Promises and challenges of senolytics in skin regeneration, pathology and ageing

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Highlights

  • Senescent cells have been detected in skin in conditions such as development, regeneration, disease and ageing.

  • Skin cell types including fibroblasts, keratinocytes and melanocytes can acquire the phenotype of cellular senescence.

  • Cellular senescence of skin cells can be studied and measured also indirectly using with dermal open flow microperfusion.

  • Senolytics eliminate senescent skin cells with cell type specificity, but should be used with caution in clinical settings.

Abstract

The research of the last two decades has defined a crucial role of cellular senescence in both the physiology and pathology of skin, and senescent cells have been detected in conditions including development, regeneration, aging, and disease. The pathophysiology of cellular senescence in skin is complex as the phenotype of senescence pertains to several different cell types including fibroblasts, keratinocytes and melanocytes, among others. Paradoxically, the transient presence of senescent cells is believed to be beneficial in the context of development and wound healing, while the chronic presence of senescent cells is detrimental in the context of aging, diseases, and chronic wounds, which afflict predominantly the elderly. Identifying strategies to prevent senescence induction or reduce senescent burden in the skin could broadly benefit the aging population. Senolytics, drugs known to specifically eliminate senescent cells while preserving non-senescent cells, are being intensively studied for use in the clinical setting. Here, we review recent research on skin senescence, on the methods for the detection of senescent cells and describe promises and challenges related to the application of senolytic drugs. This article is part of the Special Issue - Senolytics - Edited by Joao Passos and Diana Jurk.

Introduction

Cellular senescence is defined as a process initiated with an induction of cell cycle arrest and entailing changes on the levels of the epigenome, transcriptome, proteome and secretome. Some of the most common markers of senescent cells are derived from these changes and include an expansion of the lysosomal compartment characterised by high activity of so-called senescence-associated-β-galactosidase (SA-β-gal) (Dimri et al., 1995), elevated levels of intracellular damage forms such as lipofuscin and DNA double strand breaks (DSBs) (Ogrodnik et al., 2019a,b) an increase in levels of cell cycle inhibitors p16INK4a (INK4a) and p21 (Cip1/Waf1) (Collado et al., 2007) and a pro-inflammatory phenotype termed the senescence-associated secretory phenotype (SASP) (Coppe et al., 2008). Described for the first time for human fibroblasts that were kept in long-term in vitro cell culture (Hayflick, 1965), cellular senescence has come a long way and currently is considered a major player in development and progression of many age-related diseases (Baker et al., 2016, 2011; Collado et al., 2007) with a robust pipeline of anti-senescence interventions planned (Kirkland and Tchkonia, 2017). Out of those, a group of drugs termed "senolytics" were selected to specifically induce cell death in senescent compared to non-senescent cells and are currently at the stage of clinical trials (Wissler Gerdes et al., 2020). Another group of senotherapies termed "senomorphics" (and “senostatics”) leave senescent cells intact, but mitigate their negative effects on surrounding tissues, for instance by SASP inhibition (Birch and Gil, 2020). Due to the availability of several excellent reviews summarizing senotherapies in more general terms (Birch and Gil, 2020; Kirkland and Tchkonia, 2020; Niedernhofer and Robbins, 2018; Short et al., 2019; von Kobbe, 2019), this article focusses on the current state of research and development of senolytic drugs with a specific angle into their applications to skin pathology, ageing and regeneration (Table 1).

Induction of cellular senescence occurs in conditions with a functional overlap of pro-growth (or pro- “expansion”) and damage-inducing factors (Ogrodnik et al., 2019b). Skin is a regenerative organ with some of its structures (such as epidermis and hair follicles) displaying proliferative activity, which acts as an outer protective layer of animal bodies and is therefore at a constant risk of damage. Skin damage is exacerbated by conditions such as ageing and photoageing, while an increase in the proliferative rate of skin cells is observed during development and wound healing (Gu et al., 2020; Ivanova et al., 2005). Not surprisingly, all these conditions are known to coincide with an increase in the amount of senescent cells (Demaria et al., 2014; Drosten et al., 2014; Fitsiou et al., 2021; Wang and Dreesen, 2018). Such an increase is also observed for skin pathologies such as psoriasis (Mercurio et al., 2020) and pigmentary disorders (Bellei and Picardo, 2020), as well as during obesity - a condition which drives both damage and expansion signalling (Aoki and Murase, 2019). During the transition of research on the characterization of cellular senescence from in vitro to in vivo conditions, skin was one of the first organs to be investigated. This was likely due to the fact that skin samples from bigger mammals can be easily excised; in fact, skin is the source of dermal fibroblasts, the cells which represent the predominant model for senescence research in vitro. For example, Dimri et al., assessed skin samples from human head for activity of β-galactosidase under sub-optimal pH (SA-β-gal), which has been found to show a difference between dermal fibroblasts of early and late passages (Dimri et al., 1995). While the study identified several skin structures and appendages positive for SA-β-gal staining (including hair follicles and sweat glands), an age-dependent increase in the number of SA-β-gal-positive cells was observed only in epidermis and lower dermis. Specifically, the observed skin cell types were identified as keratinocytes and dermal fibroblasts respectively (Dimri et al., 1995). Since then multiple studies have confirmed these findings (Lewis et al., 2011; Ressler et al., 2006).

Several years later, another study assessed skin and muscle of nonhuman primates (baboons) and showed an age-dependent accumulation of cells positive for a senescence marker: DSBs colocalizing with signal from telomeres, termed telomere-induced foci (TIF) (Herbig et al., 2006; Jeyapalan et al., 2007). The study showed an exponential increase in the number TIF-positive cells in skin, but in contrast found an overall low frequency of TIF-positive cells in muscle, with no age-dependent increase (Herbig et al., 2006; Jeyapalan et al., 2007). Another study has proven that both of the above senescence markers (SA-β-gal and DSBs) can be found not only in skin of ageing nonhuman primates, but also in mice (Wang et al., 2009). Several other studies have shown an age-dependent increase in markers of senescence including a high level of oxidative damage, inflammatory phenotype and elevated expression of cell cycle inhibitors, among others (Wang and Dreesen, 2018).

Based on the evidence of a correlation of cellular senescence with both age and age-related-disease, it has been hypothesised that cellular senescence causally contributes to age-related skin dysfunction (Gruber et al., 2020a; Waaijer et al., 2012). In summary, the recognition of the significance of cellular senescence in skin ageing has progressed in the past decades creating a therapeutic niche for development of drugs targeting dermal senescent cells.

Another historical angle on the characterization of skin senescence in vivo pertains to wound healing. Since the late 90's and mid-00's, an elevated level of senescence markers has been reported for chronic wounds including pressure, diabetic and venous ulcers (Mendez et al., 1998; Stanley and Osler, 2001; Tomic-Canic and DiPietro, 2019; Vande Berg et al., 2005; Wilkinson et al., 2019). This has been linked to several known features of senescent fibroblasts, including high expression levels of metalloproteinases (MMPs) and their inhibitors (TIMPs), proteins which are causally involved in skin healing (Telgenhoff and Shroot, 2005). It thus came as a surprise that senescent cells can also be responsible for efficient skin regeneration and that their elimination results in a significant delay of the wound healing process (Demaria et al., 2014). To tackle this contradiction a hypothesis was devised: senescent cells arising at the early stages of skin regeneration (acute senescence) contribute to the process of healing, while complete wound closure requires an elimination of senescent cells by the immune system (Wilkinson and Hardman, 2020). If elimination fails, senescence becomes chronic and by sustaining inflammation of the wound environment it prevents skin from returning to its anatomical integrity in a timely fashion. Thus, the impact of cellular senescence on skin anatomy and function is pleiotropic and often depends on which type of cells have undergone transition to senescence.

Section snippets

Cellular senescence: induction and heterogeneity

Stressors inducing senescence include telomere exhaustion from repeated cell divisions (replicative senescence), various forms of damaging factors, such as ionizing radiation (IR), UV light, genotoxic drugs and activation of specific oncogenes (oncogene-induced senescence (OIS) (Munoz-Espin and Serrano, 2014). The common feature of many of these factors is that they lead to DNA damage, which if not cleared, will elicit a prolonged DNA damage response (DDR) (Di Micco et al., 2021). The

Relationship between cellular senescence and the immune system of skin

While the subject of the immunity in skin has already been extensively explored (Chambers and Vukmanovic-Stejic, 2020), little is known about the interaction between senescent cells and the immune system. In general, ageing leads to a change in skin structure that is associated with a decline in its anti-microbial properties. The thinning of the epidermis renders the skin more exposed to bacteria, which constantly stimulates the innate immune system, especially macrophages (Chambers and

Cell types showing a signature of cellular senescence in the skin

The skin consists of an exterior stratified epidermal layer containing epithelial cells and an inner dermal layer containing mostly mesenchymal cells. Below the dermis, a layer of white adipose tissue containing adipocytes forms the hypodermis. These layers cooperate to form the skin's appendages, including hair follicles, sweat glands, sebaceous glands, and nails. Cooperation is further needed to sustain the skin's main functions as the physical and biological barrier to the outside, an immune

Human skin equivalents as in vitro models of skin structure in senescence research and senolytic development

When advancing in vitro research in the field of skin ageing and related conditions, investigators are confronted with several drawbacks of the commonly used in vivo models. Rodents are the most popular experimental animals in the field of ageing research but are of limited use when investigating the effects on skin due to the fundamental differences in skin structure between rodents and humans. The murine epidermis consists of only three layers of keratinocytes compared to six to ten layers in

Advances in the development of senolytics for skin conditions

In a follow-up of a study that provided evidence on the clearance of senescent cells as a measure to extend the healthspan and the average lifespan of mice (Baker et al., 2011), the search for senolytics embarked. The first senolytic strategies exploited the pro-survival networks of senescent cells, which are collectively called senescent cell anti-apoptotic pathways (SCAPs), by targeting PI3K/AKT, p53, serpines receptor/tyrosine kinases (Baar et al., 2017; Zhu et al., 2015) and members of the

Methods for sample collection in senescence research and senolytic development

An important aspect of senescence research on skin and the use of senolytic drugs is the lack of a robust tool for sample collection in a minimally invasive fashion to measure senescence markers in the skin. Classic methods using histology and biochemistry are affected by the low reliability of specific senescence markers that can be displayed by cells before cell cycle arrest establishment (Ogrodnik, 2021), and thus quantitative assessment of senescence, especially in vivo can be challenging.

Concerns about the usage of senolytic drugs for targeting senescence in the skin

As described above, an increase in the number of senescent cells has been reported not only for conditions related to damage accumulation and skin pathology, but also for processes of increased expansion signalling such as development and healing. In fact, high expansion stimulation of skin is maintained during human growth and development and present in conditions of skin stretching, such as obesity (Black et al., 1971; Choo et al., 2010) and pregnancy (Cordeiro et al., 2010). As the

Inhibition of negative aspects of skin cell senescence with senomorphic therapies – a viable alternative to senolytic drugs?

Therapies that mitigate fundamental properties of senescent cells and are referred to as "senomorphics" could potentially alleviate safety concerns related to the removal of senescent cells by senolytics. By targeting the SASP, for example, many of the negative attributes of senescence and ageing will be abrogated.

One of the most promising targets for senomorphic therapies is the mTOR pathway, which regulates cell growth, metabolism, protein synthesis, and autophagy in response to nutrients.

Declaration of Competing Interest

Authors declare no conflict of interest.

Acknowledgements

Research Group Senescence and Healing of Wounds is a collaboration between the Ludwig Boltzmann Gesellschaft GmbH and the Austrian Workers' Compensation Board (AUVA).

Support by the Federal Ministry for Digital and Economic Affairs of Austria, by the National Foundation for Research, Technology and Development of Austria and by CHANEL Fragrance Beauty, Research & Innovation to the Christian Doppler Laboratory for Biotechnology of Skin Aging and the Christian Doppler Laboratory for Skin

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      However, any loss of full-thickness skin more than 4 cm diameter in case of deep injuries and burns can't be restored totally, leading to a chronic wound, and thus needs grafting or other adequate treatment. [4] Surgical procedures available for skin wound repair often suffered limited availability of healthy donor tissue, potential infection and immune rejection risk of foreign tissue, as well as several limitations from the commercial skin substitutes (i.e., Integra®, Apligraf®, Biobrane®, Pelnac®, Dermagraft®, AlloDerm®, Epicel®, etc.), like reduced vascularization, poor mechanical integrity, failure to integrate, scarring, and immune rejection. [5–7] In particular, wound healing with varying degrees of scar contracture and deformity not only limit the flexibility of new skin but also affect the appearance and life quality of patients. [8,9]

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