo, the best currently available models for senescent cell detection are reporter-harbouring transgenic mice (p16-LUC, p16 tdTom)^199,200 and transgenic mouse strains used for selective senescent cell removal (INK-ATTAC, p16-3MR).²⁰¹

Secondly, a better understanding of the role of cellular senescence in vascular disease development is needed. Studies in cell cycle inhibitor knock-out models have raised questions about the complex relationships between senescence, proliferation, and apoptosis in different phases of atherosclerosis. p16 INK4a, p14 ARF (p19 ARF in mice), p21, and p53 enable cell cycle arrest in senescence. However, polymorphisms that hinder their activity promote atherosclerosis in humans.²⁰² Knock-out of p16 INK4a, p19 ARF, p21, and p53 had a pro-atherosclerotic effect in mice.^203–205 On the other hand, cellular senescence promotes atherosclerosis in each developmental phase.^9,101 Selective removal of p16 INK4a-positive senescent cells prevented atherosclerosis, while stabilizing plaques and decreasing plaque inflammation.⁹ This discrepancy can be explained by other critical roles of cell cycle inhibitors in cell physiology and disease development, all of which are afflicted by the abovementioned genetic polymorphisms and knock-outs in a nondiscriminatory way.

Thirdly, special care must be taken in selecting patients for possible anti-senescence clinical trials. Experimental evidence shows that cellular senescence has roles in limiting myocardial²⁰⁶ and liver²⁰⁷ fibrosis, promoting skin healing,⁸² as well as in embryonal development,²⁰⁸ which have to be considered. However, these studies focused on the function of senescence in acute organ injury, not in chronic processes such as atherosclerosis. The aim of anti-senescent therapies would be to clear chronic senescent cells or suppress their noxious effects, while not permanently abrogating the senescence programme. Nevertheless, these findings may imply that initial anti-senescence/anti-SASP therapeutic trials need to be applied to patients with a clinically stable phase of vascular disease, with high residual CVD risk,² and not in a setting of acute injury. Another concern would be that inducing senescence in the short term initially slows cancer growth due to the cell cycle arrest induction,⁸¹ which raises questions about oncogenesis as an adverse effect. However, chronic senescence promotes cancer.⁹² These issues remain to be carefully addressed and analysed in future studies exploring anti-senescence strategies.

To track the effect of anti-senescence therapies in a clinical setting, it would be important to specifically detect total or localized senescent cell burden.⁷ Using a matrix-biomarker approach of 48 SASP-associated cytokines and miRNAs⁸⁸ seemed not to be an effective strategy in evaluating the effects of senolytic therapy in IPF. The possible explanation is that immune cells can also produce these factors, making senescence-specific detection problematic. An alternative to the matrix approach may be the discovery of senescence-specific soluble antigens, such as oxidized vimentin.²⁰⁹

Anti-senescence approaches may be a crucial component of precision medicine therapy. The development and decreasing cost of –omics methods or imaging can lead to a clinically applicable biomarker set, which combines senescence-associated soluble biomarkers and cellular compartments to identify patients with a high senescence burden, and thus the best candidates for anti-senescence therapies. For example, patients with a high burden of Bcl-2 overexpressing senescent T-EMRA cells may be good candidates for therapy with a Bcl-2-targeting senolytic, navitoclax. On the other hand, if there is a dominance of endothelial senescence, SASP inhibitors may be more beneficial than senolytics to avoid endothelial erosion. Decision-making can be further tailored with individual genetic profiling. Speculatively, a patient with a down-regulating rs2802292 FOXO3 T; T polymorphism²¹⁰ may benefit more from FOXO/SIRT-1 agonists and NAD boosters than from other forms of anti-senescence therapy. Ultimately, developing next-generation biomarkers and imaging methods seems essential to translate anti-senescence therapeutics to the clinic.

Conclusions

Current therapeutic strategies targeting known risk factors for CVD, such as hypertension, diabetes mellitus, and cholesterol levels lower CVD risk. However, the disease risk remains high and will increase in the ageing western population. Targeting specific upstream inflammatory processes involved in deleterious local plaque pathophysiology has emerged as a novel approach to limit atherosclerosis. Accumulation of senescent cells promotes inflammation and negatively affects plaque remodelling. Translating novel anti-senescence strategies into the clinic has potential to causally and effectively prevent and treat atherosclerosis and/or CVD.