Cellular Senescence and Frailty in Transplantation

Abstract

Purpose of review.

To summarizes the literature on cellular senescence and frailty in solid-organ transplantation and highlight the emerging role of senotherapeutics as a treatment for cellular senescence.

Recent findings.

Solid-organ transplant patients are aging. Many factors contribute to aging acceleration in this population, including cellular senescence. Senescent cells accumulate in tissues and secrete proinflammatory and profibrotic proteins which result in tissue damage. Cellular senescence contributes to age-related diseases and frailty. Our understanding of the role cellular senescence plays in transplant-specific complications such as allograft immunogenicity and infections is expanding. Promising treatments, including senolytics, senomorphics, cell-based regenerative therapies, and behavioral interventions, may reduce cellular senescence abundance and frailty in patients with solid-organ transplants.

Summary.

Cellular senescence and frailty contribute to adverse outcomes in solid-organ transplantation. Continued pursuit of understanding the role cellular senescence plays in transplantation may lead to improved senotherapeutic approaches and better graft and patient outcomes.

Introduction.

The world is aging. According to the World Health Organization, the number of people aged 60 or over is expected to grow to 2 billion, or 22% of the global population, by 2050.¹ With increased aging comes increased disease burden. Age is a risk factor for numerous chronic illnesses, such as nonalcoholic fatty liver disease,² chronic obstructive pulmonary disease,³ heart failure,⁴ and end-stage kidney disease.⁵ Given that many patients with these diseases progress to needing organ replacement therapy, this epidemic of aging and age-related comorbidities directly impacts the field of transplantation where the average age and of liver,⁶ lung,⁷ heart,⁸ kidney⁹ transplant recipients is steadily increasing.

Our understanding of the aging process has evolved dramatically over the past fifty years. We now know that aging has many pathophysiologic drivers, including shortening of telomeres, genomic instability, stem cell exhaustion, and cellular senescence abundance.¹⁰ Cellular senescence refers to the process in which cells enter a state of irreversible growth arrest in response to perturbations, including DNA damage and oxidative stress.¹¹ Senescent cells are resistant to apoptosis,¹² accumulate with age,¹³ and contribute to tissue dysfunction, age-related disease,¹⁴ and frailty.¹⁵ The purpose of this review is to outline recent advances in our understanding of cellular senescence abundance and frailty in solid-organ transplantation and discuss potential therapeutics.

Cellular Senescence.

Although senescent cells have may identifying characteristics, they display significant heterogeneity, and no one single marker identifies them all.¹⁴ Morphologically, senescent cells tend to be flatter and larger than healthy cells and are often multinucleated. Histochemically, they stain for senescence-associated β-galactosidase (SA-β-gal), a lysosomal enzyme that catalyzes the hydrolysis of carbohydrates. They exhibit increased levels of DNA damage (e.g. p53)¹⁶ and cell cycle inhibitors (e.g. p16 and p21).¹⁴ Senescent cells can also be indirectly identified by the products they secrete into the circulation. Senescent cells upregulate production of various cytokines, growth factors, chemokines, and other proteins referred to as the senescence-associated secretory phenotype (SASP), levels of which can be detected in the blood (Table 1).¹⁷ In a recent study, Saul et al.¹⁸ sought to develop a more accurate way of identifying senescent cells. Their team developed a panel of genes capable of identifying senescent cells across tissues and species which may be useful in future studies evaluating senescent cell burden.

Table 1.

Examples of Senescence-Associated Secretory Phenotype (SASP) components (adapted from Kirkland et al.²²)

Cytokines

Interleukin-6 (IL-6) Tumor necrosis factor-α (TNF-α) Transforming growth factor-β (TGF-β) Vascular endothelial growth factor-A (VEGF-A) Platelet-derived growth factor-AA (PDGF-AA)

Chemokines

Monocyte chemoattract protein-1 (MCP-1/CCL-2) Chemokine (C-X-C motif) ligand 2 (CXCL2) Macrophage inflammatory protein-1α (MIP-1α) Eotaxin

Extracellular matrix proteases and remodeling factors

Matrix metalloproteinases (MMPs) Tissue inhibitor of metallopeptidase (TIMPs)

Receptors

Tumor necrosis factor receptor-1 (sTNFR1) Tumor necrosis factor receptor-2 (sTNFR2) Urokinase plasminogen activator receptor (uPAR)

Reactive metabolites

Reactive oxygen species

Senescent cell accumulation contributes to aging and chronic disease throughout the body.¹⁴ Senescent cells accrue in diseased tissue, such as the lungs in chronic obstructive pulmonary disease, adipose and kidney tissue in type 2 diabetes mellitus, and the heart in cardiovascular disease. Cellular senescence is abundant in the transplanted organ¹⁹ and reduces allograft survival in mouse models.²⁰ Senescent cells induce the formation of other senescent cells locally and systemically via the SASP and contribute to tissue dysfunction.¹⁴ SASP proteins, such as interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon gamma (IFN-γ), are proinflammatory and can induce matrix remodelling, fibrosis, and apoptosis.^21,22 These cytokines and chemokines attract and activate immune cells within tissue.²¹ The resulting low-grade, chronic inflammation associated with the SASP is often referred to as ‘inflamm-aging.’²³ In adipose tissue, senescent cells can decrease replication of preadipocytes and decrease insulin sensitivity.²⁴ In the liver, they can induce hepatic steatosis.²⁵ In bone, they can lead to decreased fracture repair.²⁶ In the immune system, they can cause dysregulation by increasing the number of immunosuppressive cells (i.e. regulatory T cells) and decreasing the efficacy of CD4 and CD8 T cells, NK cells, and B cells, a process referred to as immunosenescence.^27,28 Senescent cell abundance can also promote malignant transformation in tissues throughout the body.²⁹ In organ transplantation, senescent cells accumulate due to immune activation and reduced immune clearance from ongoing immunosuppressive therapy.^30–32

Cellular senescence and frailty.

Cellular senescence has emerged as a potential driver of frailty. The concept of frailty was initially described by Linda Fried and colleagues. In her landmark paper published in 2001, Fried defined frailty as a syndrome of decreased physiologic reserve and increased susceptibility to stress.³³ The Fried frailty phenotype, also known as the physical frailty phenotype, consists of five criteria: slow gait speed, unintentional weight loss, exhaustion, low self-reported physical activity, and weak grip strength. Patients meeting three or more of the criteria are classified as frail, while patients meeting one or two of the criteria are classified as pre-frail.³³ Other measures of frailty include tests of physical performance, the cumulative deficit model, and surveys (Table 2).³³ Emerging digital measures of frailty include pendant and upper extremity sensors and accelerometers.^34–37 These digital measures of frailty have shown to predict falls,³⁸, functional mobility,³⁹ and cardiovascular events after vascular surgery.⁴⁰ These digital measures have the advantage of being able to be assessed using telemedicine, monitored remotely, and do not require weight bearing.^41,42

Table 2.

Commonly utilized measures of frailty in solid-organ transplantation

Category	Description	Examples
Performance-based	Tests of physical performance	Short Physical Performance Battery111 6-Minute Walk Test112 Timed Up and Go Test113 Sit-to-Stand Test114 Liver Frailty Index115
Phenotype model	Combination of tests of physical performance and self-report	Physical Frailty Phenotype33
Cumulative deficit model	Comorbidities, disabilities, and symptoms determined from the medical record	Rockwood Frailty Index116,117
Self-report	Questions answered by the patient	Short Form 12 Physical Component Scale118 PROMIS Physical Function Short Form119,120
Provider-based	Assessed by healthcare provider	Clinical Frailty Scale121 Karnofsky Score122

Frailty is associated with numerous adverse health outcomes across patient populations, including disability, falls, hospitalization, and death.^43,44 Frailty reflects derangements in multiple physiologic systems, including the cardiopulmonary system, the nervous system, and the musculoskeletal system.⁴⁵ Cellular senescence is believed to contribute to the muscle dysfunction observed in frailty in several different ways. Senescent cells accumulate in muscle fibers,⁴⁶ reduce muscle hypertrophy after exercise,⁴⁷ contribute to muscle atrophy and decreased contractility,⁴⁸ and lead to sarcopenia.⁴⁹ In support of the causal role cellular senescence plays in muscle dysfunction and frailty, studies have shown that circulating levels of SASP are associated with frailty,⁵⁰ including worse grip strength, gait speed, chair rise time, and balance.⁵¹ In fact, transplanting senescent cells into mice has been shown to decrease hanging endurance and gait speed.⁴⁸

Treating cellular senescence and frailty.

The growing understanding of adverse implications of cellular senescence has led to extensive research on how best to halt or reverse the process. Promising pharmacologic treatments, termed senotherapeutics, are emerging. Senotherapeutics include senolytics which selectively deplete senescent cells and senomorphics which inhibit the SASP. One of the first studied senolytics involves a combination of dasatinib (a tyrosine kinase inhibitor) plus quercetin (a naturally occurring flavanoid).²² Together dasatinib and quercetin have been shown to improve cardiac ejection fraction and pulmonary function in older mice.^52,53 In the first-in-human study, intermittent oral administration of dasatinib plus quercetin in patients with idiopathic pulmonary fibrosis, a cellular senescence-associated disease, was associated with improved 6-minute walk distance, gait speed, and chair stand time.⁵⁴ The first evidence that senolytics cleared senescent cell abundance in humans was demonstrated in a phase I trial applying dasatinib plus quercetin in older patients with diabetic kidney disease.⁵⁵ In addition to reducing senescent cell burden, senolytics also improve grip strength, muscle endurance, and exercise capacity in mice.⁴⁸

Senomorphic drugs that suppress the SASP without eliminating senescent cells require continuous administration and are another potential avenue of therapy.²² One such drug, rapamycin, an mTOR inhibitor, is commonly used as an immunosuppressive agent in the transplant population. Rapamycin has been shown to improve lifespan in mice.^56,57 Moreover, Hoff et al.⁵⁸ demonstrated in a rat model of kidney transplantation that early post-transplant rapamycin therapy prevented accumulation of p16 positive cells in kidney compartments (tubules, interstitium, and glomeruli). However, the effects of rapamycin on cellular senescence or SASP in solid-organ transplant patients is unclear. Metformin, an antidiabetic medication, is another promising SASP suppressor and the most commonly studied senomorphic.^59,60 Similar to senolytics, SASP suppressors have been shown to improve muscle performance and physical function in animal models.^61,62

A recent addition to the armamentarium of senotherapeutic agents are stem cells and the extracellular vesicles that they produce. Mesenchymal stem cell derived-extracellular vesicles reduce senescence burden and extend lifespan in aging mice.⁶³ Another study by Sahu et al.⁶⁴ demonstrated the anti-geronic effect of extracellular vesicles. They showed that extracellular vesicles within the serum of young animals contribute to the regeneration of aged skeletal muscle in older animals.

The study of senotherapeutics in humans and particularly in solid organ transplant recipients remains in its infancy. Special consideration must be given to the safety of using these agents in immunocompromised patients. For example, adverse effects of the senolytic drug dasatinib include myelosuppression and infection.^65,66 The use of senotherapeutics could promote malignant transformation given that the SASP has been shown to play a physiological role in tumor suppression.¹⁴ This issue is especially relevant to solid-organ transplant recipients who are already at an increased risk of malignancy.

In addition to pharmacologic therapy, behavioral interventions can play a role in treating cellular senescence and frailty. The most commonly studied behavioral intervention is exercise.^15,67 Englund et al. demonstrated that a 12-week exercise intervention not only improved grip strength, sit-to-stand time, and the timed up and go test but also decreased expression of the senescent markers (i.e. p16 and p21) and decreased circulating levels of SASP.⁶⁸ Furthermore, several pilot studies have examined the impact of exercise on frailty in patients with advanced liver and kidney disease, including transplant candidates. These studies have demonstrated that exercise is safe and associated with improvements in frailty parameters.^15,69–72 Further studies examining the impact of prehabilitation, or exercise prior to surgery, on quality of life, healthcare utilization, and mortality are needed.⁶⁹

Cellular senescence, frailty, and transplantation.

Cellular senescence is believed not only to contribute to frailty in transplantation but also to other adverse outcomes such as organ dysfunction, decreased quality of life, and death (Figure 1). Our emerging understanding of the transplant-specific impact of cellular senescence will hopefully lead to better interventions capable of improving transplant outcomes. The following paragraphs will highlight data regarding frailty and cellular senescence in liver, lung, heart, and kidney transplantation.

Figure 1.

Potential impact of cellular senescence in solid-organ transplantation

In liver transplantation, frailty associated with waitlist mortality,^73,74 longer hospital length of stay,⁷⁵ rehospitalization after transplantation,⁷⁶ acute cellular rejection,⁷⁷ worse post-transplant global functional and physical health,⁷⁸ and post-transplant mortality.⁷⁶ A recent study examined markers of cellular senescence in liver allograft biopsies with rejection and found increased expression of senescent cell markers, including p16 and p21, in biopsies showing late acute cellular rejection and chronic rejection.⁷⁹

In lung transplantation, frailty has been associated with increased mortality in transplant candidates,⁸⁰ post-operative delirium,⁸¹ rehospitalizations,^82,83 lower quality of life,⁸⁴ disability,⁸⁵ and post-transplant mortality. Few studies have examined cellular senescence in patients with lung transplants. However, senescent cells have been found in the airway epithelium of lung transplants.¹⁹

In heart transplantation, frailty has been associated with lower rates of waitlisting, transplantation, and mortality in transplant candidates.⁸⁶ Candidates who are frail are also more likely to have depression and anemia and require more frequent hospitalizations.⁸⁷ Lastly, frailty has been associated with longer hospital length of stay and mortality after heart transplantation.⁸⁸ Cellular senescence is now being recognized as a potential explanation for why older allografts are associated with more inflammation, alloreactivity, and adverse outcomes than younger allografts.^89,90 Specifically, a study by Iske et al.⁹¹ demonstrated that cardiac allografts from older donors contain higher numbers of senescent cells. These senescent cells produce cell-free DNA that promotes immunogenicity after heart transplantation. Promisingly, treating older mice with the senolytics dasatinib and quercetin led to fewer senescent cells within the allograft, decreased levels of cell-free DNA, and better graft survival after subsequent cardiac transplantation.⁹¹ Research into whether older donor organs can transfer senescent cells into the recipient is ongoing.⁹²

In kidney transplantation, frailty has been associated with many adverse outcomes both before and after transplantation.¹⁵ Specifically, frailty has been associated with waitlist mortality,^93,94 delayed graft function,⁹⁵ delirium,⁹⁶ longer hospital length of stay,^97,98 and rehospitalizations.^97,99 It has also been associated with immunosuppression intolerance,¹⁰⁰ cognitive decline,¹⁰¹ lower quality of life,¹⁰² and post-transplant mortality.¹⁰³

Numerous studies have investigated the impact of cellular senescence in kidney transplantation. First, cellular senescence may contribute to mortality. In kidney transplant candidates, SASP components, including IL-6, TNF-α, and tumor necrosis factor receptor-1 (sTNFR1) were associated with increased risk of waitlist and post-transplant mortality.^94,104 Second, cellular senescence may contribute to donor organ quality. An animal study by He et al.¹⁰⁵ found that transplanted kidneys from older donors have different immunophenotypes than kidneys from younger donors, including increased levels of CD8 T cells. Older allografts also are associated with more inflammatory cytokine production during the first week post-transplant. However, pretreating older murine donors with senolytics prior to organ harvest appears to reduce expression of inflammatory cytokines in the older allografts suggesting a potential role for senolytics in improving donor organ function similar to the above studies in heart transplantation. Third, cellular senescence may adversely impact kidney transplant function. Transplanting senescent cells into mice has been shown to be associated with increased creatinine levels, renal tissue hypoxia, and fibrosis.¹⁰⁶ Fourth, cellular senescence may be involved in renal allograft rejection. Extracellular vesicles (miRNA) from kidney transplant recipients with antibody-mediated rejection have been shown to induce renal tubular senescence in vitro via complement activation and fibrosis.¹⁰⁷ Lastly, immunosenescence may increase infection risk in kidney transplant recipients. T cell senescence and impaired-cytomegalovirus (CMV) specific response have been associated with increased vulnerability to infections after kidney transplantation.¹⁰⁸ Fortunately, a recent study demonstrated that early post-transplant administration of rapamycin protects against cellular senescence in a murine model of kidney transplantation.⁵⁸

Conclusions.

Our understanding of the role cellular senescence plays in aging and frailty is expanding. The study of frailty is complicated by the numerous ways used to measure it. Digital measurements of frailty may allow for better remote diagnosis and monitoring of frailty. Senotherapeutics and exercise have been shown to decrease cellular senescence abundance and improve frailty in non-transplant patients. While numerous studies have highlighted the relationship between frailty and adverse health outcomes before and after solid-organ transplantation, less is known about the role and impact of cellular senescence in transplant outcomes. Recent studies suggest that cellular senescence may play a role in the alloreactivity seen with older allografts and in rejection. Future research examining how to safely and effectively reverse cellular senescence and frailty in solid-organ transplant patients before and after transplantation is needed. As donor age boundaries are pushed, further research into the promising role of senotherapeutics in expanding the organ pool by improving the function of older organs would also be beneficial.^109,110