Cellular Senescence: Ageing and Androgenetic Alopecia Free

Androgenetic alopecia (AGA), also called male pattern baldness, is the most common type of hair loss, especially involving the frontotemporal area and vertex of the scalp in men and over the crown in women [1]. It features a progressive miniaturization of hair follicles, which has long been thought to be driven by androgen and leads to a vellus transformation of terminal hair. This premature baldness can have a definite negative impact on the self-image and even cause psychological disturbances such as anxiety, depression, and distress in these patients [2]. Although research has shown that the occurrence and development of AGA depend on the interaction of endocrine factors and genetic predisposition, the exact pathogenesis of AGA remains to be clarified, limiting available treatments [3]. In recent decades, increasingly, data proposed that ageing and AGA are intimately linked.

Firstly, the incidence and prevalence of AGA do increase with age, although they are not confined to older people [4‒8]. In Singaporean males, the prevalence of AGA was reported from 32% at 17–26 years to 100% after 80 years [9]. Histologically, follicular miniaturization occurs in both AGA and senescent alopecia as they undergo an increased latency period between the hair growth cycles [10, 11]. Whiting even suggested that hair loss in elderly subjects is actually androgen driven [10]. Besides, patients with premature AGA were found to be susceptible to prostate cancer (PCa), cardiovascular diseases, metabolic syndrome, insulin resistance, diabetes mellitus, and hypertension [12, 13], indicating that AGA itself might represent a pro-ageing state.

As a central hallmark of ageing, senescence is a cell fate that occurs in response to damage such as telomere damage, DNA damage, and mitochondrial dysfunction in ageing and involves growth arrest, resistance to apoptosis, and secretion of a host of proinflammatory factors [14]. Interestingly, the capacity of hair follicles to regenerate was not lost (except in cases of follicle destruction) in AGA; neither does the follicle cycling. Instead, the regenerated hair cycles are compromised by extended telogen and shortened anagen phases [10]. The same feature occurs not only in AGA but also in hair loss caused by premature ageing, such as senescent alopecia, Werner syndrome, and Hutchinson-Gilford progeria [15, 16]. All the above data suggested a mechanism of AGA related to cellular senescence. This review aimed to provide dermatologists with an overview of the biological links between ageing and AGA, specifically focussing on cellular senescence, and discuss the therapeutic potential of targeting cellular senescence in AGA.

The hair follicle is composed of epithelial and dermal cells from the two dermal layers. Hair follicle morphogenesis results from directed movement and differentiation of these cells attached to the stroma induced by relevant signals. During hair follicle morphogenesis and periodic hair growth, HFSCs (hair follicle stem cells) and dermal papilla cells (DPCs) play a leading role [17, 18]. Throughout the literature, there was directly and indirectly increasing evidence of senescent HFSCs and DPCs associated with the development of AGA.

DPCs are hair follicle-derived cells, forming dermal papilla located at the base of the hair follicle, in which a large proportion of androgen receptors (ARs) were located [19]. Therefore, DPCs are the action sites of dihydrotestosterone (DHT), the most active androgenic compound on hair growth, which mediates the stimulating signals by secreting numerous growth factors and/or extracellular matrix factors [20, 21]. Generally speaking, the released compounds from DPCs include hair growth-stimulating factors, such as vascular endothelial growth factor and insulin-like growth factor 1, and inhibitory factors, such as transforming growth factor-beta 2 (TGF-β2) and Dickkopf 1 (DKK-1) [1, 21]. A DHT-induced alteration will cause progressive miniaturization of hair follicles, thereby shortening the anagen phase and extending the telogen phase, consequently leading to a bald appearance [1, 19, 22].

Recent studies showed that DPCs from the balding scalp of AGA patients underwent premature senescence in vitro, rapidly showing senescent phenotype and high levels of p16INK4α/pRb protein induced by AR overexpression [23, 24]. In contrast, DPCs from the non-bald scalp required several passages of culture in vitro before they gradually developed the senescent phenotype and subsequently lost the ability of hair follicle induction [23, 25, 26]. Interestingly, DPCs isolated from hair follicles of p53 knockout mice could be continuously cultured for 29 generations in vitro, and no senescence phenotype appeared in any generation [27]. In fact, prematurely senescent DPCs still preserve key dermal papilla signature gene expression but lose the ability to induce new hair follicles and, instead, promote epidermal differentiation while inhibiting follicular differentiation. Huang WY et al. [28] found that senescent DPCs could produce more interleukin IL-6, inhibiting both proliferation of follicular keratinocytes and colony formation of HFSCs, as well as blocking telogen to anagen transition. This result agreed with previous reports that the conditioned medium from balding DPCs delayed the onset of anagen in mice [29], and overexpression of IL-6 under the control of keratin 14 (K14) promoter caused retardation of hair growth in transgenic mice [30].

Although no articles confirmed the causal relationship between the senescence of HFSCs and AGA occurrence, there were some indirect hints that aged HFSCs displayed an impaired ability to enter the hair growth phase, hair growth signalling of Wnt inhibition in ageing HFSCs, DNA damage triggered HFSC ageing leading to hair follicle miniaturization, and changed subsets of HFSCs found in AGA. Future research should focus on whether aged HFSCs increased in AGA and the underlying mechanism of aged HFSCs promoting AGA development.

The HFSCs are thought to be localized in the bulge and the outermost layer of the outer root sheath [31]. Those stem cells within the hair bulge display a high degree of plasticity, contributing to hair regeneration, which remains roughly constant normally [32]. Upon proper stimuli, quiescent HFSCs become activated to drive anagen onset and begin a new hair cycle [33]. For example, under the joint action of a variety of complex signalling pathways represented by the Wnt signalling pathway, HFSCs are activated to become “transient amplification cells” to proliferate, differentiate, and transmit signals to initiate hair growth [34]. On the other hand, aged hair follicles typically display impaired ability to enter the hair growth phase [34]. Aged HFSCs are eliminated cyclically from the skin through terminal epidermal differentiation, resulting in prominent ageing characteristics such as greying, thinning, and hair loss [35]. In fact, whether the number of HFSCs changes with age still remains controversially discussed [35‒37], while there is consensus that the function of aged HFSCs is reduced.

Wnt signalling has long been thought to drive hair cycle induction and maintenance [38], and the persistent activation causes DNA damage response in HFSCs [39], whereas inactivation of Wnt signalling has been implicated in the pathological process of many alopecia diseases such as AGA [40]. To investigate the possible involvement of Wnt signalling in HFSC ageing, Matsumura H et al. performed gene set enrichment analysis for telogen HFs (tHFSC) from young versus aged mice and found that Wnt signalling is less activated in aged tHFSCs than in young tHFSCs [35]. Moreover, expression of canonical Wnt target genes like Axin-2, Lef-1, Lgr-6, and c-Myc was decreased in HFSCs with ageing. Upon activation of canonical Wnt signalling, β-catenin stabilizes and translocates to the nucleus to initiate transcription of Wnt target genes. The nuclear localization of β-catenin was reduced in aged HFSC, and the canonical Wnt signalling was antagonized by non-canonical Wnt5a-Cdc42 signalling [41]. It was reported that the activity of Cdc42 can be specifically inhibited by CASIN (Cdc42 activity-specific inhibitor) [42, 43]. Aged HFSCs treated with CASIN presented with a youthful percentage of HFSCs polar for the distribution of Cdc42 as well as numb. Aged CASIN-treated HFSCs also presented with increased nuclear localization of β-catenin and elevated expression of Axin-2. By HE staining of the black area of the back skin, anagen areas (black patches) were about 3-fold more frequent in CASIN-treated aged mice in comparison to untreated ones. In addition, the duration of telogen in aged CASIN-treated animals was reset to the duration of telogen in young (D-50) mice [37].

It was suggested in a previous study that HFSC ageing is not simply explained by altered Wnt signalling. Yet, Wnt signalling is likely to indirectly mediate the accumulation of DNA damage via replicative stress in HFSCs [35]. Accumulation of DNA damage has been implicated in tissue ageing [44, 45]. In other domains, the induction of DNA double-strand breaks in the mouse liver has been reported to cause characteristic tissue senescence [46]. The DNA damage markers, including heat shock protein-27 (HSP-27), superoxide dismutase (SOD) catalase, ataxia-telangiectasia-mutated kinase (ATM), and ATM- and Rad3-related protein, were upregulated in the AGA scalp [23]. In vivo fate analysis of HFSCs revealed that the DNA damage response in HFSCs causes proteolysis of type XVII collagen (COL17A1/BP180), a critical molecule for HFSC maintenance, to trigger HFSC ageing, characterized by the loss of stemness signatures and epidermal commitment. Aged HFSCs are cyclically eliminated from the skin through terminal epidermal differentiation, causing hair follicle miniaturization. The ageing process can be recapitulated by Col17a1 deficiency and prevented by the forced maintenance of COL17A1 in HFSCs, demonstrating that COL17A1 in HFSCs orchestrates the stem cell-centric ageing program of the epithelial mini-organ [35].

Lineage analysis showed that the hair follicle structure originated from integrin α6, β1, β4, CK14, CK15, and CK19 are the surface markers of carina stem cells. In addition to that, it is also characterized by upregulated CD200 expression and downregulated CD34 expression [47, 48]. In the case of AGA, KRT15-positive bulge stem cells are retained. In contrast, CD200- and CD34-positive hair progenitor cells are diminished, suggesting the presence of a defunct stem cell population, whereby the activation and conversion of bulge cells into a more proliferative state are impaired [49]. Luis A.G et al. [49] found that the number of HFSCs in AGA patients did not decrease, but the reduction of CD200- and CD34-positive HFSCs with the ability to differentiate into hair follicles caused their hair loss. It may be related to the affected microenvironment for the survival of HFSCs in these parts. Studies have found that in AGA patients, hair follicles in the alopecia area have mild chronic inflammation and surrounding fibrosis [50], which may affect the microenvironment for the survival of HFSCs.

Ageing is a necessary and progressive stage in metabolism, of which the hallmarks include mitochondrial dysfunction, cellular senescence, altered intercellular communications, and loss of proteostasis [51]. On the other hand, the production and elimination of reactive oxygen species (ROS) are kept in balance during normal growth, which is affected by the function of the antioxidant system. Under pathological conditions, oxidants exceed the clearance capacity of the antioxidant system, and the balance is disturbed, leading to the excessive production of ROS, which is called oxidative stress, resulting in damage to lipids, proteins, membranes, and DNA. In addition to oxidative stress, the more important function of ROS is to act as an information molecule to regulate cellular functions such as cell growth, transformation, apoptosis, transcription, and senescence through oxidative modification of proteins [52, 53]. In 1956, Harman first proposed that free radicals are likely the key factor in the ageing process [54]. The activity of free-radical scavenging enzymes diminishes with advancing age and contributes to the adverse effects of oxidative stress with ageing [55]. Free radicals, including ROS and reactive nitrogen species (NO), are crucial to cellular damage. ROS are the main free radicals that cause cell damage, while most ROS are produced by mitochondria (mt), which are the centres of cell respiration and oxidation [56]. In senescent cells, a decrease in mitochondrial efficiency leads to increased ROS production to maintain ATP production.

In support of the opinion that oxidative stress is an inducer of ageing, experts mentioned that treatment with antioxidants can prolong life spans and benefit ageing-related diseases [52]. A low dose of dietary supplement with antioxidants partially mimics the effects of caloric restriction and delays ageing in mice [57]. It is well known that long-term caloric restriction can reduce the rate of mtROS generation and attenuate the effects of ageing on different tissues [58, 59]. On the other hand, accumulating evidence in recent years has proved that AGA is a common stressful form of hair loss partly caused by exposure to oxidative stress, which could be considered an important link between ageing and AGA.

It was found that oxidative stress could induce hair follicles to enter the catagen in advance, inhibit hair growth, promote hair follicle keratinocyte apoptosis in a dose-dependent manner, and participate in the pathogenesis of AGA [60, 61]. ROS is a key promoter of oxidative stress [62]. During the normal metabolism of hair follicles, mitochondria produce ROS to maintain the normal operation of their signalling pathways [63]. ZHU Hong-liu reported that the ROS and MDA levels in AGA mice significantly increased [64]. Bahta et al. [23] even showed that balding DPC in AGA patients expressed various markers of oxidative stress and DNA damage, such as HSP-27, catalase, ATM, and the ATM- and Rad3-related protein (ATR). It suggested that balding DPC was more susceptible to oxidative stress than non-balding DPC, with ROS increasing hair growth inhibitor (TGFβ1/TGFβ2) secretion and DPC senescence [61]. A series of experiments by Upton et al. [61] confirmed that oxidative stress is an important factor in the premature senescence of DPCs in the bald area of patients with AGA. In turn, senescent cells also develop the senescence-associated secretory phenotype (SASP), thereby secreting abnormal levels of interleukins (ILs), matrix metalloproteinases, monocyte chemotactic proteins, and growth factors and inducing inflammation and senescence in neighbouring cells [65, 66]. It can be seen from above that ROS in AGA microenvironment contributes to DPC senescence which in turn promotes the accumulation of oxidative stress products, and this vicious cycle would indeed affect the conduction of hair growth signals and consequently result in hair miniaturization.

Besides increased oxidative stress, antioxidant defence indicators such as total antioxidant activity, SOD activity, native thiol, total thiol, and disulfide levels were significantly lower in AGA than in the healthy control [67‒69]. Oxidative stress arises not only from an imbalance in the formation of ROS but also from the limited endogenous defence systems, including enzymatic and nonenzymatic antioxidants. SOD2 is one of the main mitochondrial antioxidant proteins. The genetic ablation of SOD2 leads to early postnatal death in mice [70]. Treating SOD2 knockout mice with a synthetic SOD mimetic can rescue their mitochondrial defects and prolong their survival [71]. Besides, mice lacking SOD1 can survive but have numerous pathological changes and a reduced life span [72]. A diet rich in the building-block nutrients of antioxidant enzymes, including manganese, zinc, and copper, called cofactors for SOD, has beneficial effects on delaying ageing. Interestingly, the disturbed metabolism of copper and zinc was observed in the serum, urine, and hair of the patients with AGA [73]. Moreover, the Mediterranean diet, which means eating plenty of fresh vegetables such as carrots, tomatoes, and fresh herbs, is good for reducing the risk of developing AGA. This may be because of the antioxidant and anti-ageing properties of phytochemicals present in plant foods [74].

Various factors have been identified to affect and alter the regenerating hair follicle with advancing age. Androgen is the most extensively studied regulator of the hair follicle, which can transform the short, thin, vellus hair into long, thick, black hair and change a terminal follicle into a vellus follicle in contrast [75]. In the pathogenesis of AGA, androgen is also thought to be the known important causative factor. The main circulating androgen in men is testosterone. However, AGA appears more related to the most active androgenic compound, namely, DHT, which could translocate the AR from the cytoplasm to the nucleus through binding to the AR, functioning as a transcriptional factor to control the expression of target gene [76]. The sensitivity and distribution of AR vary with anatomic location, resulting in increased hair in the pubic and armpit areas and decreased hair on the scalp vertex [77]. It is reported that the expressions of AR and DHT in the balding DPCs were higher than in the non-balding DPCs, and AR-5α-DHT compounds and their trans-activation activity are reasons for the miniaturized hair follicles in AGA [19]. In parallel, the balding DPC also showed the senescence manifestations such as changes in cell morphology, expression of senescence-related β-galactosidase, and upregulation of p16^INK4a/pRb [23, 61]. In fact, DHT induces premature ageing of DPCs in balding and non-balding frontal scalp migratory areas but not beard DPCs [78]. These implied some unveiled relationship between the elevated androgen, AR, and cell senescence in AGA, which has been extensively investigated in other ageing-related diseases.

Previous research has established that AGA might be a sign of increased risk of early PCa, an ageing-related disease promoted by androgen and AR [79‒81]. In PCa, the link between tumour suppressor proteins and AR-induced cellular senescence has long been discussed. AR-induced cellular senescence is associated with increased localization of PML (promyelocytic leukaemia) tumour suppressor to senescence-associated heterochromatic foci [82]. Additionally, AR also interacts with the tumour suppressor genes ING1 and ING2, leading to cellular senescence [83]. ING1b inhibits PCa cell proliferation and migration and induces cellular ageing by interacting with AR and decreasing the expression of the AR target gene in both androgen-dependent and castration-resistant PCa cells [84]. Importantly, the AR-p16^INK4A-pRb tumour suppressor pathway seems essential for AR-mediated cellular senescence in PCa [85].

Surprisingly, PCA was reported to exhibit a biphasic growth response according to androgen concentrations [85‒87]. Low physiological androgen levels are associated with increased PCa risk, whereas supraphysiological androgen levels inhibit PCa growth [85]. Besides AR antagonists, androgens at supraphysiological androgen levels are used in clinical trials as so-called bipolar androgen therapy of PCa [85, 88, 89]. Interestingly, both could trigger cellular senescence in PCa in cell culture and ex vivo tumour samples [85]. As a response to cancer therapies, senescent growth arrest of PCa cells induced by androgen deprivation therapy leads to stable growth abrogation, as well as the potential for immune system activation via SASP, and consequently results in tumour shrinkage [83, 90‒94]. For example, atraric acid, a natural AR antagonist, has been reported to induce cellular senescence in PCa cells by the p16/pRb involving the Src/Akt signalling [90]. As a first-generation AR antagonist, bicalutamide treatment could also induce the expression of both cyclin-dependent kinase inhibitors p16^INK4A and p27^KIP1 and cellular senescence in PCa cell lines [83]. There were findings that both AR antagonist enzalutamide and darolutamide increased SA-β-Gal positive cells in both LNCaP and C4-2 cell lines and the expression of CDKN2A encoding p16 [92]. On the other hand, supraphysiological levels of androgens have also been demonstrated to induce cellular senescence through Src-AKT and AR-lncRNASAT1-AKT-p15^INK4b signalling axis, consequently leading to the inhibition of PCa cell proliferation [85, 87]. Moreover, senescence has also been thought to be another specialized cell state that can contribute to cancer progression or recurrence, resulting in unfavourable treatment outcomes such as castration resistance in PCa [95].

It is well known that the incidence of many cancer types strongly correlates with ageing, which was thought to be linked with stromal fibroblast senescence and concomitant cancer-associated fibroblasts (CAFs) [96]. It was found that AR expression is downregulated in dermal fibroblasts under precancerous skin cancer lesions and in CAF of three main skin cancer types: squamous cell carcinomas, basal cell carcinomas, and melanomas. Downregulation of AR has been reported to induce the early steps of CAF activation, accompanied by induction of CAF effector genes and P53-dependent cellular senescence [97]. An in vitro cell model transfecting AR into thyroid cancer cells showed that adding DHT to the cells leads to the increase of the cell cycle inhibitors, p27 and p21, and induced a G1 arrest in thyroid cancer cells [98]. When AR was transduced into AR-negative oesophageal cancer cell lines, DHT could inhibit the proliferation of oesophageal cancer cell lines by inducing cell cycle arrest and senescence [99]. Interestingly, immune senescence was possibly affected by age-related decline in androgen levels [100]. It was found that the frequency of naive CD4 and CD8 T cells was decreased, while the frequency of memory T cells was increased in aged macaques which were not supplemented with androgen. However, the frequency of naive and memory T cells held steady and had a smaller increase in inflammatory cytokine levels [101].

Above all, all of the androgen, AR, and AR antagonists could induce senescence in different microenvironments through distinct molecular mechanisms, leading to contradictory outcomes of disease producing, therapeutic effect, and treatment resistance. In future studies, more evidence is needed to determine whether DHT and AR induced senescence in AGA pathogenesis and its underlying mechanism.

Although cellular senescence can be beneficial in certain circumstances, for example, in the defence against cancers, it is the aetiology of numerous diseases such as diabetes, idiopathic pulmonary fibrosis, and cardiovascular disease, for which disrupting or preventing senescence could be served as the promising treatment [102‒104]. So far, there are two types of anti-ageing drugs. One approach would be targeting the SASP without killing senescent cells, including the mechanistic target of rapamycin complex 1 (mTORC1), JAK1/JAK2, STAT3, NF-κB, p38, and mitochondrial dysfunction [105]. Such SASP inhibitors include rapamycin, ruxolitinib, and metformin [105]. The other approach is referred to as senolytics and works by selectively eliminating senescent cells, which includes the combination of dasatinib, an FDA-approved tyrosine kinase inhibitor, and quercetin, a flavanol present in many fruits and vegetables (D + Q) [105‒107]. Although the molecular mechanism of ageing and senescence was not well unveiled in AGA pathogenesis, there was increasing literature showing the antioxidant and anti-ageing effects of drugs in treating this disease.

Quercetin, well known as an effective senolytics, has been considered the main active ingredient among Chinese herbal formulas for the treatment of AGA [108‒110]. In DHT-induced DPCs and AGA mouse model, cyanidin 3-O-arabinoside could also effectively reduce the mtROS accumulation in DPCs and reverse the cellular senescence through inhibiting the expression of p38-mediated voltage-dependent anion channel, consequently restoring the DHT-induced hair growth deceleration [111]. Besides, both arctiin and troxerutin pretreatment were reported to have a protective effect on H2O2-induced senescence in human DPCs in vitro [112]. In turn, the above identification of senolytic drugs presents an opportunity to directly test the mechanisms by which senescence is involved in AGA pathogenesis.

AGA, which is attributed to genetic and androgen predisposition and increases with age, has widespread physical and mental impacts, and novel strategies to improve AGA are needed. The evidence for the role of cellular senescence in AGA is growing; however, how senescence impacts AGA remains to be determined. Elevated senescent cell burden and androgen and oxidative stress-induced senescence mechanisms that occur in ageing may be initial targets to improve AGA. Developing methods to inhibit DPCs and HFSCs senescence driven by androgen and ROS during AGA may prove to be a beneficial therapeutic approach to rescue hair follicle miniaturization. Senescent cell removal and SASP reduction are potential therapeutic strategies for improving AGA.

There is a link between AGA and cellular senescence. However, how senescence impacts AGA remains to be determined.

The authors have no conflicts of interest to declare.

This study was funded by the project of Southwest Medical University (Grant No. 2021ZKMS030) and Sichuan Medical Association (Grant No. S21022).

Y.Q.D. and M.X.W. are joint first authors and equal contributors to the work. Y.Q.D., M.X.W., Y.X.H., F.M.L., L.N.C., and X.X. all contributed to drafting and revision of the manuscript and approved the final version for submission.