Senolytic Treatment Alleviates Corneal Allograft Rejection Through Upregulation of Angiotensin-Converting Enzyme 2 (ACE2)

Hao Chi; Li Ma; Fanxing Zeng; Xiaolei Wang; Peng Peng; Xiaofei Bai; Ting Zhang; Wenhui Yin; Yaoyao Yu; Lingling Yang; Qingjun Zhou; Chao Wei; Weiyun Shi

doi:10.1167/iovs.66.2.15

Abstract

Purpose: Allograft rejection remains a major cause of failure in high-risk corneal transplants, but the underlying mechanisms are not fully understood. This study aimed to investigate the contribution of transplantation stress–induced cellular senescence to corneal allograft rejection and to elucidate the associated molecular mechanisms.

Methods: Age-matched murine corneal transplantation models were established. Cellular senescence was evaluated using senescence-associated β-galactosidase (SA-β-Gal) staining, western blot, and immunofluorescence staining. The role of cellular senescence in corneal allograft rejection was analyzed using p16 knockout mice and adoptive transfer experiments. Senolytic treatment with ABT-263 was administered intraperitoneally to evaluate its effects on corneal allograft rejection. RNA sequencing and pharmacological approaches were employed to identify the underlying mechanisms.

Results: Surgical injury induced a senescence-like phenotype in both donor corneas and recipient corneal beds, characterized by an increased accumulation of SA-β-Gal–positive cells in the corneal endothelium and stroma and elevated expression of senescence markers p16 and p21. Using genetic and adoptive transfer models, transplantation stress–induced senescence was shown to exacerbate corneal allograft rejection. Importantly, clearance of senescent cells by ABT-263 significantly suppressed ocular alloresponses and immune rejection. Mechanistically, RNA sequencing and loss-of-function experiments demonstrated that the anti-rejection effects of senolytic treatment were closely dependent on angiotensin-converting enzyme 2 (ACE2).

Conclusions: These findings highlight transplantation stress–induced senescence as a pivotal pathogenic factor in corneal allograft rejection. Senolytic therapy emerges as a potential novel strategy to mitigate transplant rejection and improve corneal allograft survival.

Corneal transplantation is a highly effective approach for restoring vision in cases of blindness, with a high success rate in low-risk scenarios due to the ocular immune privilege.¹^,² However, in high-risk conditions, such as corneal vascularization or repeated transplantation, the rejection rate exceeds 65% despite the administration of local or systemic immunosuppressive therapy. This rate is comparable to or greater than that of other commonly transplanted solid organs.¹^,³^,⁴ Consequently, immune rejection remains the major cause for corneal transplant failure.

Cellular senescence, an irreversible process triggered by external and internal stressors, has been implicated in various pathological conditions. It operates through mechanisms such as the senescence-associated secretory phenotype (SASP) and other damage-associated molecular patterns (DAMPs). Senescence contributes to aging, age-related diseases,⁵^,⁶ neurodegenerative disorders,⁷ COVID-19–related complications,⁸ and ocular disorders.⁹^,¹⁰ Senolytic therapies have been employed to counteract aging and associated pathologies¹¹^,¹² and to address other diseases.¹³^,¹⁴ Accumulating evidence suggests that senescent cells within aged organs are closely associated with poor transplantation outcomes, including allograft rejection. These effects are mediated by the enhancement of immunogenicity and alloimmune responses through the SASP and DAMPs.¹⁵^–¹⁷ For example, biliary senescence during cold storage has been shown to impair biliary architecture and regenerative capacity following transplantation.¹⁸ Notably, the presence of senescent cells in rabbit corneal grafts undergoing immune rejection has been observed.¹⁹ This phenomenon has also been documented in donor livers and kidneys post-transplantation.²⁰^,²¹ Although the adverse effects of donor age–related or cold storage–induced senescence on transplantation outcomes are well documented,¹⁶^,¹⁸ the impact of stress-induced senescence during age-matched allografts remains largely unexplored.

This study investigated the role of stress-induced senescence in corneal allograft rejection using an age-matched murine corneal transplantation model. Senescent cells induced by transplantation stress were identified in corneal grafts at early stages post-transplantation. Genetic and adoptive transfer experiments have highlighted the critical role of stress-induced senescence in promoting allograft rejection. Pharmacological clearance of senescent cells using ABT-263 significantly prolonged corneal allograft survival and suppressed intraocular alloimmune responses. Mechanistic analyses revealed that the anti-rejection effects of ABT-263 were mediated by angiotensin-converting enzyme 2 (ACE2). These findings underscore the importance of stress-induced senescence as a key driver of corneal allograft rejection and provide a rationale for senolytic therapy in mitigating transplant rejection.

Materials and Methods

Animals

All animal experiments were approved by the Ethics Committee of Shandong Eye Institute (approval nos. G-2015-020 and 2019S007). All procedures were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male wild-type (WT) C57BL/6 mice (n = 126) and BALB/c mice (n = 308) were obtained from Charles River Laboratories (Beijing, China) and housed in a specific pathogen-free facility under a 12-hour light/12-hour dark cycle. Cdkn2a/p16 knockout (KO) mice (n = 16, 01XB1:129-Cdkn2a^tm1Rdp) were procured from the Frederick National Laboratory for Cancer Research (Frederick, MD, USA) and were used as corneal donors. All mice were given ad libitum access to commercial rodent chow and water.

Murine Corneal Transplantation and Drug Treatment

Murine penetrating keratoplasty was conducted between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice under a surgical microscope (Carl Zeiss Microscopy, Oberkochen, Germany). Recipient mice were randomly divided into three groups: normal (Nor), BALB/c mice without surgery; syngeneic (Syn), BALB/c mice receiving corneal grafts from BALB/c donors; and allogeneic (Allo), BALB/c mice receiving corneal grafts from C57BL/6 donors.

The surgical procedures were followed established protocols.²² Briefly, mice were anesthetized by intraperitoneal injection of 0.6% pentobarbital sodium (75 mg/kg) and placed on a flat surgical bed. Central corneas (2.25-mm diameter) from C57BL/6 donors were excised and sutured onto graft beds (2.00-mm diameter) prepared in BALB/c recipients using a 2.00-mm trephine. Donor corneas were secured with eight interrupted 11-0 nylon sutures (Mani, Inc., Tochigi, Japan). Ofloxacin eye ointment (Santen Pharmaceutical, Osaka, Japan) was applied postoperatively to prevent ocular infection. Grafts with severe complications were excluded. All surgeries were performed by the same experienced surgeon.

Corneal allografts were evaluated twice weekly using slit-lamp microscopy. Rejection was assessed based on an opacification scoring system.²³ The scoring system was as follows: 0, clear; 1, minimal superficial opacity with pupil margin and iris vessels clearly visible; 2, minimal deep (stromal) opacity with the pupil margin and iris vessels visible; 3, moderate stromal opacity with only the pupil margin visible; 4, intense stromal opacity with only a portion of the pupil margin visible; and 5, maximum stromal opacity with the anterior chamber not visible. An opacity score ≥ 3 after suture removal was considered indicative of rejection. The scoring was performed by two independent observers in a double-blind manner, and the mean score was recorded.

To investigate the effect of transplantation stress–induced senescence on allograft rejection, the senolytic drug ABT-263 (50 mg/kg; Selleck Chemicals, Houston, TX, USA)²⁴ or vehicle was administered intraperitoneally on postoperative days 2, 4, 6, 8, 10, and 12. Additionally, a genetic approach was employed to evaluate the impact of donor corneal senescence. Corneal allografts from WT or p16 KO C57BL/6 mice were transplanted onto BALB/c recipient beds, and rejection was assessed via slit-lamp microscopy.

To determine the role of ACE2 in ABT-263–mediated anti-rejection effects, the ACE2 inhibitor MLN-4760 (10 µM; MedChemExpress, Monmouth Junction, NJ, USA)²⁵ was administered subconjunctivally, with or without ABT-263, on postoperative days 3, 6, 9, 12, and 15. Corneal allograft survival was evaluated using slit-lamp microscopy twice a week.

Additional Materials and Methods

Details regarding senescence-associated β-galactosidase (SA-β-Gal) staining, adoptive transfer experiments, immunofluorescence staining, western blot, RNA sequencing and analysis, real-time PCR, flow cytometry (FC) analysis, and Luminex cytokine assays are provided in the Supplementary Materials.

Statistical Analysis

Data are presented as mean ± SD. Statistical analyses were conducted using Prism 5 (GraphPad, Boston, MA, USA). Multiple comparisons were performed using one-way ANOVA followed by Tukey–Kramer post hoc tests. Graft survival was analyzed using Kaplan–Meier curves. A two-tailed Fisher's exact test was used to assess functional enrichment of differentially expressed genes (DEGs). All experiments were duplicated three times. P < 0.05 was considered statistically significant. The data that support the findings of this study are available from the corresponding authors upon reasonable request. RNA-sequencing data supporting the findings of this study have been deposited in National Center for Biotechnology Information (accession code CRA014662).

Results

Stress-Induced Senescence Is Increased in Corneal Allografts During Transplantation Rejection

Previous studies have demonstrated that cellular senescence caused by donor aging or cold storage is strongly associated with poor organ transplantation outcomes,¹⁶^–¹⁸ suggesting that cellular senescence is a significant contributor to graft failure. However, the extent to which surgical injury induces stress-induced graft senescence and subsequent immune rejection remains unclear. Using an age-matched murine corneal transplantation model, we evaluated the effect of transplantation injury on allograft senescence. Compared with normal and syngeneic controls, corneal allografts on postoperative day 10 exhibited a senescence-like phenotype, characterized by increased SA-β-Gal–positive staining in both the corneal endothelium and stroma (Figs. 1A, 1B), as well as elevated expression of senescence markers p16 and p21 (Figs. 1C, 1D). Double immunofluorescence staining further identified the senescent cell types as vimentin⁺ fibroblasts and CD45⁺ immune cells in the allogeneic group (Fig. 1E). These findings indicate the presence of stress-induced senescence in age-matched corneal allografts at an early stage post-transplantation, consistent with observations in high-risk rabbit corneal allografts.¹⁹

Figure 1.

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Stress-induced senescence in corneal allografts significantly increased during rejection progression. (A) Images of SA-β-Gal staining (a marker of senescence) in corneal grafts from the normal (Nor), syngeneic (Syn), and allogeneic (Allo) groups on postoperative day 10, obtained via whole-mount staining (n = 4/group). Scale bar: 50 µm, lower panel. (B) Photographs of SA-β-Gal staining in the Nor, Syn, and Allo groups on postoperative day 10, captured through frozen section staining (n = 4/group). Scale bar: 50 µm. (C) Protein levels of senescence-associated markers (p16 and p21) in corneal grafts from the Nor, Syn, and Allo groups on postoperative day 10, detected via western blot (n = 9, in pools of 3). (D) Quantification of p16 and p21 in corneal grafts using Image J software (cf. panel C). (E) Co-localization of SA-β-Gal staining with immunofluorescence staining for vimentin (corneal stromal cells) or CD45 (immune cells) in the Allo group on postoperative day 10, obtained through frozen section staining (n = 4/group). Scale bar: 10 µm. **P < 0.01.

Stress-Induced Senescence Accelerates Corneal Transplantation Rejection

To assess the impact of stress-induced senescence on corneal allograft rejection, WT and p16 KO C57BL/6 mice were used as allogeneic donors. Recipient mice with p16 KO corneal allografts exhibited reduced immune rejection and prolonged graft survival compared with recipients of WT corneas (Figs. 2A, 2B). These results suggest a pathogenic role of senescent donor corneas in allograft rejection. To further investigate this, adoptive transfer experiments were performed to mimic graft senescence. Senescent donor mouse corneal fibroblasts (MCFs) were transferred into the anterior chamber and coated onto recipient corneal endothelia. This procedure accelerated corneal graft senescence (Supplementary Fig. S1A) and exacerbated rejection, as indicated by reduced survival time and severe edema (Figs. 2C, 2D). However, when senescent MCFs pretreated with ABT-263 were transferred, the pro-rejection effects were significantly reversed (Figs. 2C, 2D). These findings demonstrate that transplantation stress–induced senescence aggravates corneal allograft rejection.

Figure 2.

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Stress-induced senescence in age-matched corneal grafts exacerbated rejection. (A) Representative slit-lamp photographs of recipient mice transplanted with WT or p16 KO donor corneal grafts on postoperative day 20. (B) Graft survival curves of recipient mice receiving WT and p16 KO donor corneas, evaluated using the Kaplan–Meier method (n = 16/group). (C) Representative slit-lamp photographs of corneal grafts in different groups following adoptive transfer on postoperative day 11. (D) Graft survival curves for groups with adoptive transfer, analyzed using the Kaplan–Meier method (n = 10/group). Allo, allogeneic recipient mice with PBS transfer; Allo+Ctrl, allogeneic recipient mice with transfer of control donor MCFs; Allo+Sene, allogeneic recipient mice with transfer of senescent donor MCFs; Allo+Sene+ABT, allogeneic recipient mice with transfer of senescent donor MCFs treated with ABT-263. ***P < 0.001.

Targeted Clearance of Senescent Cells Significantly Mitigates Allograft Rejection

The potential of selective senescent cell clearance to mitigate corneal transplantation rejection was subsequently investigated. The senolytic drug ABT-263 was administered intraperitoneally following a specified protocol (Fig. 3A). As shown in Supplementary Figure S2, corneal grafts treated with ABT-263 exhibited reduced cellular senescence, as indicated by fewer SA-β-Gal–positive cells in the endothelium and stroma, alongside decreased expression of senescence-associated markers (p16 and p21). These findings confirmed the efficacy of ABT-263 in eliminating senescent cells in corneal allografts. These findings confirmed the efficacy of ABT-263 in eliminating senescent cells in corneal allografts.

Figure 3.

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Elimination of senescent cells significantly mitigated corneal allograft rejection. (A) Schematic representation of corneal transplantation and treatment with intraperitoneal injection of ABT-263 (50 mg/kg). (B) Representative slit-lamp photographs of corneal grafts in ABT-263–treated and vehicle-treated groups on postoperative day 20. (C) Graft survival curves for the two groups, analyzed using the Kaplan–Meier method (n = 14/group). (D) FC plots of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ T cells in corneal grafts on postoperative day 20 (n = 12, in pools of 4). (E) Quantitative representation of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ T cells in corneal grafts (cf. panel D). (F) Transcriptional levels of pro-inflammatory cytokines in corneal grafts on postoperative day 20, determined via quantitative PCR (n = 9, in pools of 3). Allo, allogeneic recipient mice treated with PBS; Allo+ABT, allogeneic recipient mice treated intraperitoneally with ABT-263 (50 mg/kg). **P < 0.01; ***P < 0.001.

Building on these results, the anti-rejection effects of ABT-263 were evaluated. Corneal grafts in vehicle-treated mice displayed more severe immune rejection than those in ABT-263–treated recipients, characterized by pronounced corneal opacity and edema (Fig. 3B) and shortened graft survival time (Fig. 3C). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and FC revealed increased levels of pro-inflammatory cytokines, including IL-1β, IL-17A, TNF-α, and IFN-γ, in the rejected grafts (Figs. 3D–3F), contributing to aggravated rejection. In contrast, ABT-263–treated recipients exhibited reduced immune rejection, as evidenced by transparent corneas without noticeable edema (Fig. 3B), prolonged graft survival time (Fig. 3C), and significantly decreased levels of pro-inflammatory cytokines in the grafts (Figs. 3D–3F). Additionally, treatment with ABT-263 lowered the production of pro-inflammatory cytokines in intraocular tissues (Supplementary Fig. S3). These findings collectively demonstrate that senolytic therapy represents a promising strategy for mitigating corneal graft rejection.

Corneal Allografts Following Senolytic ABT-263 Treatment Show Significant Pathological Alterations on Postoperative Day 10

To further assess the impact of senolytic treatment on corneal allograft rejection, corneal samples under different conditions were subjected to RNA sequencing. Principal component analysis (PCA) revealed that the gene expression profiles of corneal grafts in the allogeneic group differed significantly from those in normal corneas (Supplementary Fig. S4A), indicating clear group partitioning. Analysis identified 7727 DEGs in corneal allografts on postoperative day 10 in the allogeneic versus normal group, comprised of 5469 upregulated genes and 2258 downregulated genes (Supplementary Fig. S4B). Biological process (BP) analysis showed that the upregulated genes were enriched in inflammation- and immunity-related pathways, such as inflammatory response, immune response, positive regulation of angiogenesis, neutrophil chemotaxis, and chemokine-mediated signaling (Supplementary Fig. S4C). Gene set enrichment analysis (GSEA) further confirmed the enrichment of immune/inflammatory pathways, including antigen processing and presentation, leukocyte migration, and vasculogenesis (Supplementary Fig. S4D). These results suggest that heightened immune and inflammatory responses in corneal allografts at early stages post-transplantation likely contribute to subsequent rejection.

Following ABT-263 treatment, PCA analysis indicated distinct gene expression profiles in corneal grafts compared to the untreated allogeneic group (Fig. 4A). In the ABT-263–treated group, 338 upregulated and 292 downregulated genes were identified (Fig. 4B). Functional analysis revealed significant enrichment of inflammation- and immunity-related BPs in the downregulated genes, including inflammatory response, neutrophil chemotaxis, lymphocyte chemotaxis, leukocyte chemotaxis, and monocyte chemotaxis (Fig. 4C). GSEA identified five significantly enriched pathways in the downregulated genes, such as inflammatory response, chemotaxis, leukocyte chemotaxis, and angiogenesis (Fig. 4D). Moreover, decreased expression of several inflammation-associated factors was observed in corneal tissues following ABT-263 treatment (Fig. 4E). Notably, in addition to cellular response to IFN-β and activation of innate immune response, the upregulated genes in ABT-263–treated grafts showed enrichment in negative regulation of innate immune responses, suggesting reduced inflammatory activity (Fig. 4F). These findings indicate that ABT-263 treatment facilitates inflammation resolution at an early stage post-transplantation, thereby attenuating corneal allograft rejection.

Figure 4.

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Corneal allografts treated with senolytic ABT-263 exhibited significant transcriptional changes on postoperative day 10. (A) PCA demonstrated transcriptional differences between the Allo group and Allo+ABT group. (B) Volcano plot shows the distribution of DEGs with thresholds of P < 0.05 and |log₂FC| > 1. (C) Significantly enriched BPs related to inflammatory and immune responses among downregulated DEGs in ABT-263-treated corneal grafts. (D) GSEA highlighted major BPs implicated in inflammation, immune response, and corneal vascularization in downregulated DEGs of ABT-263–treated corneal grafts. (E) Heatmap showing major downregulated DEGs associated with inflammation and chemotaxis. (F) Enrichment of immune- and inflammation-related pathways in upregulated DEGs of ABT-263–treated corneal grafts.

Pharmacological Inhibition of Transplantation Stress–Induced Senescence Suppresses Early Ocular Alloimmune Responses

Based on RNA sequencing findings, it was hypothesized that senolytic therapy could attenuate ocular alloimmune responses during early post-transplantation stages. As shown in Supplementary Figure S2, ABT-263 effectively cleared senescent cells. FC analysis revealed a lower proportion of MHCⅡ⁺CD11c⁺ dendritic cells (DCs) in ABT-263–treated corneal allografts compared to the untreated group (Figs. 5A, 5B). Quantitative RT-PCR analysis confirmed reduced transcriptional levels of DC activation–related genes, including CD80, CCR7, IL-6, IL-12p40, and S100A8, in the grafts after ABT-263 treatment (Fig. 5C). Additionally, pro-inflammatory cytokines in the aqueous humor, such as granulocyte-colony stimulating factor (G-CSF), IFN-γ, IL-1β, IL-6, IFN-γ-inducible protein 10 kDa (IP-10), keratinocyte-derived cytokine (KC), monocyte chemoattractant protein-1 (MCP-1), and RANTES, were significantly reduced in ABT-263–treated recipients (Fig. 5D). Furthermore, the expression of DC activation–related genes in inflamed corneal tissues from ABT-263–treated mice was pronouncedly reduced when compared to untreated allogeneic controls (Fig. 5E).

Figure 5.

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Clearance of stress-induced senescent cells reduced early-stage alloimmune responses. (A) FC plots of MHC II⁺CD11c⁺ DCs in corneal grafts on postoperative day 10 from ABT-263–treated and vehicle-treated groups (n = 12, in pools of 4). (B) Quantification of MHC II⁺CD11c⁺ DCs in corneal grafts (cf. panel A). (C) Transcriptional levels of DC activation-associated genes in corneal grafts on postoperative day 10, determined using quantitative PCR (n = 9, in pools of 3). (D) Protein levels of pro-inflammatory cytokines and chemokines in aqueous humor (AH) on postoperative day 10, measured after ABT-263 treatment (n = 10/group). (E) mRNA levels of DC activation–related genes in inflamed corneal (I-C) tissues following ABT-263 treatment. *P < 0.05; **P < 0.01;***P < 0.001.

Notably, adoptive transfer of senescent donor MCFs into the anterior chamber of recipient mice elevated the expression of DC activation–associated genes, including CD80, CCR7, and S100A8 (Supplementary Fig. S1B). However, transferring ABT-263–pretreated senescent donor MCFs significantly reduced the expression of these genes compared to untreated senescent MCFs (Supplementary Fig. S1B). These findings demonstrate that targeting transplantation stress–induced senescence effectively downregulates ocular alloimmune responses during the early post-transplantation period.

The Anti-Rejection Effect of Senolytic ABT-263 Depends on Elevated ACE2

Although ABT-263 treatment significantly ameliorated allograft rejection, the underlying mechanism remained unclear. Gene Ontology (GO) analysis revealed substantial enrichment of cellular components among the upregulated DEGs in ABT-263–treated corneal allografts, including the symbiont-containing vacuole membrane, omegasome, extracellular region, plasma membrane, and basement membrane (Fig. 6A), These findings likely reflect the notable enhancement of corneal allografts following senolytic treatment. Among these top five terms, the heatmap primarily illustrated the DEGs associated with the extracellular region and plasma membrane, notably demonstrating higher ACE2 expression in ABT-263–treated corneal allografts compared to untreated counterparts (Fig. 6B).

Figure 6.

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Increased ACE2 expression improved corneal allograft outcomes following senolytic treatment. (A) Top five enriched cellular component (CC) terms of upregulated DEGs in ABT-263–treated corneal grafts, identified via GO analysis. (B) Heatmap of common DEGs associated with the extracellular region and plasma membrane. (C) PPI network of ACE2 within DEGs, constructed using STRING with high confidence (score = 0.70). (D) ACE2 expression in corneal grafts with different treatments, assessed via western blot (n = 9/group, in pools of 3). Upper panel: Western blot bands. Lower panel: Quantification of ACE2 using Image J. (E) Representative slit-lamp photographs of corneal grafts after ABT-263 treatment with or without ACE2 inhibitor (10 µM) on postoperative day 20. (F) Graft survival curves for three groups analyzed using the Kaplan–Meier method (n = 14/group). (G) Transcriptional levels of pro-inflammatory cytokines in corneal grafts with different treatments on postoperative day 20, determined using quantitative PCR (n = 9, in pools of 3). Allo, allogeneic recipient mice treated with PBS; Allo+ABT, allogeneic recipient mice treated with ABT-263 (50 mg/kg); Allo+ABT+ACE2i, allogeneic recipient mice treated with ABT-263 and ACE2 inhibitor. *P < 0.05; **P < 0.01;***P < 0.001.

Additionally, protein–protein interaction (PPI) analysis indicated that ACE2 was closely linked to several DEGs with immunomodulatory properties, including immunity-related GTPase family M member 1 (IRGM-1) (Fig. 6C).²⁶^,²⁷ We also observed higher protein levels of ACE2 in senolytic-treated corneal allografts than in untreated counterparts (Fig. 6D). Based on these findings and the known role of ACE2 in mitigating inflammation,²⁸ ACE2 was identified as a critical molecule contributing to the anti-rejection effect of ABT-263.

To further investigate the role of ACE2 in this context, we employed a pharmacological approach. In recipient mice treated with ABT-263, topical application of the ACE2 inhibitor MLN-4760 markedly reversed the anti-rejection effect of ABT-263, manifesting as increased corneal opacity, edema, and reduced graft survival time (Figs. 6E, 6F). Quantitative RT-PCR analysis revealed elevated levels of pro-inflammatory cytokines, such as IFN-γ, IL-17A, and IL-1β, in ABT-263–treated corneal allografts subjected to ACE2 inhibition compared to those without MLN-4760 (Fig. 6G). These cytokines are known contributors to severe corneal transplantation rejection.²²^,²³ Collectively, these results demonstrate that the anti-rejection effect of ABT-263 is functionally dependent, at least partially, on increased ACE2 expression.

Discussion

Previous studies have demonstrated that aging and cold storage contribute to poor allograft outcomes via cellular senescence.¹⁶^–¹⁸ Our findings revealed that transplantation stress induces cellular senescence even in age-matched corneal allografts, and the pharmacological and genetic elimination of senescent cells effectively prevents immune rejection. Through transcriptomic analysis and loss-of-function experiments, we identified a close association between the anti-rejection effect of senolytic ABT-263 and elevated ACE2 expression. Thus, these findings suggest that transplantation stress–induced senescence is a key pathological driver of corneal allograft rejection, highlighting senolytic therapy as a novel approach to mitigate transplant rejection.

Extensive evidence has shown that older organs, burdened with senescent cells, exhibit reduced functionality and heightened immunogenicity, exacerbating adverse outcomes post-transplantation.¹⁵^,¹⁷ Senescent CD4⁺ T cells with enhanced glutaminolysis exhibit greater activation potential, increased pro-inflammatory cytokine production, and proliferation, thereby driving rejection of aged organs.²⁹ Similarly, overactive senescent dendritic cells promote Th1 and Th17 responses, accelerating the rejection of aged cardiac and skin transplants.¹⁶^,¹⁷ Cold storage has also been identified as a significant catalyst for donor organ and tissue aging, ultimately contributing to transplantation failure.¹⁸ In this study, we identified stress-induced senescence-like phenotypes in age-matched corneal grafts post-transplantation, indicating that transplantation stress accelerates cellular senescence. This phenomenon aligns with findings in other organ transplants²⁰^,²¹^,³⁰ and various pathological contexts.⁸^,³¹ Both genetic and pharmacological approaches underscored the pathogenic role of stress-induced senescence during corneal transplantation. Accordingly, transplantation stress–induced senescence should be recognized as a critical factor in transplantation failure, alongside donor/recipient aging and organ preservation.

The activation and propagation of innate immune response cascades, including NLRP3 inflammasome, cGAS/STING signaling, and Toll-like receptor pathways, are integral to adaptive alloimmune responses and subsequent allograft rejection.³²^–³⁵ However, the mechanisms sustaining these responses remain unclear. According to the “danger hypothesis,” DAMPs released from donor cells establish a direct link between innate immunity and allograft failure.³²^,³⁶ Increased release of DAMPs and inflammatory cytokines under various preservation conditions has been linked to allograft rejection and dysfunction, including during machine preservation and cold ischemia storage.³⁷^,³⁸ Mitochondrial DNA from senescent cells of aged donors has been implicated in worsened cardiac transplantation outcomes.¹⁶ In contrast, early senolytic treatment of corneal allografts reduced innate immune responses and pro-inflammatory cytokine levels. Given their pro-inflammatory nature and secretion of SASP, stress-induced senescent cells amplify allograft rejection; however, the precise mechanisms warrant further investigation.

ACE2 was initially identified as a key regulator of the renin–angiotensin system through its ability to hydrolyze angiotensin II (Ang II), influencing angiogenesis, inflammation, and fibrosis.³⁹ Subsequent studies expanded its roles to multiple organs, including regulating intestinal amino acid homeostasis⁴⁰ and serving as the primary receptor for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2.⁴¹ ACE2 deficiency has been linked to pathological conditions such as cardiac dysfunction, diabetic kidney injury, and acute lung injury.³⁹ In this study, we observed increased ACE2 expression in ABT-263–treated corneal allografts compared to untreated counterparts, and ACE2 inhibition reversed the anti-rejection effect of ABT-263. Previous studies have also linked ACE2 deficiency to spontaneous corneal clouding and inflammation,⁴² as well as accelerated aging,⁴³ potentially explaining our findings.

Senolytic drugs such as ABT-263 eliminate senescent cells by inducing apoptosis, addressing their anti-apoptotic properties.¹¹^,²⁴ Donor apoptotic cells have been shown to promote antigen-specific immune tolerance in allografts.⁴⁴^,⁴⁵ Thus, immune tolerance induction may contribute to the anti-rejection effects of senolytic treatment. Nevertheless, further studies are required to elucidate the relationships among cellular senescence, allograft rejection, and allograft tolerance.

Although this study has demonstrated the efficacy of targeting stress-induced senescent cells to mitigate corneal allograft rejection, several limitations remain. First, although transplantation stress–induced senescence was observed in the age-matched murine corneal transplantation model, its underlying mechanisms are yet to be elucidated. Second, although RNA sequencing established a link between reduced inflammatory immune responses and senolytic therapy, further investigations are necessary to clarify the underlying mechanisms. Third, additional mechanisms, such as the involvement of IRGM-1, require exploration to provide a more comprehensive explanation of the anti-rejection effects of ABT-263. Finally, although the anti-rejection effects of ABT-263 were achieved via intraperitoneal injection, future studies should aim to develop formulations, such as eye drops, to enhance the solubility and corneal permeability of the drug.

Despite these limitations, this study provides proof-of-concept evidence that stress-induced senescence plays a critical pathogenic role in the age-matched murine corneal transplantation model. Targeting stress-induced senescence significantly alleviates allograft rejection through ACE2 upregulation. Integrating our findings with prior studies, we propose that cellular senescence, induced by donor age, organ preservation, and transplantation stress, constitutes a major risk factor for transplantation failure and allograft rejection (Fig. 7). Collectively, this study underscores stress-induced senescence as a key pathogenic mechanism in corneal allograft rejection and highlights senolytic therapy as a promising strategy for mitigating transplant rejection in corneal and potentially other organ transplants.

Figure 7.

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Senolytic therapy as a novel approach against corneal transplant rejection. Based on prior studies and current findings, cellular senescence caused by donor age, storage conditions, and transplantation stress probably contributes significantly to allograft failure and rejection. Senolytic therapy was shown to effectively prevent and treat such outcomes. (Created with biorender.com).

Acknowledgments

The authors thank Ping Lin for her linguistic and editorial assistance. We also thank OE Biotech Co., Ltd. (Qingdao) for RNA sequencing and bioinformatics analysis.

Supported by grants from the National Natural Science Foundation of China (82271130, 81530027, 82271058, 82000851), Natural Science Foundation of Shandong Province (ZR2020MH1175, ZR2019ZD37), Taishan Scholar Program (tsqn202103185, tspd20221110), Shandong Provincial Key Research and Development Program (2022CXGC010505, 2021LCZX08), Academic Promotion Program of Shandong First Medical University (2019ZL001, 2019PT002), Innovation Project of Shandong Academy of Medical Sciences, and Qingdao Shinan District Science and Technology Plan Project (2022-2-018-YY).

Disclosure: H. Chi, None; L. Ma, None; F. Zeng, None; X. Wang, None; P. Peng, None; X. Bai, None; T. Zhang, None, W. Yin, None; Y. Yu, None; L. Yang, None; Q. Zhou, None; C. Wei, None; W. Shi, None

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