The molecular signature of cellular senescence

Cellular senescence is defined by a complex molecular signature, including a pool of cellular markers. From this pool, different cellular markers have been used to define and demask cellular senescence, depending on histological specificity, preservation modality, and the dichotomy of in vitro versus in vivo analytical frameworks [22]. Senescent cells have been demonstrated to display an overexpression of endogenous lysosomal beta-galactosidase, termed senescence-associated beta-galactosidase (SA-βgal) [23]. For example, Xu et al. transplanted 1 × 106 radiation-induced senescent (SEN) pre-adipocytes into 6-month-old mice by intraperitoneal injection. This transplantation led to a marked decrease in physical functionality—such as reduced maximal walking speed, hanging endurance, and grip strength—within just one month post-transplantation, compared to controls receiving non-senescent (CON) cells. The study further showed that even smaller doses of SEN cells in older mice similarly induced physical decline and were associated with reduced survival. Interestingly, the functional decline correlated with increased systemic levels of SA-βgal+ and p16Ink4a mRNA levels. p16INK4A is a cyclin-dependent kinase inhibitor that serves as a cell cycle regulator. Moreover, the introduction of senolytic drugs, dasatinib plus quercetin, effectively cleared senescent cells, reduced proinflammatory cytokine secretion in human adipose tissue explants, and improved physical function and survival in both senescent cell-transplanted and naturally aged mice [24]. Paralleling with other regulators such as p21Cip1 and p53, the accumulation of p16INK4A+ cells has been correlated with senescence [25]. More precisely, p16INK4A and p21Cip1 have been shown to be upregulated in skin and cardiac tissue of aging mice. In a supporting manner, transplanting murine senescent pre-adipocytes has been found to increase the number of p16INK4A+ cells in the recipient mice [18]7/19/2025 8:01:00 AM. The independent upregulation of p16 and p21Cip1 has been investigated through the generation of a human dermal fibroblast (HDF) cell strain with a fluorescent reporter for p21. This research revealed that as cells approach senescence, the expression of p16 and p21, alongside SA-βgal, increases, with a corresponding decrease in DNA synthesis as indicated by BrdU incorporation [26]. Notably, Herbig et al. demonstrated that, at 10 remaining population doublings (RPD), 79% of cells were actively cycling and 80% were negative for both p16INK4A and p21Cip1 expression. As the cells progressed closer to senescence (reaching two RPDs), the fraction of non-cycling cells rose from 21 to 63%, yet 70% of these cells remained positive for only one of p16INK4A or p21Cip1, highlighting the need for moving beyond isolated towards holistic analysis of cellular senescence [27]. Further research disclosed that, while approximately 10% of early passage cells exhibited γ-H2AX foci, this percentage increased to 90% in senescent cells. Conversely, the immortalization of cells with telomerase (hTERT) led to a marked decrease in γ-H2AX-positive cells, dropping to 7% [28]. Therefore, γ-H2AX foci can also help determine cellular senescence. In addition, various cytokines such as IL‐1α, IL‐1β, IL-6, IL-8, and TNF-α can define senescent cell states [29]. For example, experimental murine models showed that transplantation of senescent renal scattered tubular-like cells not only led to increased creatinine levels and renal tissue hypoxia but also induced IL-6 gene expression [30]. Besides cytokines, CXCR2, IGF2, chemokines, and metalloproteases have been used to determine cellular senescence [31].

Molecular, genomic, and cellular senescence drivers

Since the seminal findings by Hayflick and Moorfield in 1961, research has identified different drivers of senescence, including molecular, genomic, and cellular triggers [32]. For example, DNA damage has been proposed as a leading cause of cellular senescence [33]. More precisely, in actively dividing cells, the progressive shortening of telomeres has been demonstrated to culminate in the exposure of the chromosome's unprotected end. This so-called “replicative senescence” or “telomere-induced senescence” triggers an enduring DNA damage response [34]. The DNA damage sensor protein, ATM, binds to these uncapped telomeres. This results in the stabilization of the tumor suppressor protein p53 and the increased activity of the p53-regulated gene p21Cip1. Consequently, p21Cip1 inhibits the activity of CDK2, crucial for preventing the deactivation of the Rb protein, blocking the cell’s progression into the S phase of its cell cycle, ultimately hindering DNA replication [35]. Interestingly, recent studies on bone marrow progenitor cells have highlighted that reoxygenation stress also activates the ATM-p53-p21Cip1 pathway, leading to a marked increase in cellular senescence. For example, Zhang et al. showed that reoxygenated bone marrow cells demonstrated a significant increase in ATM and p53 activation, with ~ 20% of stainings being positive for ATM-Ser1981 and p53-Ser15. Similarly, 22% of these cells expressed p21, revealing reoxygenation stress as another driver of DNA damage and cellular senescence [36]. Additionally, UV light, gamma irradiation, chemotherapy drugs (e.g., cyclophosphamide), and overactive oncogenic Ras proteins also cause DNA damage, leading to activation of the ATM-p53-p21Cip1 pathway [37]. Conversely, the p53-p21Cip1 pathway can be induced not only by responses to DNA damage but also through the expression of the p53 stabilizing protein p19Arf (known as p14 in humans), loss of the tumor suppressor protein PTEN, overexpression of the S phase transcription factor E2F3, or the upregulation of oncogenic Ras in human breast cells [38]. Further, inflammatory pathways have been identified as senescence drivers. Pro-inflammatory cytokines like IL-6 and TNF-α activate NF-κB signaling, promoting the transcription of SASP genes [39]. This phenotype is defined by different pro-inflammatory and tissue-destructive molecules, including a broad spectrum of chemokines, cytokines like IL-1α, IL-1β, and IL-8, the canonical inflammatory factor IL-6, and growth factors such as IGF2. SASP factors, especially IL-1β, IL-8, and MCP-1, create an autocrine loop reinforcing cellular senescence [40]. Moreover, hypoxia has been demonstrated to mediate senescence via the IL-6 axis in murine renal tissue hypoxia models. It is noteworthy that transplant surgery itself may also drive senescence through ischemia–reperfusion injury and the mechanosurgical trauma, among other pathways [41]. In experimental liver transplant models, for example, cellular senescence has been shown to be induced during organ retrieval and further exacerbated during static cold storage [2]. Other research has shown that cooling of cultured cells alone leads to increased production of reactive oxygen species (ROS), which cause double-strand breaks in DNA, subsequently inducing cellular senescence. These findings could be resembled when applying a standard clinical kidney transplant procedure to porcine kidneys [3]. In a murine cardiac IRI model, increased ROS levels mediated senescence of both cardiomyocytes and interstitial cell populations, contributing to impaired cardiac function and adverse tissue remodeling [4].

The effects of modulating senescence in solid organ transplantationReducing and dampening rejection reactions

Donor age has long been described as a significant risk factor for adverse outcomes after organ transplantation including delayed graft function and rejection [42, 43]. Mechanistically, this has been linked to increased immunogenicity of older organs. On a cellular level, high numbers of senescent cells have been found in old donor organs [13]. It has been shown that senescent cells are highly effective in triggering dendritic cells (DC) and antigen-specific CD8+ T cells [44]. In addition, impaired monocyte clearance of damaged necrotic cells has been observed with aging and may fuel inflammatory responses [45]. Ischemia–reperfusion injury (IRI), an event inevitably linked to transplantation, is also more pronounced in old organs. IRI has been shown to promote sterile inflammation by upregulating pro-inflammatory cytokines and inducing the formation of ROS that cause mitochondrial dysfunction [13]. A major cytokine released by senescent cells upon IRI is cell-free mitochondrial DNA (cf-mtDNA), which further promotes age-specific inflammatory responses. Congruent findings come from clinical studies of kidney transplant recipients, which showed that increased levels of mt-DNA had been linked to higher rates of acute rejection and delayed graft function [46]. Administering senolytics to old donor animals led to an attenuated cf-mtDNA increase after transplantation, subsequently dampening immune responses and promoting allograft survival in experimental models [18].

Leveraging synergistic effects with standard immunosuppressants

In addition to beneficial effects by directly targeting senescent cells and their products, synergistic effects of senolytics and standard immunosuppressive agents have been described. Calcineurin inhibitors such as tacrolimus and CsA are widely used immunosuppressants in SOT. Mechanistically, these drugs inhibit calcineurin phosphatase activity by binding to calcineurin, thereby preventing activation of nuclear factor of activated T cells (NFAT). This transcription factor, in turn, initiates IL-2 gene transcription, which induces T cell activation48. Interestingly, panabinostat, a senolytic drug and histone deacetylase inhibitor used in multiple myeloma therapy, has also been shown to target calcineurin. When tested in vivo, cotreatment with panabinostat and tacrolimus resulted in enhanced anti-tumor effects, thus suggesting synergistic effects when used for immunosuppression [47]. Furthermore, the tyrosine kinase inhibitor dasatinib has been observed to interfere with T cell receptor signaling by targeting the Src family. Moreover, additive effects with glucocorticoids such as dexamethasone on T cell suppression have been observed [48,49,50]. In addition, flavonoids such as quercetin and fisetin have been shown to inhibit the mTOR pathway, thus implying synergistic effects with mTOR inhibitors like rapamycin on T cell suppression [51]. Rapamycin is a senomorphic itself and has been shown to inhibit cellular senescence in vitro, improve lifespan in vivo, and suppress the SASP [52]. Moreover, Song et al. demonstrated direct suppressive effects of fisetin on T cell differentiation and proliferation in a dose-dependent manner [53]. Translating these findings into the clinic, co-administering immunosuppressive drugs and senolytics could allow for reduced dosages of immunosuppressants, thus ameliorating side effects such as increased susceptibility to infections and malignancies [54, 55].

Preserving long-term graft function

Long-term outcomes of older organs have been shown to be compromised with reduced graft survival and increased rates of chronic allograft dysfunction across all types of transplants [56]. In kidney transplantation, for example, Oppenheimer et al. showed a linear increase of long-term graft failure and patient death with increasing donor age [57]. Histologically, markers of cellular senescence such as p16INK4a have been associated with interstitial fibrosis and tubular atrophy, which contributed to late graft loss. Conversely, reducing the number of senescent cells by knocking out INK4a attenuated these changes and has been associated with conservation of nephron mass and prolonged allograft survival. In addition, IRI was less pronounced in INK4a deficient mice, supporting the important role of senescent cells in mediating IRI-induced damage [58, 59]. Supporting these findings, kidney biopsies from patients with tubular atrophy and interstitial fibrosis showed increased expression of p16INK4a [60]. Moreover, investigating the long-term histology of transplanted livers, Rifai et al. described that donor age has been associated with higher levels of ductopenia and fibrosis in biopsies from patients ten years after transplantation. In contrast, donor age < 36 was a predictor for normal graft histology [61]. Taken together, these findings highlight that age and cellular senescence may not only pose a risk factor for short-term transplant outcomes but may also profoundly impair long-term graft function and survival rates. Eliminating senescent cells prior to transplantation by administering senolytics may thus improve long-term transplant outcomes.

The senescence cascade in VCA tissues—identifying potential cellular levers and key molecules Skin tissue

Skin tissue represents an integral part of VCA allografts, including a heterogenous set of alloreactive and immunotolerance-inducing cells [12]. Similar to the mucosa, skin tissue carries a high alloreactive potential [62,63,64]. Different senescence drivers can lead to distinct changes in skin composition and architecture. In the context of skin tissue senescence, particularly in VCA, several pathways interact to influence graft viability and function.

One such pathway is the JAK/STAT signaling pathway, which is closely linked to inflammatory responses in senescent skin cells. Activation of JAK/STAT by pro-inflammatory cytokines like IL-6 contributes to chronic inflammation in a senescent skin tissue. In particular, STAT3 activation amplifies the secretion of SASP components, reinforcing the pro-inflammatory state that characterizes senescent cells. This persistent inflammation not only compromises wound healing but also increases the antigenic profile of the skin graft, further heightening the risk of rejection [65]. In VCA settings, the JAK/STAT pathway plays a dual role—while it facilitates immune surveillance and host defense, its prolonged activation in senescent skin cells undermines the integrity and regenerative capacity of the graft, making it a critical target for modulating inflammation post-transplantation [66, 67].

Another key pathway involved in skin senescence is the mTOR signaling pathway, which regulates cell growth and metabolism. In senescent skin cells, mTOR is upregulated, contributing to the metabolic dysfunction often observed in aging tissues. mTOR activation promotes the production of SASP components, further driving inflammation and tissue degradation [68]. Additionally, mTOR signaling has been linked to delayed autophagy in senescent skin cells, impairing the clearance of damaged proteins and organelles. This accumulation of cellular debris accelerates the aging process, weakening the structural integrity of the skin [69]. The inhibition of mTOR has been explored as a potential therapeutic strategy to reduce senescence and enhance graft survival, particularly through the use of mTOR inhibitors like rapamycin. For instance, in murine models, combining low-dose IL-2 with rapamycin significantly prolonged skin allograft survival. This combination therapy extended graft survival from a median of 10 days in untreated controls to approximately 30 days in treated mice, effectively tripling the graft lifespan [70].

The p53/p21 pathway, known for its role in regulating the cell cycle and inducing senescence, also plays a critical role in skin tissue within VCA grafts. In response to DNA damage or oxidative stress, p53 is activated and promotes the expression of p21, a cyclin-dependent kinase inhibitor that halts the cell cycle and induces senescence [71]. In skin grafts, this pathway is particularly important for maintaining the balance between cellular proliferation and senescence. However, chronic activation of the p53/p21 axis in senescent skin cells can lead to a reduction in keratinocyte proliferation, weakening the skin barrier and impairing the graft’s ability to repair itself post-injury. This contributes to the thinning of the skin and makes the tissue more susceptible to mechanical stress, infections, and immune-mediated damage. In VCA patients, excessive activation of p53/p21 may also exacerbate graft fragility, leading to premature failure of the transplanted skin [72].

Mucosal tissue

Mucosal tissue plays a pivotal role in VCAs, especially in facial VCAs (fVCAs), where it constitutes a significant immunological interface and is hypothesized to influence both acute and chronic alloreactive rejection episodes. Recent studies have underscored the high alloreactive potential of mucosal tissue in VCA, demonstrating that the mucosal immune landscape can actively drive graft rejection processes in recipients [62,63,64, 73]. This dynamic may further be exacerbated by the SASP, which is a hallmark of senescent mucosal cells. Senescent mucosal cells secrete a plethora of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6) [74], chemokines like CXCL1 and CXCL8 [40], and matrix-degrading enzymes like MMP-1 and MMP-3, creating a chronically inflamed microenvironment [75]. This inflammatory milieu is closely linked to the diminished regenerative capacity of senescent epithelial cells, contributing to tissue dysfunction and increasing susceptibility to rejection [29, 76].

A central mediator in mucosal senescence is interleukin-8 (IL-8), a chemokine known for its dual role in promoting both inflammation and regeneration in epithelial tissues. In senescent mucosal cells, IL-8 interacts with the CXCR1 and CXCR2 receptors, initiating a signaling cascade through the NF-κB pathway. This pathway plays a pivotal role in driving the SASP and is responsible for recruiting immune cells—particularly macrophages and neutrophils—to sites of senescent mucosal tissue [74, 77]. The influx of these immune cells exacerbates local inflammation and promotes tissue damage, creating a feedback loop that may intensify graft rejection risk in VCA recipients. In the context of mucosal grafts, this IL-8-mediated immune cell recruitment has been implicated in acute rejection episodes, where the robust inflammatory response initiated by senescent cells accelerates tissue damage and graft deterioration [78]. However, IL-8 is not purely destructive in mucosal tissues. It has been shown to promote epithelial cell proliferation and contribute to wound healing by interacting with CXCR1/2 receptors on mucosal progenitor cells. This dual role highlights the complexity of senescence in mucosal tissues, where IL-8 can paradoxically drive both inflammatory damage and regenerative processes [79]. In senescent tissues, the balance between these opposing forces can cause persistent inflammation, impairing the tissue’s ability to regenerate effectively and leading to a decline in mucosal barrier function post-transplant [80].

TGF-β is another critical factor in mucosal senescence, particularly through its dual role in promoting inflammation and fibrosis while also facilitating tissue repair. In senescent mucosal tissues, TGF-β is activated via Smad-dependent and non-Smad pathways, both of which drive fibrotic responses and chronic inflammation [81]. While TGF-β can enhance epithelial cell proliferation and differentiation under normal circumstances, its chronic activation in senescent cells leads to excessive ECM deposition and fibrosis, which are hallmark features of long-term graft dysfunction in VCA recipients [81, 82]. Moreover, chronic TGF-β signaling disrupts normal mucosal architecture, thickening the ECM and reducing epithelial cell turnover, which is essential for maintaining tissue homeostasis [83]. Persistent activation of TGF-β also triggers the secretion of additional SASP factors, further perpetuating the senescence-associated inflammatory state in the mucosa [84, 85]. Studies have shown that Smad2/3 phosphorylation, a key step in TGF-β signaling, is significantly elevated in senescent mucosal cells, correlating with increased fibrosis and reduced tissue function in transplanted mucosal tissues. This chronic activation of TGF-β also leads to a loss of epithelial stem cell function, thereby limiting the mucosa’s ability to regenerate and repair post-transplant, ultimately increasing the risk of long-term graft failure [86,87,88].

Another key pathway that is altered in senescent mucosal tissues is the Notch signaling pathway. In the context of senescent mucosal tissues, disruptions in Notch signaling lead to impaired differentiation and proliferation of mucosal progenitor cells, which are essential for maintaining epithelial barrier integrity and promoting tissue repair [89,