TGF-β2 levels in the aqueous humor are elevated in the second eye of high myopia within two weeks after sequential cataract surgery

Transforming growth factor-β (TGF-β) is the critical regulator of physiological and pathological conditions in lens. The TGF-β signaling pathway is closely associated with high myopia patients. Thirty eyes from fifteen patients with high myopia who received sequential cataract surgery were enrolled in this prospective study. Ten cataract patients with non-myopia were chosen as a control group. Aqueous humor (AH) samples were used to detect the levels of TGF-β1, TGF-β2, and TGF-β3 in both groups. Compared with the non-myopic cataracts patient group, the highly myopic cataracts group had a significantly higher TGF-β2 (P = 0.019). Besides, the level of TGF-β2 of the second eye was significantly higher than that in the first eye in high myopia cataract patients group (P = 0.037). And TGF-β1 showed significant differences with age and axial length of high myopia cataract patients. Therefore, TGF-β2 may contribute to the development of high myopia and cataract surgery increased the expression of TGF-β2.

Aqueous humor sample collection. Samples were obtained at the beginning of clear lens extraction or cataract extraction surgery. A 27-gauge needle was used to aspirate AH samples (100-200 μl) from the central pupillary area without touching the iris, lens, or corneal endothelium to avoid trauma and contamination of AH samples. Specimens were placed inside sterile Eppendorf tubes that were immediately stored below − 80 °C until analysis.
Analysis of TGF-β1, TGF-β2, and TGF-β3 concentrations in AH samples. All analytical procedures were performed according to the manufacturer's instructions. Proprietary techniques were used to internally color-code microspheres with two fluorescent dyes. After concentrating the dyes, each dye was coated with a specific capture antibody. Next, an analysis from a test sample was captured by the bead to introduce the detection antibody. A Luminex® analyzer (MAGPIX®) was used to identify each microsphere, and the result of its bioassay was quantified based on fluorescent reporter signals. EMD Millipore combines the streamlined data acquisition power of Luminex® xPONENT® acquisition software with the sophisticated analytic capabilities of the new MILLIPLEX® Analyst 5.1, integrating data acquisition and analysis seamlessly with all Luminex® instruments.
Statistical analysis. All data are mean ± standard deviations. The χ2 tests was used to examine differences in categorical variables. Shapiro-Wilk tests were employed to assess whether the data were normally distributed. If the criteria for normal distribution were satisfied, an unpaired/paired t-test was used for comparison; if not, the nonparametric Mann-Whitney-U-test was adopted. The student's t-test was used to detect differences between the first-and second eye surgery groups. unpaired t-tests were used to detect differences between the high myopia cataract patients and non-myopia cataract patients. The linear regression test was used to analyze the relationship between TGF-β1, TGF-β2, and TGF-β3 concentrations and AL, ages, and gender. The results of each statistical test were shown in Supplementary Table 1. All analyses were performed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA). A two-tailed P < 0.05 was considered statistically significant for this analysis.

Results
Baseline characteristics of all cataract patients. The study comprised a total of 30 eyes (15 patients) who satisfied the inclusion criteria. Demographic characteristics of the patients are present in Table 1 and  Table 2. There were no statistically significant differences among the cataract (control) group and surgery eyes group in terms of age and sex. And the two surgery eyes group differences in operated eye, AL and ACD were not statistically significant (all P > 0.05, unpaired/paired t-test for age, AL and ACD; χ2 tests for sex and eye[s] operated on). Table 3 shows the statistics of best-corrected visual acuity, the visual acuity without correction, and spherical equivalent diopter of both eyes during the preoperative time and 1 week and 1 month after surgery.
Concentrations of TGF-β1, TGF-β2, and TGF-β3. The comparison of TGF-β1, TGF-β2, and TGF-β3 concentrations in AH between high myopia cataract patients and non-myopia cataract patients, and the first and second operated eyes are summarized in Table 4. The concentration of TGF-β2 in high myopia cataract patients was significantly greater than in cataract patients (P = 0.019), whereas TGF-β1 and TGF-β3 concentrations were www.nature.com/scientificreports/ not significantly different between these two groups (P = 0.509, P = 0.985). Furthermore, the level of TGF-β2 was significantly higher in the second operated eye than in the first operated eye (P = 0.037). However, the levels of TGF-β1 (P = 0.324) and TGF-β3 (P = 0.76) did not exhibit significant differences between these two groups of eyes.

Discussion
In the present study, we identified elevated TGF-β2 levels in high myopic cataract patients. The results showed that the levels of TGF-β2 were significantly increased in patients undergoing cataract surgery with high myopia on the second eye than those on the first eye. Meanwhile, the level of TGF-β2 was positively higher in high myopia cataract patients than in non-myopia cataract patients. TGF-β1 concentration in the AH was positively correlated  www.nature.com/scientificreports/ with AL and age, on the other hand, the level of TGF-β2 decreased with age. We did not find a significant correlation between the levels of TGF-β1, TGF-β2, and TGF-β3 with gender. TGF-β2 is an important factor in modulating the growth and development of the eyeball 12 . Animal models demonstrated that the sclera level of TGF-β2 protein was highly correlated with the high myopia 13,14 . In addition, clinical researches have shown that the concentration of TGF-β2 in AH is more elevated in high myopia cataract patients than in non-myopia cataract patients 11,15 . However, there are inconsistencies in the TGF-β2 expression during myopia formation as some studies have reported a decrease or do not significantly change 13,16,17 . It appears that the TGF-β2 expression alterations during the development of myopia appear to be species and tissue specific 18 . Our study found that TGF-β2 levels were increased in AH sample in high myopia patients compared to non-myopia patients. However, although TGF-β2 is a key factor in the progression of high myopia development, it is still unclear what role TGF-β2 plays in the process.
Interestingly, our study showed that the TGF-β2 concentration was more significant in the second eye compared with the first eye in high myopia cataract patients. Because the microenvironment conditions of the second eye can be affected by cataract surgery in the first eye 19 . Meanwhile, the TGF-β family is an important tissue repair mediator that shows a distinct pattern during normal acute wound healing 20 . According to Thompson et al., surgery-induced epithelium damage could increase TGF-β2 levels twofold 21 , therefore the higher TGF-β2 levels in the second operated eye could represent a response to the first eye operation. In addition, our previous study found a higher level of TGF-β2 in the second cataract surgery eye compared to the first surgery eye 9 . Both of our studies identified that cataract surgery increased the level of TGF-β2 in cataract patients and high myopia cataract patients. Furthermore, we compared the extent of increase of TGF-β2 in both groups of patients. For cataract patients, the level of TGF-β2 in the second eye increased by 23.49%, from 2419.84 pg/ml to 2988.19 pg/ml. For high myopia cataract patients, the level of TGF-β2 in the second eye increased by 17.27%,   www.nature.com/scientificreports/ from 3346.67 pg/ml to 3924.73 pg/ml. Although the increase rate of high myopia cataract patients was lower than cataract patients, which may contribute to that high myopia patients already shown a higher level of TGF-β2. However, high level of TGF-β2 may increase the risk of capsular contraction syndrome (CCS) and posterior capsular opacification (PCO), which are the complication of cataract surgery 18 . Further research is needed to investigate whether TGF-β2 inhibitors could decrease the risk of these complications. The relationship between the level of TGF-β and AL has been compared in numerous in vivo and in vitro studies 22,23 . In a chick model, Rohrer and Stell observed that TGF-β1 was a potent inhibitor of basic fibroblast growth factor, which restrains the progression of myopia 22 . Furthermore, clinical investigation, Haiso et al. found that TGF-β1 was increased in eyes with excessive elongation of AL, which was consistent with our results 23 . Some studies have also shown a relationship between TGF-β2 and AL. For example, Jia et al. reported that the concentrations of TGF-β2 in 65 patients were positively correlated with AL 15 . In our study, we did not find a significant correlation between the level of TGF-β2 and AL. However, the AL in that study ranged from 24 to 29 mm, while our subjects ranged from 26 to 34 mm. In addition to the AL, we discovered substantial variations in TGF-β1 and TGF-β2 levels with age, with TGF-β2 being adversely linked with age. Studies on glaucoma patients reported that the TGF-β2 concentration in AH decreased with age 24,25 . The cause is thought to be the decreased ability with age of the tissues to produce TGF-β2 24 .
In conclusion, the results of the present study revealed that the concentration of TGF-β2 in the AH samples was significantly higher in the second surgery eye than in the first surgery eye in high myopia cataract patients, and the high myopia group had significantly higher TGF-β2 concentration than non-myopia cataract group. This finding suggests that eyes with high myopia may have a special internal microenvironment and TGF-β2 may contribute to the development of high myopia. Meanwhile, it has been recommended that during binocular surgery for high myopia cataract patients, the contralateral eye should be adequately delayed for surgery time.
Although it may cause problems of binocular balance, it might be able to avoid the complications caused by the increase in TGF-β2 concentration caused by short-term second-eye surgery.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.