TGF-β induces corneal endothelial senescence via increase of mitochondrial reactive oxygen species in chronic corneal allograft failure

Increased cellular senescence in the human CE with chronic corneal graft failure

Alizarin Red Staining showed that the original hexagonal structure of endothelial cells with chronic graft failure was destroyed compared to that in healthy donor (Fig.1A). Accumulative evidence indicated that cellular senescence is closely related to organ transplant and involved in the pathogenesis of chronic graft failure [11,12]. Therefore, we speculate that cellular senescence was associated with the changes of corneal endothelial cells in chronic corneal graft failure. Our results showed that senescence-related genes p16 and p21 were dramatically increased at transcription level or protein level (Fig.1B, D, E), suggesting that decreased corneal endothelial cells may result from increased cellular senescence in the CE with corneal graft failure.

Figure 1. Increased senescence associated markers and TGF-β1 in the human corneal buttons CE with chronic graft rejection. (A) Clinical evaluation of corneal endothelial cells with chronic failure using alizarin red staining. (B-C) The mRNA expression of p16, p21 (B) and TGF-β1 (C) in the CE with or without chronic graft failure (**P<0.01,*P<0.05). (D-E) Representative photographs (D) and histopathology scores (E) for the IHC staining of p16 and TGF-β1 in the CE with chronic failure.

Elevated expression of TGF-β1 in the human CE with chronic graft failure

Next, we questioned what triggered cellular senescence. A large body of evidence indicates that TGF-β signaling is involved in ocular diseases and cellular senescence. Therefore, we analyzed the expression of TGF-β in the CE. We found that TGF-β1 expression was largely elevated at the transcriptional level (Fig.1C) and protein level (Fig. 1D, E) in senescent CE compared to control. These results suggest that the increased TGF-β found in the CE with chronic graft failure may correlate to the increased cellular senescence.

TGF-β1 induces senescence of B4G12 cells

To evaluate the effect of TGF-β1 on corneal endothelium senescence, CE cell line B4G12 was used in vitro. Cellular senescence is characterized as an irreversible arrest of mitotic cells at G1 phase, while some cancer cells enter senescence at G2 or S phase. Our results showed that the B4G12 accumulated at G1 phase (from 53.01% to 65.89%) with a concomitant depletion of S phase cells (from 16.95% to 11.66%) upon TGF-β1 treatment (Fig. 2A, B), suggesting that cell cycle arrest during TGF-β1 induced CE cells senescence occurred at G1 phase. In addition, percentage of SA-β-gal–positive cells was upregulated upon TGF-β1 treatment (Fig. 2C, D). Moreover, TGF-β1 could increase the expression of p16 and p21 at mRNA and protein level (Fig. 3A, B, C). Taken together, our findings suggested that TGF-β1 was capable of inducing human CE cells senescence.

Figure 2. TGF-β1 induces cellular senescence in B4G12 cells. (A-B) The G1 phase arrest was induced by TGF-β1 treatment. Control and TGF-β1–treated B4G12 cells were subjected to cell cycle analysis after 48h-treatment. A representative flow cytometric analysis of the DNA content was shown in (A) and the values are mean±SD in (B). (C-D) SA-β-Gal activity was measured in B4G12 cells treated with 10 ng/ml TGF-β1 alone, or in combination with NAC (10mM) for 72h. NAC treatment alone (10mM) did not affect the cell cycle status and SA-β-Gal activity. Bar graphs represent mean±SD. *P<0.05, **P<0.01. All the experiments were independently repeated at least three times.

Figure 3. TGF-β1 treatment of B4G12 cells causes the induction of p16 and p21. B4G12 cells were treated with 10 ng/ml TGF-β1 alone, or in combination with NAC (10mM) or NAA (10mM) for 72h. The mRNA (A) and protein (B-C) expression of p16 and p21 were induced by TGF-β1. NAC or NAA treatment alone (10mM) did not change the levels of p16 and p21. Bar graphs represent mean±SD. *P<0.05, **P<0.01.

Senescent cells will develop a complex senescence-associated secretory phenotype (SASP). It has been demonstrated that increased production of inflammatory cytokines, such as interleukin (IL)-6 and -8, during senescence play substantial roles in the establishment and maintenance of senescent phenotype. To test whether SASP happens during TGF-β1-induced human CE cells senescence, we further measured the key inflammatory mediators. In TGF-β-induced senescent CE cells, elevated monocyte chemotactic protein-1 (MCP-1), IL-6 and tumor necrosis factor-α (TNF-α) gene expression (Fig. S1A) and secretion (Fig. S1B) was detected.

TGF-β1 treatment enhances mtROS production in B4G12 cells

Mitochondrial ROS has been demonstrated to be involved in cellular senescence. Our results showed that mtROS were dramatically augmented in B4G12 after treatment of 10 ng/ml TGF-β1 for 48 hours detected with MitoSox Red by flow cytometer (Figure 4A and B). Similar results were obtained by fluorescent microscope (Fig. 4C). We also used the hydrogen peroxide- specific peroxy orange 1(PO1) probe to detect H₂O₂ molecule, and the use of this ROS probe confirmed the induction of ROS by TGF-β1(Fig. 4C). Next, we tested the expression of mitochondrial matrix enzyme Superoxide Dismutase2 (SOD2), which has been indicated as a mitochondria protector against oxidative radicals by suppressing mtROS production. Our results demonstrated that SOD2 expression was substantially decreased upon TGF-β1 treatment for 48 hours in B4G12 (Fig. 4D), which suggested its involvement in cellular senescence.

Figure 4. mtROS production in B4G12 cells after exposure to 10ng/ml TGF- β 1 for 48 hours. (A-B) Control and TGF-β1–treated B4G12 cells were subjected to MitoSOX Red Indicator staining after 48h of culture. A representative flow cytometric analysis of mtROS was shown in (A) and the values are mean±SD (B). (C) The MitoSOX Red and peroxy orange 1 fluorescence was imaged with a fluorescent microscope. (D) SOD2 protein expression was tested in CE cells upon 10 ng/ml TGF-β1 treatment. *P<0.05, **P<0.01. All the experiments were independently repeated at least three times.

NAC rescues TGF-β1 induced B4G12 cells senescence by repressing mtROS production

We further used N-acetyl-L-cysteine (NAC) as a physiological mtROS scavenger to rescue TGF-β1-induced senescence. Our data showed that 10 mM NAC could effectively inhibit mtROS production induced by TGF-β1 in B4G12 cells, while 10mM N-acetyl-L-alanine (NAA), a compound with similar chemical structure to NAC, was with no anti-ROS activity (Fig. 4A, B and C). In addition, NAC also reversed TGF-β1-induced G1 arrest (Fig. 2A, B), and markedly inhibited TGF-β1-induced SA-β-Gal activity (Fig. 2C, D), p16 and p21 expression (Fig. 3 A, B, C), as well as SASP production (Fig. S1A, B), whereas NAA failed to protect B4G12 cells from the senescence induced by TGF-β1 (Fig. 3 A, B, C). Taken together, our results suggest a key role for mtROS in TGF-β1-induced cellular senescence.

Presence of immune cells on mouse CE with chronic graft failure

Using a murine chronic allograft failure model in which graft failure occurs more than 90 days post-engraftment (Fig. 5A, B), we not only found the destroyed hexagonal structure of corneal endothelial cells from failed corneal grafts (Fig. 5C), but also found increased transcriptional levels of p16, p21 (Fig. 5D) and TGF-β1 (Fig. 5E) in the CE cells with graft failure, as well as shown in protein levels using immunofluorescent staining (Fig. 5F). In addition, in late failed corneal allograft group sporadic leukocytes were observed in proximity of CE (Fig. 6A), which were not found in the syngenic group. We speculated that CE cells may function as chemoattractant. Next, we measured pro-inflammatory factors associated with SASP by qPCR in CE cells from chronic failure and syngenic mouse. Elevated IL-6, MCP-1 and TNF-α gene expression (Fig. 6B) were observed in the chronic failure CE cells. Collectively, these results suggest that TGF-β could induce CE cell premature senescence and thus, contribute to CE cell accelerated loss in chronic corneal graft failure.

Figure 5. Premature senescence and elevated TGF-β1 in the murine CE with chronic failure. (A-C) Corneal graft phenotype in syngenic and chronic failure mouse model (D-E) p21, p16 (D) and TGF-β1 (E) expression was measured at transcription level in the murine CE from syngenic and chronic failure group. (F) Representative photographs for the IF staining of TGF-β1 in the CE with chronic graft failure. **P<0.01,*P<0.05.

Figure 6. Leukocytes in proximity to murine CE of chronic graft failure. (A) H&E staining showing immune cell infiltration in the murine CE. (B)The expression of senescence-associated secretory phenotypefactor in RNA pooled from murine CE from chronic failure group compared with syngenic group. *P<0.05, **P<0.01. All the experiments were independently repeated at least three times.

Human cornea tissue

This study was approved by the Affiliated Hospital of Qingdao University Review Board and Shandong Eye Institute Review Board according to the tenets of the Declaration of Helsinki. Ten fresh chronic dysfunction corneal buttons more than 5 years after the first corneal transplantation were obtained prospectively. The sex-matched healthy corneas were obtained from International Federation of Eye Banks, Eye Bank of Shandong (Qingdao, China) and the donor corneal rims (residual tissues) were collected by penetrating keratoplasty (PKP) in this study.

Animals

The animal experiment procedure was approved by Institute Animal Care and Use Committee of Qingdao University according to National Institutes of Health guide for the care and use of Laboratory animals. Male BALB/c and C57BL/6 (B6) mice were obtained from Shanghai Experimental Animal Center. All mice were cultivated under SPF condition. Mice used for corneal transplantation were between 8 and 12 wk of age.

Corneal transplantation and evaluation of grafted mouse corneas

B6 corneas were grafted onto the right eye of BALB/c recipients for allogenic, as described previously [22]. In brief, Donor grafts were placed into the recipients and the sutures were taken out in one week after corneal transplant (Mani, Tochigi, Japan). Clinical examination was performed two times once a week. The grafts were scored for opacity as previously described [22]. At each time point, graft opacity needs to be evaluated on a scale of 0–5. Grafts were considered to be undergoing acute rejection with persistent opacity score > 2 within 2–5 weeks postoperatively, while grafts which were clear through 5–6 weeks postoperatively but developed opacity at > 90 d were considered as chronic graft failure.

Cell culture and treatment

Immortalized CE cell line B4G12 was maintained in K-SFM (Gibco, Grand Island, NY) containing 2% FBS within 5% CO₂ at 37°C. Medium was replaced by fresh serum-free K-SFM with 10 ng/ml recombinant human TGF-β1 (Peprotech) alone, or in combination with NAC (a potent ROS scavenger, 10 mM, Sigma) or NAA (10 mM, Sigma) after overnight starvation.

Immunohistochemistry and immunofluorescence assay

Immunohistochemical and immunofluorescence assays were conducted as previously described [23]. In brief, paraffin embedded corneas was sectioned for 4 μm and antigen retrieval was possessed (Maixin Biotech, Fujian, China). TGF-β1 antibody (sc146, Santa Cruz) was used as primary antibody and naive IgG of appropriate specie was used as negative control.

Cell Cycle analysis

Cell cycle status in the B4G12 cells was detected by measuring nuclear DNA content. The cells were collected 48 hours after exposure to TGF-β1 and were fixed in 70% ethanol at 4°C for more than 2 hours. The pellet was collected by centrifugation before the RNase A solution was added (10 mg/ml). After a 1-hour incubation at 37°C, the cells were stained with propidium iodide (PI; final concentration 100 μg/ml) at 37°C for 30 minutes. The samples were analyzed using a FACS Calibur flow cytometer (BD Bioscience).

Mitochondrial ROS measurement

Mitochondrial ROS (mtROS) were measured using a MitoSOX ™ Red mitochondrial superoxide indicator (Invitrogen), or hydrogen peroxide- specific peroxy orange 1(PO1, APExBIO) probe as the manufacturer’s instructions. Briefly, TGF-β1 treated B4G12 cells were incubated with 5 μM MitoSOX red for 10 min, or 5 μM PO1 for 30 min in the incubator. After removing the reagents and washing twice, fluorescence was detected with a FACS Calibur flow cytometer (BD Bioscience) or a fluorescent microscope.

Quantitative RT-PCR

Total RNA was extracted from the cornea endothelium or cultured B4G12 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, cDNAs were obtained with PrimeScript RT reagent Kit (TOYOBO, Osaka, Japan). All the qPCR was done with SYBR GREEN reagents at ABI prism 7500 (Applied Biosystems, Foster City, CA, USA). Primers were shown in the Table1.

Western blot

For whole cell extraction, the B4G12 cells were lysed with RIPA buffer (Beyotime, P0013B, Beijing, China). The detailed procedure has been previously described [9]. The primary antibodies were used as following: α-tubulin (11224-1, Proteintech), p16 (ab108349, Abcam), p21 (ab109520, Abcam) and SOD2 (ab13533, Abcam).

SA-β-Gal staining and ELISA

SA-β-gal activity was detected with Staining Kit according to manufacturer’s instructions (Beyotime, RG0039, Beijing, China). Levels of IL-6 and MCP-1 in cell culture supernatants were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience).

Statistical analyses

All experiments were performed at least three times independently, and data was presented as mean±SD. Two-tailed Student’s t-test and one-way analysis of variance (ANOVA) were used for two groups and more was used for statistical analyses. p<0.05 was considered statistically significant.