Figures & data
Table 1. Target sequences of Smad2, Smad3, gremlin and non-sense control (NC) shRNA.
Table 2. PCR primers used in the study.
Table 3. List of antibodies used for western blot.
Table 4. PCO grade in different groups of rats at 56 days after surgery.
Table 5. Reaction in the anterior chamber after surgery.
Figure 1. Occurrence of PCO in rats treated with gremlin.shRNA AAV, NS or BSS on different days after ECCE. (a) Control group injected with BSS; (b) Negative control group injected with NC.shRNA AAV; (c) Group injected with gremlin.shRNA AAV.
Table 6. PCO grading standard.
Figure 2. Comparison of PCO scores by EPCO software. The PCO scores from the BSS group (a and b), NS group (c and d) and gremlin.shRNA group (e and f) were compared using the EPCO software program. One way ANOVA was used for statistical analyses and significant differences were observed in the PCO scores between the BSS and gremlin.shRNA AAV groups (**p < 0.01) and between the NS and gremlin.shRNA AAV groups (##p < 0.01).
Figure 3. Expression of gremlin and α-SMA protein in the lens capsule, as detected by immunohistochemistry (IHC). Expression of gremlin in the posterior capsule and anterior capsule of the BSS group (a and e), NS group (b and f), gremlin.shRNA group (c and g) and non-surgery group (d and h). Expression of α-SMA in the posterior capsule and anterior capsule of the BSS group (i and m), NS group (j and n), gremlin.shRNA group (k and o) and non-surgery group (l and p). Scale bar: 50 µm.
Figure 4. Gremlin promotes LEC proliferation and induces EMT-specific protein expression in a capsular bag model. A capsular bag model was established and cultured with or without gremlin. (a) Control group on day 14; (b, c, d, e) The expression of α-SMA in capsular bags in the control group; (f) Capsular bags that were cultured with 200 μg/L gremlin on day 14; (g, h, i, j) Expression of α-SMA in capsular bags after treatment with gremlin. (b, g) Natural light; (c, h) IHC of α-SMA; (d, i) DAPI staining; (e, j) Merged. The experiments were repeated three times with five samples in each group each time.
Figure 5. Effect of gremlin on the expression of BMPRs and BMPs in HLECs. (a, b) BMPRIA and BMPRII expression in HLECs. HLECs were treated with different concentrations of gremlin or with 200 μg/L gremlin for different lengths of time. (c) Detection of BMPRIA and BMPRII expression; (d) Detection of BMP4 protein expression. (e) Detection of BMP7 protein expression.
Figure 6. Effects of Gremlin on the levels of Bmp/Smad1/5. (a, b, c) Western blot assay was used to detect the expression of p-Smad1/5, Smad1 and Smad5 proteins induced by 200 μg/L gremlin, 10 μg/L BMP4 and 200 μg/L gremlin+10 μg/L BMP4 for different durations. (d, e) Real-time PCR and western blot assay were used to detect the expression of α-SMA, Fn and COL-1 following treatment with gremlin, BMP4, gremlin+BMP4, and gremlin+BMP4+ Smad1/5.siRNA.
Figure 7. Effects of Gremlin on the levels of AKT, ERK and Smad2/3. HLECs were treated with 200 μg/L gremlin for different lengths of time (0, 5, 15, 30, 60, 120, 240 and 480 min), (a) Detection of p-Smad2, Smad2, p-Smad3 and Smad3 protein expression by western blot; (b) Detection of p-ERK1/2, ERK1/2, p-AKT and AKT protein expression by western blot; (c) p-p38, p38, p-FAK and FAK protein expression as detected by western blot. Effects of the MEK inhibitor U0126 on gremlin-induced expression of α-SMA, Fn, COL-1, p-AKT, AKT, p-ERK and ERK in HLECs. (d) mRNA levels of α-SMA, Fn and COL-1 in HLECs treated with or without gremlin and/or LY294002; ** p < 0.01 versus the controls; ##p < 0.01 versus the group treated with gremlin or gremlin+LY294002. (f) Protein expression levels of α-SMA, Fn, COL-1, p-AKT, AKT, p-ERK and ERK in HLECs treated with or without gremlin and/or LY294002. (e) mRNA levels of α-SMA, Fn and COL-1 in HLECs treated with or without gremlin and/or U0126; *p < 0.05 and **p < 0.01 versus the controls; ##p < 0.01, statistically significant difference between the group treated with gremlin and the group treated with gremlin+U0126. (g) Protein expression levels of α-SMA, Fn, COL-1, p-ERK, ERK, p-AKT and AKT in HLECs treated with or without gremlin and/or U0126. (h) Protein expression levels of p-AKT, AKT, p-ERK and ERK in HLECs treated with or without gremlin and/or Smad2.shRNA. (i) Protein expression levels of p-AKT, AKT, p-ERK and ERK in HLECs treated with or without gremlin and/or Smad3.shRNA.
Figure 8. Schematic diagram of the proposed mechanism by which gremlin regulates EMT and ECM protein expression in LECs. The expression level of gremlin was increased in the aqueous humor after cataract surgery. Gremlin promoted EMT and ECM synthesis by activating the Smad2/3, ERK and AKT signaling pathways and suppressing the BMPs/Smad1/5 signaling pathway. Accumulation of EMT and ECM proteins led to the development of PCO.
