Research ArticleHEALTH AND MEDICINE

Pathological processes in aqueous humor due to iris atrophy predispose to early corneal graft failure in humans and mice

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Science Advances  13 May 2020:
Vol. 6, no. 20, eaaz5195
DOI: 10.1126/sciadv.aaz5195
  • Fig. 1 Condition of iris and AqH determines the prognosis of corneal transplantation.

    (A) CEnCs form a monolayer tissue in the posterior surface of the cornea and play a pivotal role in maintaining corneal transparency. Decreased CEnC number causes irreversible corneal edema, leading to loss of transparency. (B) Slit-lamp anterior segment microscopy imaging in A-R syndrome. Loss of transparency due to endothelial dysfunction–induced corneal edema. Pupil is distorted due to iris atrophy. In these eyes, aqueous humor (AqH) is inflamed. Photo credit: Takefumi Yamaguchi, Tokyo Dental College Ichikawa General Hospital. (C) Long-term graft survival stratified by iris atrophy severity after endothelial keratoplasty. The prognosis of eyes with severe iris atrophy score (IAS3 to IAS4) was significantly poor (n = 172; log-rank Mantel-Cox test, P < 0.0001). (D) Protein levels in AqH increase with iris atrophy severity in human eyes (Spearman’s correlation analysis, r = 0.468, P < 0.0001). (E) Correlation between preoperative protein levels in AqH and the CECD at 12 months after endothelial keratoplasty (Spearman’s correlation analysis, r = −0.408, P < 0.0001). Other time points are given in table S2. (F) Graft survival was significantly shortened in eyes with high preoperative AqH protein levels compared to those with lower protein levels (log-rank Mantel-Cox test, P < 0.0001). (G to J) Representative transmission electron microscopy (TEM) of healthy CEnCs (G and H) and CEnCs of bullous keratopathy (I and J). In bullous keratopathy (I and J), TEM reveals mitochondrial vacuolization, electron-dense deposits, and loss of cristae (red arrowheads). (K and L) JC-1 staining representing mitochondrial membrane potential in healthy CEnCs (K) and CEnCs of bullous keratopathy (L). Scale bars, 20 μm. (All samples depicted are human). (M) Human CEnCs were cultured either in normal AqH cocktail (protein level, 0.466 mg/ml) or AqH cocktail from bullous keratopathy (2.40 mg/ml) for 48 or 24 hours, respectively. (N to Q) JC-1 staining. CEnCs were cultured in normal AqH for 48 hours (N), in normal AqH cocktail for 24 hours, and then transferred into AqH cocktail from bullous keratopathy for 24 hours (O), vice versa (P), and in AqH cocktail from bullous keratopathy for 48 hours (Q). Scale bars, 50 μm.

  • Fig. 2 Proteomic analysis of AqH in bullous keratopathy.

    (A) Venn diagram displaying the number of statistically significant identified altered proteins in eyes with bullous keratopathy. (B) The results of FDR ≤ 0.05 (five healthy eyes versus five eyes with bullous keratopathy) depicted on a volcano plot. CFB, complement factor B; GAPD, glyceraldehyde-3-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; TPI, triosephosphate isomerase; TKT, transketolase; TSP1, thrombospondin1; FC, fold change. (C and D) Complement factors were significantly elevated in bullous keratopathy as compared to healthy controls (C), whereas the glycolysis-related proteins were significantly decreased (D). IgG, immunoglobulin G; acetyl-coenzyme A, CoA. (E) Heat map illustrating quantitative alterations of representative proteins (BK, bullous keratopathy; CT, healthy controls). (F and G) Canonical biological processes which were significantly up-regulated (F) and down-regulated (G) in bullous keratopathy were characterized using DAVID GO analysis. (H and I) Functional association network of up-regulated (H) and down-regulated (I) proteins by STRING classification with high confidence (score = 0.700).

  • Fig. 3 Transcriptomic analysis of CEnCs in bullous keratopathy.

    (A) Venn diagram displaying the number of statistically significant identified altered genes in CEnCs with bullous keratopathy. (B) The principal components analysis results clearly divided healthy and dysfunctional CEnCs (seven eyes with bullous keratopathy versus four healthy eyes). (C) The results of FDR < 0.05 (healthy eyes versus bullous keratopathy) depicted on a volcano plot. (D) Heatmap illustrating quantitative alterations of representative genes. (E and F) Canonical biological process were significantly up-regulated (E) and down-regulated (F) in CEnCs of bullous keratopathy were characterized using DAVID GO analysis. (G) Significant alterations in the Wnt/MGM2/PGAM2 and SIRT1/NAMPT/SLC12A8 pathways, such as up- and down-regulation of the CEnC senescence markers, p27Kip1 and p16INK4a, implicated stress-induced senescence in bullous keratopathy. (H and I) Inflammatory cytokine (H) and 8-OHdG (I) levels were significantly up-regulated in eyes with bullous keratopathy (cytokine, n = 194 eyes; 8-OHdG, n = 33 eyes) as compared to healthy controls (cytokine, n = 51 eyes; 8-OHdG, n = 27 eyes; all P ≤ 0.0051). Unpaired Student’s t test. (J) Immunohistochemistry of 8-OHdG was positive only in CEnCs of bullous keratopathy. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 4 The DBA2J murine model mimics the human bullous keratopathy pathology.

    (A) Iris structure is normal in DBA2J mice at 6 weeks (red arrowheads); however, iris stromal atrophy spontaneously develops at 20 weeks (red arrows). Scale bars, 100 μm. Photo credit: Kazunari Higa, Tokyo Dental College Ichikawa General Hospital. (B) Immunohistochemistry of ZO-1 in DBA2J mice. CECD significantly decreased at 20 weeks in DBA2J mice, whereas no significant difference was noted in BALBc mice (all, n = 5 mice). Scale bars, 50 μm. (C) Electron microscopy revealed mitochondrial vacuolization at 20 weeks (right, red arrowheads) similar to what was observed in human CEnCs of bullous keratopathy, whereas mitochondria appeared normal at 6 weeks (left). Scale bars, 500 nm. CEnC mitochondrial mass (by MitoTracker) was significantly decreased at 20-week compared to 6-week mice. Scale bars, 20 μm. (D) JC-1 staining was decreased in 20-week DBA2J compared to 20-week BALBc mice. Scale bars, 20 μm. (E) Protein levels were significantly elevated from 6- to 20-week DBA2J mice (all, n = 6 mice, P < 0.0001), whereas no statistical difference was observed in BALBc mice. (F) AqH IL6, monocyte chemotactic protein-1 (MCP-1), and LIF protein levels were significantly elevated at 20-week compared to 6-week DBA2J mice. Iris IL-6, MCP-1, and LIF protein levels were significantly elevated at 20-week compared to 6-week DBA2J mice (all, n = 12 mice, P < 0.0001), whereas corneal IL-6, MCP-1, and LIF protein levels were elevated at both 6- and 20-week DBA2J, as compared to BALBc mice. ND, not detected. (G) A significant increase in the number of immune cells in the AqH was observed in 20-week compared to 6-week and BALBc mice. Scale bars, 50 μm. Unpaired Student’s t test, *P < 0.01. (H) Immune cells were positive for CD10 and MPO immunostaining. Scale bar, 10 μm.

  • Fig. 5 Convalescence of DBA2J CEnCs when transplanted into BALBc mice.

    (A) Corneal endothelial tissues were transplanted from 6- and 20-week DBA2J into 6-, 20-week DBA2J, or BALBc mice to evaluate whether CEnC damage in DBA2J mice is due to the pathological AqH microenvironment secondary to the atrophic iris. Unexpectedly, 6-week DBA2J mice developed severe inflammation after syn-/allogeneic grafts, and all ended in graft failure. (B) Representative images of corneal grafts. Failed grafts became white and edematous. DBA2J (syngeneic) and BALBc (allogeneic) grafts into DBA2J mice failed within a week despite the absence of apparent immunological rejection, whereas grafts into BALBc mice successfully survived (n = 10 to 12 mice, log-rank Mantel-Cox test, P < 0.0001). Photo credit: Kazunari Higa, Tokyo Dental College Ichikawa General Hospital. (C and D) To confirm whether graft failure was due to corneal endothelial decompensation, we assessed graft CEnCs by mitochondrial staining (MitoTracker). Failed grafts exhibiting mitochondrial vacuolization (red arrowheads) and decreased number of CEnCs. Notably, CEnCs transplanted from 20-week DBA2J to BALBc mice were normal (yellow arrowheads). Scale bars, 50 μm. NS, not significant. (E) Cytokine levels in AqH after corneal transplantation were measured using a multiplex immunobead assay. Cytokine levels were significantly elevated in eyes with failed grafts (DBA2J/BALBc to DBA2J), whereas in eyes with survived grafts (DBA2J to BALBc). Unpaired Student’s t test, *P < 0.01. (F) Immune cells numbers in AqH after corneal transplantation remained at the same levels in 20-week DBA2J mice compared to those in a steady state, whereas they were significantly increased in eyes with allogeneic transplantation (BALBc to DBA2J), in which cytokine levels were extraordinarily elevated. Scale bars, 50 μm. (G) JC-1 staining revealed that mitochondrial membrane potential was reversed in grafts transplanted from 20-week DBA2J to BALBc compared to naïve 20-week DBA2J mice. Scale bars, 20 μm.

  • Fig. 6 Comprehensive mechanism of bullous keratopathy development.

    (A) Healthy iris pigmentary epithelial cells in a steady state exhibit immunomodulatory property. Protein and cytokines levels in AqH are normal, and no immune cells are present. Thus, CEnCs are kept healthy. (B) In bullous keratopathy eyes with atrophic iris, iris tissue produces high levels of cytokines, such as IL-6, MCP-1, and LIF, which are released into AqH, and, in turn, recruit immune cells into AqH. Atrophic iris also results in the breakdown of the BAB. Subsequently, proteins, cytokines, and complement factor levels in AqH increase. Chronic exposure to this AqH environment leads to up-regulation of cytokine receptors expression in the surface of CEnCs. Such pathological microenvironment alterations lead to mitochondrial vacuolization, increased oxidative stress, cellular senescence, and reduced glycolysis inside CEnCs, which lastly ends in loss of CEnCs.

Supplementary Materials

  • Supplementary Materials

    Pathological processes in aqueous humor due to iris atrophy predispose to early corneal graft failure in humans and mice

    Takefumi Yamaguchi, Kazunari Higa, Yukari Yagi-Yaguchi, Koji Ueda, Hisashi Noma, Shinsuke Shibata, Toshihiro Nagai, Daisuke Tomida, Ririko Yasu-Mimura, Osama Ibrahim, Ryo Matoba, Kazuo Tsubota, Pedram Hamrah, Jun Yamada, Kohsuke Kanekura, Jun Shimazaki

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