Potential Clinical Implications of Senotherapies for Cardiovascular Disease

Abstract

Aging is a major risk factor for cardiovascular diseases (CVDs) and accumulating evidence indicates that biological aging has a significant effect on the onset and progression of CVDs. In recent years, therapies targeting senescent cells (senotherapies), particularly senolytics that selectively eliminate senescent cells, have been developed and show promise for treating geriatric syndromes and age-associated diseases, including CVDs. In 2 pilot studies published in 2019 the senolytic combination, dasatinib plus quercetin, improved physical function in patients with idiopathic pulmonary fibrosis and eliminated senescent cells from adipose tissue in patients with diabetic kidney disease. More than 30 clinical trials using senolytics are currently underway or planned. In preclinical CVD models, senolytics appear to improve heart failure, ischemic heart disease, valvular heart disease, atherosclerosis, aortic aneurysm, vascular dysfunction, dialysis arteriovenous fistula patency, and pre-eclampsia. Because senotherapies are completely different strategies from existing treatment paradigms, they might alleviate diseases for which there are no current effective treatments or they could be used in addition to current therapies to enhance efficacy. Moreover, senotherapies might delay, prevent, alleviate or treat multiple diseases in the elderly and reduce polypharmacy, because senotherapies target fundamental aging mechanisms. We comprehensively summarize the preclinical evidence about senotherapies for CVDs and discuss future prospects for their clinical application.

Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality among older people globally, claiming an estimated 17.9 million lives each year.¹ The prevalence of the majority of CVDs such as heart attacks, coronary artery disease, hypertension, atherosclerosis, stroke, heart failure (HF), valvular heart diseases, aortic aneurysms, and arrhythmias, including atrial fibrillation (AF), increases steadily with patient age. Although the pathogenesis of CVDs is multifactorial and risk factors for CVDs such as diabetes and hypertension increase with age, aging appears to be an independent risk factor.^2,3 Although CVD mortality rates are declining,⁴ despite aggressive control of risk factors as recommended in recent guidelines, the prevalence of CVDs has not improved dramatically and may be due to failure to intervene against a key underlying cause of CVD: biological aging.

Age-associated diseases and geriatric syndromes including CVDs are, in part, a consequence of biological aging. Targeting the hallmarks of biological aging may be a root-cause therapeutic approach for delaying the onset or alleviating multiple age-associated conditions; that is, the “geroscience hypothesis”.⁴ Moreover, intervening against any one of the biological aging mechanisms that underlie age-associated diseases and geriatric syndromes may affect many or most of the rest, a unitary theory of fundamental aging mechanisms.⁵

Senotherapies

The 3 approaches being investigated as potential senotherapies in preclinical models are: (1) elimination of persisting senescent cells (senolytics), (2) in vivo direct reprogramming of senescent cells, and (3) suppression of release of the senescence-associated secretory phenotype (SASP) factors by senescent cells (senomorphics) (Figure).

Figure.

Contributions of senescent cells to cardiovascular diseases (CVDs). Senescent cells accumulate during aging and are related to many forms of stress including DNA damage, mechanical/shear stress, reactive metabolites (e.g., reactive oxygen species), angiotensin II, hyperinsulinemia, and oxidized low-density lipoprotein. The cell cycle becomes arrested and so reduces the risk that damaged dysfunctional cells proliferate and give rise to cancers. However, persistent dysfunctional senescent cells can continue to release tissue-damaging SASP factors that induce inflammation, contributing to the pathology of CVDs. There are currently 3 strategies being investigated to attenuate unfavorable consequences of persistent, pro-inflammatory senescent cells: (1) senolytics that induce death of those senescent cells that are pro-apoptotic; (2) reprogramming to alter epigenetic programming through the Yamanaka factors (OSKM) to revert senescent cells into a non-senescent state; and (3) senomorphics that inhibit production of tissue-destructive SASP factors from senescent cells while not directly eliminating the senescent cells themselves. Metformin, rapamycin, and ruxolitinib are among the senomorphics in clinical use for various indications.

Senolytics was proposed as a therapeutic strategy of inducing cell death in senescent cells. The first senolytic, a combination of dasatinib and quercetin (D+Q), reduced the senescent cell burden in naturally aged mice,^6–9 and alleviated age-related metabolic dysfunction.¹⁰ A causal role for cellular senescence in the generation of age-related phenotypes was further demonstrated by showing that transplantation of small numbers of senescent cells into middle-aged mice can cause frailty-like physical dysfunction and multiple age-associated diseases.¹¹ These findings support the potential of senolytics as a root-cause treatment for many age-associated disorders and diseases. Based on the first study of senolytics, more have since been identified^4,10,12–31 (Table 1). To date, senolytics appear to alleviate multiple conditions, including cardiac dysfunction,^5,7,32–35 vascular diseases,^34,35 frailty,^36–38 cognitive impairment,^39–43 renal dysfunction,⁴⁴ pulmonary diseases,³⁸ osteoporosis,^28,45,46 and hepatic steatosis.⁴⁷

Table 1.

List of Senotherapies

Senolytic	Target molecules	Localization
Dasatinib	Src kinase, Tyrosine kinase, etc	Plasma membrane
Quercetin	Bcl-2 family, p53/p21/serpine, PI3K/AKT	Cytoplasm, Mitochondria, Nuclei
Fisetin	PI3K/AKT	Cytoplasm
Navitoclax (ABT-263)	Bcl-2 family (Bcl-2, Bcl-xl, Bcl-w)	Cytoplasm, Mitochondria
PZ15227	Bcl-xl	Cytoplasm, Mitochondria
TW-37	Bcl-2 family (Bcl-2, Bcl-xl, Mcl-1)	Cytoplasm, Mitochondria
ABT-737	Bcl-xl, Bcl-w	Cytoplasm, Mitochondria
Luteolin	Bcl-2 family (Bcl-xl)	Cytoplasm, Mitochondria
Curcumin, Curcumin analog EF24	Bcl-2 family (Bcl-xl)	Cytoplasm, Mitochondria
A1331852	Bcl-2 family (Bcl-xl)	Cytoplasm, Mitochondria
A1155463	Bcl-2 family (Bcl-xl)	Cytoplasm, Mitochondria
Piperlongumine	p53/p21, Bcl-2 family (PUMA)	Cytoplasm, Mitochondria, Nuclei
FOXO4-DRI	FOXO-p53 interaction	Nuclei
Nutlin3a	p53	Nuclei
UBX0101	p53-MDM2 / p21	Nuclei
Panobinostat	Histon deacetylase	Nuclei
ARV825	BET family protein, NHEJ	Cytoplasm
AT-406	IAP1/2/XIAP	Cytoplasm
Fenofibrate	PPARa	Cytoplasm
Alvespimycin (17-DMAG)	HSP90	Cytoplasm
Tanespimycin (17-AAG)	HSP90	Cytoplasm
Geldanamycin	HSP90	Cytoplasm
Digoxin (cardiac glycosides)	Na+/K+ ATPase pump	Plasma membrane
BPTES	GLS1	Lysosome
Galactose-modified duocarmycin (GMD)	Lysosomal β-galactosidase	Lysosome
SSK1-Gemcitabine	Lysosomal β-galactosidase	Lysosome
2-DG	Lysosomal V-ATPases	Lysosome
GPNMB vaccine	GPNMB	Plasma membrane, lysosome
CD153 vaccine	CD153	Plasma membrane
ADC against B2M	β2 microglobulin (B2M)	Plasma membrane
uPAR specific CAR T	Urokinase-type plasminogen activator receptor (uPAR)	Plasma membrane
Senomorphics	Target molecules	Localization
Rapamycin	mTOR	Cytoplasm
Metformin	AMPK	Cytoplasm
Ruxolitinib	JAK 1/2	Cytoplasm
NBD peptide/mimetics	NF-κB	Cytoplasm

2-DG, 2-deoxy-d-glucose; 17-DMAG, 17-dimethylaminoethylamino-17-demethoxygeldanamycin; NHEJ, non-homologous end joining.

Another strategy to mitigate the adverse effects of senescent cells is to reprogram them using the Yamanaka factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), the discovery of which led to a Nobel prize in 2012.⁴⁸ This approach allows cells, including senescent cells, to be converted to a state akin to pluripotency that might be applicable to counteract the effect of biological aging,^49–55 as well as regenerative medicine and development of new drugs. This field is evolving, and the long-term effects and the risk for adverse events have not yet been fully elucidated. Enabling potentially cancerous mutation-harboring senescent cells to escape replicative arrest is a concern.

The third approach to targeting cellular senescence is to use SASP inhibitors, senomorphics. This approach is different from the other two, because it does not target senescent cells themselves but rather the release of detrimental, inflammatory SASP factors by senescent cells. Several agents that are senomorphic, including metformin, ruxolitinib, and rapamycin, are in clinical use, with data available from post-marketing surveillance⁵⁶ and retrospective studies.^57–64 A clinical trial using low-dose rapamycin for coronary artery disease (Cardiac Rehabilitation And Rapamycin in Elderly [CARE]) was conducted to test the safety and feasibility of low-dose rapamycin and effects on both SASP and frailty in elderly subjects undergoing cardiac rehabilitation. Intriguingly, rapamycin suppressed some of markers of senescent cells in adipose tissue, although it failed to alleviate frailty.⁶⁵ Another clinical trial, TAME (Targeting Aging with Metformin), will test if metformin delays the appearance of a second age-associated disease.^59,66 A challenge in using senomorphic drugs is modulating potential off-target effects on non-senescent cells and potential side effects from suppressing the physiological inflammation that may be necessary for resolving certain acute diseases or promoting tissue repair. Additionally, senomorphic treatment allows senescent cells themselves to persist, potentially necessitating more continuous administration of SASP inhibitors that senolytics, which could increase off-target and side effects.

Preclinical Evidence of Senolytics for CVDs

In preclinical studies of CVDs there is mounting evidence of the potential benefits of senolytics (Table 2). D+Q,⁷ quercetin,^67,68 and navitoclax^69,70 have improved cardiac function in aged mice and several HF models. In older humans, diastolic dysfunction, also known as HF with preserved ejection fraction (HFpEF), is a major problem.^71–74 An effect of senolytics on HFpEF may be also expected. In addition, D+Q prolongs survival of cardiac allografts from old mice,⁶⁹ with a reduction in senescent cell burden and inflammation, suggesting senolytics may be of benefit in the case of organ transplantation or cell-based therapies to increase their viability.^75–77 Moreover, D+Q, by eliminating senescent cells, improved human induced pluripotent stem (iPS) cells-derived cardiomyocyte survival.⁷⁸ Senolytics may be potentially useful in ischemic heart disease. D+Q activated approximately 10% of cardiac resident cardiac progenitor cells (CPCs) and increased differentiation from CPCs,³² in association with an improvement of cardiac repair.⁷⁹ Navitoclax alleviated increased the left ventricular EF, enhanced myocardial vascularization in an ischemia-reperfusion model in mice and rats, and increased survival after myocardial infarction in aged mice.^33,80,81 Navitoclax-coated stents were associated with reduced stenotic area in rabbits on a high cholesterol diet.⁸²

Table 2.

Preclinical Studies of Senolytics for Cardiovascular Diseases

Disease targeted	Results of senolytic therapy	Senolytics	Senolytic model	Disease model
Heart failure	Alleviated impaired ejection fraction Activated resident CPCs and increased cardiomyocyte proliferation Reduced myocardial hypertrophy and fibrosis	D+Q7,32 Navitoclax33	INK-ATTAC 32,33	Aged (mouse)
	Attenuated cardiac systolic dysfunction Normalized hypertrophy Reduced cardiac fat and fibrosis Increased proliferation of CPCs and cardiac repair	Q67,68 D+Q79 Navitoclax70,119		High fat diet-induced Angiotensin II (Ang II)-induced Doxorubicin (DOX)-induced (Mouse or rat)
Heart transplantation	Prolonged the survival of cardiac allografts from aged mice	D+Q69		Aged (mouse)
Hypertension	Improved vascular relaxation Alleviated aortic calcification and improved vasomotor function	D+Q7,34	INK-ATTAC 34	Aged (mouse)
Pulmonary arterial hypertension	Improved hemodynamic and structural changes associated with severe PAH	Navitoclax97		MCT and shunt-induced PAH (rat)
Ischemic heart diseases	Reduced myocardial hypertrophy and fibrosis Alleviated impaired ejection fraction Increased myocardial vascularization Increased survival after myocardial infarction	D+Q120 Navitoclax81,121,122		Ischemia-reperfusion or myocardial infarction model (mouse or rat)
Atherosclerosis	Reduced atherosclerotic plaque formation and instability Increased thickness of the fibrous cap	Navitoclax35,91,95 GPNMB-vaccine26 Digoxin93 17-DMAG94 BPTES24	INK-ATTAC 35,95 p16-3MR 35,91,95	Ldlr KO or ApoE KO (mouse)
Coronary artery stent restenosis	Reduced stenosis area of senolytic-coated stent	Navitoclax82		Cholesterol diet (rabbit)
Abdominal aortic aneurysm	Reduced size of AAA	D+Q96		Angiotensin II (Ang II) administrated in aged (mouse)
Atrial fibrillation	Ameliorated atrial fibrosis Shorten AF duration	Fisetin89 Q88		Myocardial infarction (rat) Isoproterenol (rat)
Chronic kidney disease	Decreased senescent cells burden	D+Q99		Dialysis arteriovenous fistula (mouse)
Diabetic retinal angiopathy	Attenuated ischemic retinopathy	UBX1967100	INK-ATTAC 100	Oxygen-induced retinopathy (mouse) Eye tissue (human)

The incidence of AF increases with aging.^83,84 Senescent cell accumulation in the atrium with aging has been demonstrated in rodents and humans.^85–87 There are 2 preclinical studies with either quercetin⁸⁸ or fisetin⁸⁹ that demonstrated potential alleviation of AF and fibrosis, although in those studies the drugs were used as antifibrotics and senescent cell removal was not specifically examined. The results obtained in rodent models may not fully reflect the underlying mechanisms of AF in humans, because AF is not a naturally occurring condition in rodents.⁹⁰

Senolytic therapies have also been tested in preclinical models of vascular diseases with promising results. Senolytics such as anti-Gpnmb vaccination, navitoclax, digoxin, BPTES, and 17-DMAG (alvespimycin) have reduced atherosclerotic plaque formation and inflammation,^{22–24,35,91–94} alleviated fibrous capsule thinning of atherosclerotic plaques,⁹⁵ and attenuated vascular calcification.³⁴ D+Q might attenuate hypertension in the elderly because it improves vasorelaxation and vasomotor function in aged mice.^7,34 Also, in aged mice with abdominal aortic aneurysms (AAA), D+Q reduced the size of the AAA.⁹⁶ In addition to these effects, senolytics has been tested for efficacy in the diseases such as pulmonary arterial hypertension (PAH),^97,98 hemodialysis shunt survival,⁹⁹ and ischemic retinopathy¹⁰⁰ (Table 2).

Clinical Trials of Senolytics

More than 30 clinical trials of senolytics, including phase 2 randomized, double-blinded, placebo-controlled trials, are already underway or planned based on the promising results in preclinical experiments.⁴ D+Q was used in 2 pilot studies of senolytics because 1 approach is to use senolytic compounds already approved for other uses or natural products with established safety data in the early studies and already favorable results have been reported.^101,102 In the field of CVDs, translation of antisenescence drugs into the clinic has lagged, although, there are current studies planned or beginning with senolytic drugs targeting cardiac and arterial diseases.¹⁰³ One is the Q-CABG study, testing oral quercetin monotherapy as a senolytic in patients undergoing coronary artery bypass graft (CABG) surgery. More clinical studies are being planned, including for HF with different causes, arrhythmias such as AF, and maintaining arteriovenous dialysis fistula patency, among other CVDs.

Future Directions

Senolytics, as a novel class of drugs, might be effective in diseases for which no good treatments currently exist, such as HFpEF. Senolytics may have the potential for synergistic beneficial effects if combined with current therapies that are known to improve the prognosis of CVDs.¹⁰⁴ The effectiveness of senolytics might also be enhanced when used in combination with other interventions, such as lifestyle changes (e.g., caloric restriction, exercise), that target interlinked fundamental aging processes. Senolytics might also alleviate more than one pathology associated with aging at the same time. Such multimorbidity is often a problem for older patients with CVDs.^105,106

The senotherapy field is new and there are important steps that need to be taken to efficiently discover new, safe senolytics and senomorphics and translate them into agents for clinical use. Preclinical and early clinical trials suggested short-term safety and target engagement for senolytics, but it will be important to conduct further studies to test for potential long-term side effects. Another need is to identify gerodiagnostic biomarkers that can be tested in body fluids such as blood, urine, saliva or tears to monitor senescent cell abundance. Among such gerodiagnostics are CD3-positive T cells expressing p16^INK4a in peripheral blood,^107,108 and α-Klotho in urine.¹⁰⁹ SASP factors in circulating blood, tissue or cells might also be used for estimating senescent cell burden. Although a single SASP factor may not be highly sensitive or specific, combinations of such indicators might be better.^{69,101,102,110,111} In addition to protein and peptide SASP factors, blood non-coding nucleotide SASP factors (microRNAs [miRNA], short non-coding RNAs,^112,113 small extracellular vesicles,^114,115 and mitochondrial DNAs⁶⁹) are emerging as gerodiagnostic markers. One difficulty is that senescent cells are highly heterogeneous: they release different factors depending on the cell type that became senescent, the inducer of senescence, how long the cell has been senescent, and the tissue microenvironment.^116–118

Conclusions

Senotherapies are an emerging strategy for intervening against fundamental aging mechanisms to potentially prevent and treat multiple disorders and diseases and extend health span. Because senotherapies allow targeting of the underlying causes of aging, rather than treating disorders and diseases individually, they could reduce polypharmacy, which is a cause of the attendant risks of adverse events and drug interactions. Senolytics are particularly attractive because they seem to be effective even when given intermittently, despite having short elimination half-lives, thereby minimizing potential toxic effects.

Although clinical studies of senotherapies for CVDs are limited so far, promising evidence from preclinical studies suggests senolytic agents might become a novel strategy for delaying, preventing, alleviating or treating CVDs. Further research is needed to identify the combinations of gerodiagnostic markers for monitoring target engagement during clinical trials and potentially for identifying which senolytics to use, when, and for how long to customize treatment for each patient. It must be borne in mind that treatments that appear to be highly promising in rodents frequently fail in clinical trials, especially in the cases of disorders such as AF, which do not occur naturally in rodents. It will be important to conduct validation studies for these diseases in large animal CVD models such rabbits, dogs, pigs, and monkeys. Because clinical studies of senolytics and senomorphics have already begun for other indications, it will be helpful to closely monitor cardiovascular effects and side effects in those studies. If eventually shown to be safe and effective, senolytics might one day be indicated for a number of CVDs.