1. Cell Biology

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

  1. Lijuan Xu
  2. Xinyao Zhang
  3. Yin Zhao
  4. Xiaorui Gang
  5. Tao Zhou
  6. Jialing Han
  7. Yang Cao
  8. Binyan Qi
  9. Shuning Song
  10. Xiaojie Wang
  11. Yuanbo Liang  Is a corresponding author
  1. State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, China
https://doi.org/10.7554/eLife.81198
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Cite this article
  1. Lijuan Xu
  2. Xinyao Zhang
  3. Yin Zhao
  4. Xiaorui Gang
  5. Tao Zhou
  6. Jialing Han
  7. Yang Cao
  8. Binyan Qi
  9. Shuning Song
  10. Xiaojie Wang
  11. Yuanbo Liang
(2023)
Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals
eLife 12:e81198.
https://doi.org/10.7554/eLife.81198
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Abstract

This study aimed to investigate the protective effect of metformin on trabecular meshwork (TM) and explore its molecular mechanisms in vivo and in vitro. Ocular hypertension (OHT) mouse models were induced with dexamethasone and further treated with metformin to determine the intraocular pressure (IOP)-lowering effect. Cultured human TM cells (HTMCs) were pre-stimulated with tert-butyl hydroperoxide (tBHP) to induce oxidative damage and then supplemented with metformin for another 24 hr. The expression of fibrotic markers and integrin/Rho-associated kinase (ROCK) signals, including α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), fibronectin, integrin beta 1, ROCK 1/2, AMP-activated protein kinase, myosin light chain 1, and F-actin were determined by western blotting and immunofluorescence. Reactive oxygen species (ROS) content was analysed using flow cytometry. Transmission electron microscopy was performed to observe microfilaments in HTMCs. It showed that metformin administration reduced the elevated IOP and alleviated the fibrotic activity of aqueous humour outflow in OHT models. Additionally, metformin rearranged the disordered cytoskeleton in the TM both in vivo and in vitro and significantly inhibited ROS production and activated integrin/ROCK signalling induced by tBHP in HTMCs. These results indicated that metformin reduced the elevated IOP in steroid-induced OHT mouse models and exerted its protective effects against oxidative injury by regulating cytoskeleton remodelling through the integrin/ROCK pathway. This study provides new insights into metformin use and preclinical evidence for the potential treatment of primary open-angle glaucoma.

Editor's evaluation

The claims that metformin reduced elevated intraocular pressure in vivo and regulated cytoskeleton remodeling in vitro are supported by convincing data. This study provides a new direction for further exploration toward the treatment of primary open-angle glaucoma.

https://doi.org/10.7554/eLife.81198.sa0

Introduction

Intraocular pressure (IOP) elevation, predominantly resulting from increased resistance to aqueous humour outflow (AHO), is a major risk factor for primary open-angle glaucoma (OAG) deterioration (Casson et al., 2012; Wu et al., 2020). The only proven method is IOP lowering (Richter and Coleman, 2016). According to Bill and his colleagues (Bill and Hellsing, 1965; Bill and Svedbergh, 1972), up to 80% aqueous humour is drained via conventional trabecular meshwork (TM) pathway; however, available anti-glaucoma medications mostly act on sites other than TM and have limited efficiency. Therefore, polypharmacy has become increasingly prevalent, coupled with an increasing economic burden on society and patients (Wu et al., 2020).

IOP elevation is associated with TM stiffness (Alvarado et al., 1984; Heijl et al., 2002; Johnstone et al., 2021). Theoretically, drugs promoting the recovery of damaged TM are potentially effective in lowering IOP. Integrin and Rho-associated protein kinase (ROCK) play pivotal roles in cytoskeletal formation and maintenance (Tan et al., 2020; Yemanyi et al., 2020), and ROCK inhibitors (ROCKi) can decrease actomyosin contraction and actin crosslinking (Liu et al., 2021). ROCKi are the only drug that directly target conventional outflow function (Aga et al., 2008; Rao et al., 2001; Ren et al., 2016). It alters the architecture of AHO and expands the juxtacanalicular connective tissue region. Currently, the clinically available ROCKi include Y-27632 and ripasudil.

Metformin (MET), an oral biguanide insulin-sensitising drug, is the most widely used treatment for type 2 diabetes mellitus (DM) (Foretz et al., 2014). It is a multifunctional drug (Rangarajan et al., 2018; Zhao et al., 2021). Studies by Lin et al., 2015 and Maleškić et al., 2017 found that MET reduced the risk of OAG in patients with DM, and this effect persisted even after controlling for glycated haemoglobin. However, this effect was not observed with other hypoglycaemic medications (insulin, sulfonylureas, thiazolidinediones, and meglitinides), suggesting that the protective effect of MET on glaucoma goes beyond glycaemic improvement. However, the precise mechanisms involved remain unclear.

Excessive reactive oxygen species (ROS) in TM play an important role in the disruption of cytoskeletal integrity and apoptosis (Hu et al., 2017), leading to pathological alterations in AHO and subsequent IOP rise (Babizhayev and Bunin, 1989; Saccà et al., 2016). Reduction of intracellular ROS levels by MET via activating AMPK signal has been reported in primary hepatocytes (Ota et al., 2009), vestibular cells (Lee et al., 2014), and human immune cells (CD14+ monocytes, CD3+ T cells, CD19+ B cells, and CD56+ NK cells) (Hartwig et al., 2021). Conversely, there is also evidence on cellular ROS level increase in some cancer cells after MET treatment, including AsPC-1 pancreatic (Warkad et al., 2021), osteosarcoma (Li et al., 2020a), and breast cancer cells (Yang et al., 2021). These seemingly contradictory results suggested that MET plays different roles under different metabolic environments. To investigate the role of MET in damaged TM cells and ocular hypertension (OHT) mouse models, we used tert-butyl hydroperoxide (tBHP) to induce oxidative damage in TM cells (Tang et al., 2013; Wang et al., 2021) and topical glucocorticoids to create OHT mouse models (Li et al., 2021). The results showed that MET protected against cytoskeletal destruction in TM by activating the integrin/ROCK pathway and alleviated elevated IOP in steroid-induced OHT mouse models.

Results

Steroid-induced OHT in mice

The experiment included two steps: first, OHT modelling and the subsequent drug’s IOP-lowering effect test were performed, both of which lasted for 28 days. A steroid-induced OHT mouse model was successfully established bilaterally in the first step. The baseline IOP did not differ between dexamethasone (DEX)-treated and phosphate-buffered saline (PBS)-treated vehicle eyes; however, starting at day 5, IOP was significantly elevated in DEX-treated eyes (p < 0.01, Figure 1A) and stabilised by day 28 (defined as an increase by >3 mm Hg or 30% from the baseline in at least two of the three IOP tests [days 23, 26, and 28]). The body weight (BW) was significantly lower in DEX-treated mice than in controls from days 7 to 28 (Figure 1B). Approximately 90.0% DEX-treated eyes had elevated IOP after 28 days of steroid induction with an average increased magnitude of 4.12 ± 1.26 mm Hg (2.00–7.30 mm Hg). Moreover, the fibrotic marker (α-SMA) was obviously overexpressed in the OHT mouse models (p <0.05, Figure 1D–F).

Figure 1
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Topical ocular DEX-induced OHT in mice.

(A) Elevated IOP in DEX-treated C57BL/6J mice was induced significantly at 4 weeks (p < 0.01). (B) The body weight comparison between the DEX-treated mice and control. (C) HE staining of OHT models. Scale bar, 50 µm. (D) α-SMA and TGF-β staining in the representative OHT models. Scale bar, 20 µm, (E, F) Quantification of α-SMA and TGF-β of the models. *p < 0.05, **p < 0.01, ns: non-significance, DEX: dexamethasone, PBS: phosphate-buffered saline, HE: haematoxylin and eosin, IOP: intraocular pressure, OHT: ocular hypertension, BW: body weight, TM: trabecular meshwork, SC: Schlemm’s canal, CB: ciliary body, α-SMA: α-smooth muscle actin, TGF-β: transforming growth factor-β.

MET effectively reversed steroid-induced OHT in mice

In this step, successfully DEX-induced OHT mouse models were randomly assigned into three groups with the continued prescription of DEX: DEX withdrawal (group 2, n = 10), DEX + PBS (group 3, n = 8), and DEX + 0.3% MET (group 4, n = 18). Meanwhile, a group of mice were treated with solo PBS as control with a duration of 8 weeks (group 1, n = 8).

The experimental procedure is illustrated in Figure 2A. The IOPs after 28 days of steroid induction in all three OHT groups were similar (17.27 ± 1.08, 16.91 ± 1.01, and 17.06 ± 0.82 mm Hg, respectively). Compared with group 3, DEX-withdrawal mice had significantly reduced IOP after 17–28 days (all p < 0.05). After 5 days of MET treatment, IOPs significantly reduced in group 4 (p < 0.01, Figure 2B). In fact, MET almost completely neutralised steroid-induced OHT, returning IOP to near DEX-withdrawal levels on day 21, suggesting a therapeutic role of MET in OAG. Specific IOP values are shown in Figure 2B.

Figure 2
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Effect of MET on OHT mouse models.

(A) Experimental process overview. (B) MET effectively reversed the IOP in steroid-induced OHT mouse models. **p < 0.01 (comparison between groups 3 and 4), ##p < 0.01 (comparison between groups 2 and 4), △p < 0.05, and △△p < 0.01 (comparison between groups 1 and 4). DEX: dexamethasone, MET: metformin, PBS: phosphate-buffered saline, IOP: intraocular pressure, OHT: ocular hypertension.

MET attenuated the steroid-induced TM cytoskeletal destruction in vivo

As shown in Figure 3, MET improved fibrosis and the intensity of phalloidin labelling of F-actin in the TM tissue of steroid-induced OHT C57BL/6 mice. Quantitative comparison showed a significant difference in the number of α-SMA- and fibronectin (FN)-positive TM cells between MET-treated and control mice (Figure 3C, E). Additionally, MET treatment promoted the cytoskeleton recovery of steroid-induced TM cell damage, as confirmed by the pronounced upregulation of F-actin (Figure 3F). We concluded that the IOP-lowering effect of MET in this steroid OHT model can be largely explained by the attenuation of fibrotic alterations and rearrangement of the cell skeleton at sites of TM or trabecular outflow pathways.

Figure 3
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MET decreased the expression of fibrotic markers in steroid-induced trabecular meshwork stiffening in mice.

Representative images of HE (A) and fibrotic markers (B). Scale bar, 20 µm, (C) Quantification of α-SMA of the models. (D) Quantification of TGF-β of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. (E) Quantification of fibronectin (FN) of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. (F) Quantification of F-actin of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. *p < 0.05, ns: non-significance, DEX: dexamethasone, HE: haematoxylin and eosin, MET: metformin, PBS: phosphate-buffered saline, α-SMA: α-smooth muscle actin, TGF-β: transforming growth factor-β. White arrows indicate the representative positive cells.

Protective effects of MET on TM in vitro

To test the effect of MET on TM in vitro, we performed studies on human TM cells (HTMCs). Western blotting (WB) showed that the expression of myocilin, a glucocorticoid-inducible gene in HTMCs, increased after DEX treatment (Figure 4B), confirming that the cells had characteristics of TM cells.

Figure 4 with 1 supplement see all
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Low dose of MET reversed the disarranged morphology of HTMCs.

(A) HTMCs were treated with MET for 24 hr with or without pre-treatment of tBHP for 1 hr. Representative images of cell distribution and morphology photographed by inverted microscopy. Scale bar, 50 µm, (B) The expression of myocilin after DEX treatment in HTMCs. (C, D) The relative HTMC viability after exposure to MET with different concentrations. Cell proliferation was measured using the CCK8 assay. (E, F) The relative ROS levels were assayed via flow cytometry, and the results showed that MET reduced the ROS production of HTMC induced by tBHP. *p < 0.05, **p < 0.01. DEX: dexamethasone, HTMCs: human trabecular meshwork cells, MET: metformin, ROS: reactive oxygen species, tBHP: tert-butyl hydroperoxide, TM: trabecular meshwork.

Higher doses (≥10 mM) of MET decreased the number of HTMCs, but this inhibitory effect was less evident at lower doses (<10 mM) (Figure 4C). To verify the protective role of MET in HTMCs, cells were pre-treated with 100 μM tBHP for 1 hr to induce chronic oxidative stress (Tang et al., 2013), and subsequently exposed to low doses of MET (3, 5, and 10 mM) (shortened as L-MET in this study). The results showed that L-MET significantly reversed the inhibitory effect induced by tBHP (Figure 4A, D).

Furthermore, cells treated with L-MET showed less intracellular ROS signals compared to the control, and 1 mM MET reduced tBHP-induced ROS production. These results were confirmed by flow cytometry (FCM) analysis (Figure 4E,F) and further evidenced in DEX-induced (500 nM, 7 days) ROS increase in HTMCs (Figure 4—figure supplement 1). These results indicated that MET reversed oxidative damage to HTMCs.

MET restored tBHP-induced cytoskeletal destruction in HTMCs and activated integrin/ROCK signals

Actin filaments play key roles in cortical polarisation and asymmetric spindle localisation during phagocytosis in HTMCs. F-actin was stained to determine whether actin dynamics were involved in tBHP-induced cellular dysfunction. As shown in Figure 5A, F-actin was evenly accumulated in the cytoplasm with a robust fluorescent signal in control HTMCs. Exposure to L-MET did not change the morphology of HTMCs as observed by inverted phase-contrast microscopy. Contrarily, HTMCs exposed to 1 hr of tBHP displayed intermittent distribution of actin filaments with faded fluorescent signals. Additionally, tBHP treatment significantly destroyed HTMCs, manifesting as various morphology-aberrant cells with misaligned cytoplasm. These pathological changes could be partially rescued by L-MET, implying that MET could restore the dynamic instability from oxidative damage. Furthermore, WB results (Figure 5B, C) showed that L-MET significantly activated the integrin/ROCK pathway by upregulating integrin, ROCK, AMPK, and pAMPK in healthy HTMCs, and significantly activated integrin, ROCK, pAMPK, and F-actin in damaged HTMCs, and it was more pronounced in the 3 mM doses.

Figure 5
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MET promoted the recovery of tBHP-induced cytoskeletal destruction (A) and activated the intergrin/ROCK pathway (B) in HTMCs.

Scale bar, 20 µm. (C) The quantitative protein levels of B. *p < 0.05 (comparison with the control), #p < 0.05 (comparison with the tBHP-treated group). HTMCs: human trabecular meshwork cells, MET: metformin, tBHP: tert-butyl hydroperoxide.

The ultrastructure of HTMCs was examined by transmission electron microscopy (TEM) (Figure 6). We observed a significant reduction in the amount and density of microfilaments in the tBHP-treated cells, and these changes recovered after L-MET treatment.

Figure 6
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MET partially normalised the damaged microfilaments of HTMCs induced by tBHP (A).

Scale bar, 2 µm, Red arrows indicate the representative microfilaments imaged by transmission electron microscopy. The red rectangles embedded in the lower left part of each picture are representative microfilaments with a magnification of ×150,000. (B) The quantitative microfilaments of A. *p < 0.05 (comparison with the control), ##p < 0.01 (comparison with the tBHP-treated group). HTMCs: human trabecular meshwork cells, MET: metformin, tBHP: tert-butyl hydroperoxide.

Discussion

The major observations of the current study were the three significant phenotypic changes induced by MET in OHT mouse eyes and HTMCs. The first was the IOP-lowering effect in steroid-induced OHT mouse eyes. The second was reversal of the skeletal destruction of TM cells, both in vivo and in vitro. Third, there was a significant decrease in the accumulation of fibrotic markers, namely α-SMA and FN, in TM tissues. These results suggest a protective effect of MET in TM, probably via promoting cytoskeleton recovery through the integrin/ROCK pathway.

Steroid-induced OHT in mice was generated (Wang et al., 2018) with the characteristics of TM stiffening and elevated IOP (Li et al., 2019). In line with previous studies (Clark et al., 2001; Johnson et al., 1997; Johnson et al., 1990), our model suggested that the stiffness of AHO closely matched that of OAG in humans, including the deposition of extracellular matrix (ECM) in the TM, disordered cytoskeleton, increased AHO resistance, and elevated IOP. Similar to the study by Zode et al., 2014; Ren et al., 2022, our topical DEX elevated the IOP by 2.00–7.30 mm Hg after 4 weeks of induction.

TM tissues play an important role in the regulation of AHO function and IOP (Wang et al., 2018). TM cells have the properties of extracellular debris phagocytosis and ECM degradation. If these functions are disturbed, the balance of TM in keeping AHO free of obstructive debris is disrupted (Last et al., 2011). It is noteworthy that MET appeared to restore biomechanical properties of TM in our steroid-induced OHT model after 5 days of treatment, and this restoration was accompanied by significant downregulation of ECM proteins in TM.

Moreover, as evidenced by immunofluorescence (IF) and WB analyses, L-MET rearranged the disordered cytoskeleton of HTMCs. This finding was supported by a study conducted by Li et al., 2020b, who showed that 50 μM MET protected oocytes against cytoskeletal destruction. Interestingly, the time course of structural changes in the TM was consistent with the observed pharmacodynamics of MET on IOP, significantly decreasing 4 weeks of OHT because of MET treatment, which was consistent with the known effects of other ROCK regulators (Lin et al., 2018; McMurtry et al., 2010). Taken together, our results indicate that L-MET promotes the regeneration of abnormal HTMCs and enables the recovery of TM function; this should be confirmed in future studies.

As confirmed by WB analysis, complementation with L-MET treatment unregulated integrin, pAMPK, ROCK, and F-actin signals. These in vitro findings indicate that MET might elicit its protective effects through the regulation of the integrin/ROCK pathway in HTMCs. The in vivo observations in the OHT model again support the importance of the integrin/ROCK pathway in TM tissue protection. Generally, these signals mediate cellular biomechanical tension through actomyosin cytoskeletal tension, ECM synthesis, assembly, and degradation (Nakajima et al., 2005; Pattabiraman and Rao, 2010; Rao et al., 2001).

Inconsistent with the previous studies indicating that the AMPK activator suppressed ROCK activity, our study revealed a novel mechanism of MET action within which activation of both AMPK and ROCK signals sequentially upregulated F-actin expression in TM. We hypothesised that ROCK regulators protect TM via different mechanisms, either by inhibiting ROCK signals when increased AHO resistance is attributed to TM stiffness or by activating the ROCK pathway to promote cytoskeletal formation when it is damaged. In the current study, we presumed that MET lowered IOP through short-term changes in TM cell morphology and adhesion by regulating integrin/ROCK signalling. In addition, the steroid-induced OHT recovered its normal IOP with MET treatment, preventing further TM cell loss, whereas MET promoted the maintenance of cytoskeletal morphology and repair after injury; therefore, IOP was maintained at a normal level for an extended period.

Generally, these data emphasise the protective properties of MET in the conventional outflow pathway, consistent with a large body of literature on other tissues (Rangarajan et al., 2018; Yi et al., 2021; Zhao et al., 2021). In addition to MET-mediated changes in ECM turnover, the observed changes in ECM composition and amount may also be due to the MET-mediated opening of flow pathways and the consequential removal of ECM. Additionally, the observed reduction in ROS content in HTMC treated with L-MET was in line with earlier studies displaying its protective effect in several types of cells (Ghasemnejad-Berenji et al., 2018; Huang et al., 2015; Louden et al., 2014). In any case, therapy for steroid-induced OHT, which inhibits the cycle of damage and promotes cell remodelling by restoring function to a diseased tissue, offers a potential benefit for patients.

While experimental data were encouraging, there were limitations in our study. First, the concentration of MET in the anterior chamber was not measured. Additionally, we did not investigate its toxicity on ocular surfaces, such as tear film stability and components, which may change with the application of MET. Second, the sample size of the animals was relatively small, and a larger sample is needed to verify the protective effect of MET on TM. Third, the experimental period was too short to prove the long-term effects of MET and the presented figures were not in the same direction. Thus, additional studies are needed in the future.

In summary, promotion of TM cytoskeleton recovery by MET treatment may be an underlying mechanism for IOP reduction and AHO increase. A thorough investigation of the mechanisms by which cytoskeleton recovery is promoted will improve our understanding of the therapeutic mechanism of MET. If MET is confirmed to promote damaged cytoskeleton recovery and maintenance in the human body, it may have clear therapeutic effects on the main sites of glaucoma pathogenesis, which will broaden their application in the field of glaucoma. In conclusion, we revealed that integrin/ROCK activation reprograms metabolism in TM cells by enhancing the cytoskeleton and downregulating excessive ECM proteins.

Materials and methods

Animals

All the animals were treated in accordance with the principles of the Declaration of Helsinki and in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement. All experiments were approved by the Institutional Animal Care and Use Committee of the Wenzhou Medical University (wydw2022-0209). Healthy C57BL/6J mice (age: 6–8 weeks, male, weight: 20–25 g) were used in this study. The mice were purchased from Zhejiang Vital River Experimental Animal Technology Co. Ltd (Charles River Lab China), bred/housed in clear cages, and kept in housing rooms at 21°C with a 12:12 hr light:dark cycle.

OHT animal model and drug treatments

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The animal experiments were performed in two consecutive steps. In the first step, OHT C57BL/6J mouse model was induced by topical 0.1% DEX phosphate (dissolved in PBS twice daily, 8–9 AM and 5–6 PM) as described previously (Li et al., 2021), and sterile PBS was used as a vehicle control. The second step was conducted 4 weeks after steroid induction. The successfully created OHT mouse models with elevated IOP were randomly divided into three groups: DEX withdrawal (group 2, DEX was stopped without any additional prescribed drug, n = 10), DEX + PBS (group 3, n = 8), and DEX + 0.3% MET (group 4, n = 18). Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment. Meanwhile, PBS-only treatment was administered for 8 weeks in the control (group 1, n = 8). Drugs were prescribed twice daily with at least 15-min interval when administering 2 types of eye drops to ensure effective penetration.

IOP measurements

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Briefly, mice were anaesthetised using gaseous isoflurane (approximately 2 min) and topical alcaine (Alcon-couvreur n. v, Rijksweg, Puurs Belgium). IOP was measured using a rebound tonometer (icare TONOVET; Vantaa, Finland). Each recorded IOP was the average of five measurements and three IOP readings were recorded for the same eye to calculate the mean value. IOP measurements were conducted every 2–3 days (Monday, Wednesday, and Friday, between 2 PM and 3 PM).

Weight measurement

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The anaesthetised mice were gently placed on digital electronic scales (Electronic Scale, Kunshan, China) to measure their weight. The effective reading of BW was recorded to an accuracy of 0.01 g. The BW of each mouse was calculated from the average of three test values.

Cell culture and treatment

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HTMCs were purchased from BNCC (338506, Shanghai, China), which were authenticated by STR profiling. We confirmed that no mycoplasma contamination was detected by regular examinations. Cells were cultured in Dulbecco’s Modified EagIe’s Medium: F-12 (DMEM/F12) (Cytiva, HyClone Laboratories, Logan, UT, USA) containing 10% foetal bovine serum (BI, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Gibco, Life Technologies Corporation, NY, USA) at 37°C and 5% CO2. To identify the characteristics of HTMCs, cells were treated with 500 nM DEX (Shanghai Macklin Biochemical Co., Ltd, China) for 7 days, and the expression of myocilin was evaluated by WB (He et al., 2019). For drug testing, tBHP (Damas-beta, China) and MET (Sigma-Aldrich, St. Louis, USA) were dissolved in DMEM/F12. HTMCs were pre-treated with tBHP solution for 1 hr to induce oxidative damage, followed by 24-hr incubation in normal culture medium containing MET at certain concentrations.

Cell viability

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Cell viability was measured using the CCK8 assay kit (APE BIO), following the manufacturer’s instructions. The cells were seeded in 96-well plates and exposed to increasing concentrations of MET (0, 1, 3, 5, 10, 20, 30, 40, and 50 mM) for 24 hr. The cell viability was determined by measuring the optical density at 490 nm using an absorbance microplate reader (SpectraMax 190; version 7.1.0, Molecular Devices, CA, USA).

Detection of intracellular ROS levels

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Intracellular ROS levels were determined using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe (Beyotime Biotechnology, Shanghai, China) by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent dichlorofluorescein according to the manufacturer’s instructions and a previous study (Xu et al., 2020). Briefly, 10 μM DCFH-DA solution diluted in serum-free DMEM/F12 medium was added and incubated with the cells, then they were kept in the dark for 20 min. Excess DCFH-DA was removed by washing the cells three times with serum-free cell culture medium. ROS was detected by both IF imaging and FCM (BD Accuri C6 Plus, CA, USA), and analysed by Flow Jo (Flowjo X 10.0.7r2). The values were normalised to signals from the control group.

Western blotting

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Cells were lysed 24 hr after drug treatment as previously described (Xu et al., 2020). After gel separation and membrane transfer, myocilin (Abcam), integrin 1 (Abcam), AMPK (CST), pAMPK (CST), ROCK1/2 (Abcam), MLC1 (Abcam), and F-actin (CST) were detected. β-Actin (CST) was used as the loading control. Western blot membranes were developed using a chemiluminescence detection system (Amersham Imager 680RGE; GE Healthcare Bio-Sciences AB, Sweden, Japan).

Histology, immunostaining, and TEM

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At the time of harvest, the mice were re-anaesthetised; the eyes were fixed in 4% paraformaldehyde overnight and embedded in paraffin or optimum-cutting temperature compound in the sagittal axis. The sections were incubated overnight with primary antibodies at 4°C according to the manufacturer’s instructions (Xu et al., 2020). The secondary antibodies were goat anti-rabbit or anti-mouse (CST) at a 1:500 dilution. Sections were subsequently incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime Institute of Biotechnology, Shanghai, China) for 5 min to stain the nuclei, washed, and then mounted.

The cells were fixed with 4% paraformaldehyde for 15 min, permeabilised with 0.25% Triton X-100 for 20 min, and blocked with bovine serum albumin for 30 min at room temperature. After blocking, they were incubated overnight with primary antibodies at 4°C. The next day, the cells were rinsed and incubated with secondary antibodies conjugated to Alexa Fluor (CST) for 1 hr at room temperature. Fluorescent images were obtained using an inverted microscopy (Carl Zeiss Microscopy GmbH, Göttingen, Germany) with the luminescence of X-Cite Series 120(Lumen Dynamics Group Inc, Canada).

For electron microscopy studies, cells were fixed in 2.5% glutaraldehyde (Shanghai Macklin Biochemical Co., Ltd, China) and embedded in Epon resin, and 80-nm sagittal thin sections were cut through iridocorneal tissues using an ultramicrotome (Power Tome-XL, RMC Products, USA). Sections were stained with uranylacetate/lead citrate and examined with a transmission electron microscope (HITACHI, H-7500).

Image analysis

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Immunohistochemical images were obtained using a microscope (ECLIPSE 80i, Nikon) and analysed using the NIS-Elements Imaging Software (3.22.00; Build 700, LO, USA). For quantification, high-power fields (×400 magnification) of the AHO from each model were captured. TM region was defined as the area between the ciliary body and the inner wall of Schlemm’s canal, with anterior and posterior end points of Schwalbe line and scleral spur, respectively (Yan et al., 2022). For microfilament quantification, five fields with ×80,000 magnification of each group were captured with TEM, within which two were from the cell cortex region (defined as within 400 nm beneath the cell membrane) (Svitkina, 2020) and the remaining three were beyond this area. ImageJ (v1.52a, National Institutes of Health, USA) was used to quantify the positively stained cells, which referred to those cells stained by both blue colour from DAPI and green or red signals from the secondary antibodies.

Statistical analysis

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Data analysis was conducted using SPSS (version 22.0) software and GraphPad Prism (version 7.0). Cell- and animal-based experiments included at least three biological and technical replicates. Continuous data are summarised as mean ± standard deviation. Student’s t-test and analysis of variance were used to test the differences in continuous variables. A paired t-test was used to compare IOP changes from the baseline. Statistical significance was set at p value <0.05.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–6 and Figure 4—figure supplement 1.

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Decision letter

  1. Mishaela R Rubin
    Reviewing Editor; Columbia University Medical Center, United States
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States
  3. Mishaela R Rubin
    Reviewer; Columbia University Medical Center, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, including Mishaela R Rubin as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Please provide control data

2) Please provide information about animal numbers, sexes, biological repeats, and technical repeats

3) Please provide information about the duration of dex treatment

4) Please consider providing data using dex-treated cells to detect a metformin anti-oxidant effect

Reviewer #1 (Recommendations for the authors):

In the Introduction, consider removing sentences about the cost and availability of other ROCKi, as these are specific to a geographical location and distracting from the scientific rationale.

In Figure 2B, it looks like IOP might be rising in the MET groups towards the last few days. If possible, please provide data on the mice beyond D42.

There should be mouse control that was not steroid-treated. This would allow comparison of the parameters in the metformin-treated mice with controls.

The authors state (line 160) that the inhibitory effect of MET was not evident at lower doses, but in Figure 4C it looks like 3 mM decreased the number of HTMC.

What were the ROS signals that were measured by FCM (Figure 4E-F)?

In Figure 5, ROCK did not increase statistically in tBHP+MET at any dose, but the text states otherwise (line 192).

In Discussion, line 270, the wording is confusing- "recovery to normal IOP prevents further TM cell loss, whereas metformin…." Does this mean that IOP mice recovered in the absence of metformin?

Reviewer #2 (Recommendations for the authors):

Although the presented results were promising with translational potential in the future, there are some concerns as listed below:

1. The animal numbers and sexes in each group and the biological repeats and technical repeats were not clearly listed which must be listed in the manuscript as well as in the figure legends.

2. Page 2 Abstract: Methods: Subsequent TEM tests were not mentioned.

3. Page 4 lines 64-65: "Theoretically, TM-targeting drugs are potentially effective in lowering IOP caused by diseased TM." This is not clear what the authors meant. Please modify and add references.

4. Page 5 Introduction: Please briefly introduce the effect of MET on ROS.

5. For all the sections with immunofluorescent staining, it's not clear how the authors defined the TM region. They didn't show any specific marker expression and didn't have the same direction as all the figures.

6. Figure 1. How did the authors calculate the a-SMA and TGFb positive cells, especially TGFb is an ECM marker

7. How long did Dex treatment last? Was it stopped at 3 weeks or was it continued together with metformin treatment? If continued, how did they give the different treatments? Please describe those in detail. If Dex was not continued, IOP could possibly reduce in 3 weeks. Please compare if IOP in PBS group has statistically changed or not with that when Dex stropped.

8. Figure 2, several other control groups should also be compared, like PBS only, PBS+metformin eyedrops, and PBS+metformin oral without Dex treatment.

9. Besides the in vitro tBHP induced oxidative cell model, it would be more convincing if the authors could also use Dex-treated cells to detect the metformin anti-oxidation effect since they used Dex induced mouse model.

10. Figure 4, how many biological repeats and technical repeats? One of the characteristics of TM cells is that TM cells have increased expression of myocilin after exposure to Dex. Figure 4B didn't show myocilin expression increasing after Dex.

11. Fig6, it's not clear what the authors marked. How did they mark the microfilaments? A statistical analysis with graphs would be more clear and convincing.

12. The discussion of this study lacks further elaboration. There was no mention of the limitation of the study.

https://doi.org/10.7554/eLife.81198.sa1

Author response

Essential revisions:

1) Please provide control data.

Thank you for this important suggestion. We have conducted a repeated experiment during the submission period and have extended both the OHT modelling process and drugs’ IOP-lowering effect tests from 21 to 28 days to strengthen the power of this study. Metformin oral group was deleted because of the unstable intake amount, which may reduce the reliability of the results. New data, including the control data, have been added to the manuscript. The main points are summarised as follows:

Materials and methods (p. 10, Line 8-22 in the manuscript)

“OHT animal model and drug treatments

The animal experiments were performed in two consecutive steps. In the first step, OHT C57BL/6J mouse model was induced by topical 0.1% DEX phosphate (dissolved in PBS twice daily, 8–9 AM and 5–6 PM) as described previously (Li, et al.,2021), and sterile PBS was used as a vehicle control. The second step was conducted 4 weeks after steroid induction. The successfully created OHT mouse models with elevated IOP were randomly divided into three groups: DEX withdrawal (group 2, DEX was stopped without any additional prescribed drug, n=10), DEX+PBS (group 3, n=8), and DEX+0.3% MET (group 4, n=18). Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment. Meanwhile, PBS-only treatment was administered for 8 weeks in the control (group 1, n=8). Drugs were prescribed twice daily with at least 15-min interval when administering 2 types of eye drops to ensure effective penetration.”

Results (p. 5, Line 4-27 in the manuscript, Figure 2 and 3 in the figure legends)

“MET effectively reversed steroid-induced OHT in mice

In this step, successfully DEX-induced OHT mouse models were randomly assigned into three groups with the continued prescription of DEX: DEX withdrawal (group 2, n=10), DEX+PBS (group 3, n=8), and DEX+0.3% MET (group 4, n=18). Meanwhile, a group of mice were treated with solo PBS as control with a duration of 8 weeks (group 1, n=8).

The experimental procedure is illustrated in Figure 2A. The IOPs after 28 days of steroid induction in all three OHT groups were similar (17.27±1.08, 16.91±1.01, and 17.06±0.82 mmHg, respectively). Compared with group 3, DEX-withdrawal mice had significantly reduced IOP after 17–28 days (all p < 0.05). After 5 days of MET treatment, IOPs significantly reduced in group 4 (p < 0.01, Figure 2B). In fact, MET almost completely neutralised steroid-induced OHT, returning IOP to near DEX-withdrawal levels on day 21, suggesting a therapeutic role of MET in OAG. Specific IOP values are shown in Figure 2B.”

“Figure 2. Effect of MET on OHT mouse models. A. Experimental process overview. B. MET effectively reversed the IOP in steroid-induced OHT mouse models. **p < 0.01 (comparison between groups 3 and 4), ## p < 0.01 (comparison between groups 2 and 4), △p < 0.05 and △△p < 0.01 (comparison between groups 1 and 4). DEX: dexamethasone, MET: metformin, PBS: phosphate buffered saline, IOP: intraocular pressure, OHT: ocular hypertension”

“MET attenuated the steroid-induced TM cytoskeletal destruction in vivo

As shown in Figure 3, MET improved fibrosis and the intensity of phalloidin labelling of F-actin in the TM tissue of steroid-induced OHT C57BL/6 mice. Quantitative comparison showed a significant difference in the number of α-SMA- and fibronectin (FN)-positive TM cells between MET-treated and control mice (Figure 3C, 3E). Additionally, MET treatment promoted the cytoskeleton recovery of steroid-induced TM cell damage, as confirmed by the pronounced upregulation of F-actin (Figure 3F). We concluded that the IOP-lowering effect of MET in this steroid OHT model can be largely explained by the attenuation of fibrotic alterations and rearrangement of the cell skeleton at sites of TM or trabecular outflow pathways.”

Figure 3. MET decreased the expression of fibrotic markers in steroid-induced trabecular meshwork stiffening in mice. A-B. Representative images of HE (A) and fibrotic markers (B). C. Quantification of α-SMA of the models. D. Quantification of TGF-β of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. E. Quantification of fibronectin (FN) of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. F. Quantification of F-actin of the models after 4 weeks of DEX withdrawal or MET/PBS treatment. *p < 0.05, ns: non-significance, DEX: dexamethasone, HE: haematoxylin and eosin; MET: metformin, PBS: phosphate buffered saline. White arrows indicate the representative positive cells.”

2) Please provide information about animal numbers, sexes, biological repeats, and technical repeats.

Thanks for the suggestion. We have added the animal number in the manuscript (see p. 5, Line 6-9 or “Essential revisions 1”). The animal sexes were shown in p. 10, Line 4-5: “ Healthy C57BL/6J mice (age: 6–8 weeks, male, weight: 20–25 g) were used in this study”. Biological and technical repeats were shown in p. 13, Line 21-22: “Cell-based and animal-based experiments included at least three biological and technical replicates.”

3) Please provide information about the duration of dex treatment.

Thanks. We have added the information as follows (see p. 10, Line 16-19 in the manuscript or “Essential revisions 1”):

“Mice in groups 3 and 4 received an additional 4 weeks of supplemented drug (PBS or 0.3% MET) delivered bilaterally with the continued synchronous use of steroid, leading to an entire 8-week duration of DEX treatment.”

4) Please consider providing data using dex-treated cells to detect a metformin anti-oxidant effect.

Thanks for the suggestion. We have conducted the DEX-treated HTMC to evaluate the anti-oxidant effect of metformin. We found that after DEX treatment, the DCF fluorescence intensity was significantly increased in HTMCs, and this effect was attenuated by the additional treatment of low doses metformin. This part of result was presented as supplemental Figure 1 for Figure 4:

“Figure S1. L-MET attenuated the ROS production in HTMCs induced by DEX treatment. A. ROS signals of HTMCs were determined by labelling cells with DCFH-DA and photographed by an inverted phase-contrast microscopy. B. Quantification of DCF-positive cells in A. C. The relative ROS levels assayed by flow cytometry showed that 3mM MET reduced the ROS production of HTMC induced by DEX. **p < 0.01, *p < 0.05, ns: non-significance. DEX: dexamethasone, MET: metformin, ROS: reactive oxygen species, HTMCs: human trabecular meshwork cells, L-MET: low doses of metformin (≤ 10 mM)”

Reviewer #1 (Recommendations for the authors):

In the Introduction, consider removing sentences about the cost and availability of other ROCKi, as these are specific to a geographical location and distracting from the scientific rationale.

Thanks for the comments. We agree with the reviewer’s opinion and have deleted the above sentences from the text.

In Figure 2B, it looks like IOP might be rising in the MET groups towards the last few days. If possible, please provide data on the mice beyond D42.

Thank you for your advice. We have extended the experimental period of drugs’ IOP-lowering effect from 21 days to 28 days, and the new data have been provided in the manuscript (see Figure 2 legend, or “Essential revisions 1”).

There should be mouse control that was not steroid-treated. This would allow comparison of the parameters in the metformin-treated mice with controls.

Thank you for your advice. We have extended the experimental period of drugs’ IOP-lowering effect from 21 days to 28 days, and the new data have been provided in the manuscript (see Figure 2 legend, or “Essential revisions 1”).

The authors state (line 160) that the inhibitory effect of MET was not evident at lower doses, but in Figure 4C it looks like 3 mM decreased the number of HTMC.

Thanks. We have revised the manuscript accordingly (see p. 6, Line 3-4 in the manuscript):

“Higher doses (≥10 mM) of MET decreased the number of HTMCs, but this inhibitory effect was less evident at lower doses (<10 mM) (Figure 4C).”

What were the ROS signals that were measured by FCM (Figure 4E-F)?

Thanks. Intracellular ROS levels were determined by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent DCF. The ROS signals were referred to positive DCF cells analysed by FCM, and the explanation was added in the manuscript (p. 11, Line 25-30, p. 12, Line 1-4):

“Intracellular ROS levels were determined using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe (Beyotime Biotechnology, Shanghai, China) by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent dichlorofluorescein according to the manufacturer’s instructions and a previous study (Xu, et al.,2020). Briefly, 10 μM DCFH-DA solution diluted in serum-free DMEM/F12 medium was added and incubated with the cells, then they were kept in the dark for 20 min. Excess DCFH-DA was removed by washing the cells three times with serum-free cell culture medium. ROS was detected by both IF imaging and FCM (BD Accuri C6 Plus, CA, USA), and analysed by Flow Jo (Flowjo X 10.0.7r2).”

In Figure 5, ROCK did not increase statistically in tBHP+MET at any dose, but the text states otherwise (line 192).

Thanks for this important comment. We have replicated the experiment, analysed, and modified the text to match the Figure accordingly. Modified texts are shown as follows (p. 6, Line 28-30, p. 7, Line 1 in the manuscript):

“Furthermore, WB results (Figure 5B-C) showed that L-MET significantly activated the integrin/ROCK pathway by upregulating integrin, ROCK, AMPK, and pAMPK in healthy HTMCs, and significantly activated integrin, ROCK, pAMPK, and F-actin in damaged HTMCs, and it was more pronounced in the 3 mM doses.”

In Discussion, line 270, the wording is confusing- "recovery to normal IOP prevents further TM cell loss, whereas metformin…." Does this mean that IOP mice recovered in the absence of metformin?

Thanks. We have made revision accordingly for a clearer and more accurate expression (see p. 8, Line 26-29 in the manuscript):

“In addition, the steroid-induced OHT recovered its normal IOP with MET treatment, preventing further TM cell loss, whereas MET promoted the maintenance of cytoskeletal morphology and repair after injury; therefore, IOP was maintained at a normal level for an extended period.”

Reviewer #2 (Recommendations for the authors):

Although the presented results were promising with translational potential in the future, there are some concerns as listed below:

1. The animal numbers and sexes in each group and the biological repeats and technical repeats were not clearly listed which must be listed in the manuscript as well as in the figure legends.

Thanks. We have added the animal number in the manuscript (see p. 5, Line 6-9 or “Essential revisions 1”). The animal sexes were shown in p. 10, Line 4-5: “Healthy C57BL/6J mice (age: 6–8 weeks, male, weight: 20–25 g) were used in this study”. Biological and technical repeats were shown in p. 13, Line 21-22: “Cell-based and animal-based experiments included at least three biological and technical replicates.”

2. Page 2 Abstract: Methods: Subsequent TEM tests were not mentioned.

Thanks. We have added the information as follows (see p. 2, Line 13-14 in the manuscript):

“Transmission electron microscopy was performed to observe microfilaments in HTMCs.”

3. Page 4 lines 64-65: "Theoretically, TM-targeting drugs are potentially effective in lowering IOP caused by diseased TM." This is not clear what the authors meant. Please modify and add references.

Thanks for the suggestion. We have revised the text accordingly (see p. 3, Line 12-13 in the manuscript):

“Theoretically, drugs promoting the recovery of damaged TM are potentially effective in lowering IOP.”

4. Page 5 Introduction: Please briefly introduce the effect of MET on ROS.

Thanks for this suggestion. We have summarised the effect of MET on ROS as follows (see p. 4, Line 4-11 in the manuscript):

“Reduction of intracellular ROS levels by MET via activating AMPK signal has been reported in primary hepatocytes (Ota, et al.,2009), vestibular cells (Lee, et al.,2014), and human immune cells (CD14+ monocytes, CD3+ T cells, CD19+ B cells, and CD56+ NK cells) (Hartwig, et al.,2021). Conversely, there is also evidence on cellular ROS level increase in some cancer cells after MET treatment, including AsPC-1 pancreatic (Warkad, et al.,2021), osteosarcoma (Li, et al.,2020), and breast cancer cells (Yang, et al.,2021). These seemingly contradictory results suggested that MET plays different roles under different metabolic environments.”

5. For all the sections with immunofluorescent staining, it's not clear how the authors defined the TM region. They didn't show any specific marker expression and didn't have the same direction as all the figures.

Thanks. We have added the definition of the TM region in the Method section and marked TM (trabecular meshwork), SC (Schlemm’s canal), CB (ciliary body), and representative positively stained cells in all immunofluoresence figures (see p. 13, Line 10-12 in the manuscript).

“TM region was defined as the area between the ciliary body and the inner wall of Schlemm’s canal, with anterior and posterior end points of Schwalbe line and scleral spur, respectively (Yan, et al.,2022).”

The non-conformity with the figures’ direction was listed as a technical limitation in the revised version.

6. Figure 1. How did the authors calculate the a-SMA and TGFb positive cells, especially TGFb is an ECM marker.

Thanks. We have added the definition of positively stained cells in the Method section in the text (see p. 13, Line 16-18 in the manuscript):

“ImageJ (v1.52a, National Institutes of Health, USA) was used to quantify the positively stained cells, which referred to those cells stained by both blue color from DAPI and green or red signals from the secondary antibodies.”

7. How long did Dex treatment last? Was it stopped at 3 weeks or was it continued together with metformin treatment? If continued, how did they give the different treatments? Please describe those in detail. If Dex was not continued, IOP could possibly reduce in 3 weeks. Please compare if IOP in PBS group has statistically changed or not with that when Dex stropped.

Thanks. Please refer to “Essential revisions 1”.

8. Figure 2, several other control groups should also be compared, like PBS only, PBS+metformin eyedrops, and PBS+metformin oral without Dex treatment.

We appreciate your comments. We have added the control groups according to your suggestion. Please refer to “Essential revisions 1”.

9. Besides the in vitro tBHP induced oxidative cell model, it would be more convincing if the authors could also use Dex-treated cells to detect the metformin anti-oxidation effect since they used Dex induced mouse model.

Thanks for this important suggestion. Please refer to “Essential revisions 4”.

10. Figure 4, how many biological repeats and technical repeats? One of the characteristics of TM cells is that TM cells have increased expression of myocilin after exposure to Dex. Figure 4B didn't show myocilin expression increasing after Dex.

Thanks for the suggestion. We have added the related information (please refer to “Essential revisions 2”) and changed the Figure 4B to a representative one in the manuscript.

11. Fig6, it's not clear what the authors marked. How did they mark the microfilaments? A statistical analysis with graphs would be more clear and convincing.

Thanks for your comments and suggestions. We have marked representative microfilaments with larger magnified (150000×) figures embedded in Figure 6 and added statistical analysis according to your advice. The related methods and results have been shown in the manuscript (see p. 13, Line 12-16 in the manuscript, and Figure 6 in figure legend):

“For microfilament quantification, five fields with 80000× magnification of each group were captured with TEM, within which two were from the cell cortex region (defined as within 400 nm beneath the cell membrane) (Svitkina,2020) and the remaining three were beyond this area.”

12. The discussion of this study lacks further elaboration. There was no mention of the limitation of the study.

Thanks for this important suggestion. We have revised the Discussion and added the limitation as follows (see p.10, Line 11-18 in the manuscript).

“While experimental data were encouraging, there were limitations in our study. First, the concentration of MET in the anterior chamber was not measured. Additionally, we did not investigate its toxicity on ocular surfaces, such as tear film stability and components, which may change with the application of MET. Second, the sample size of the animals was relatively small, and a larger sample is needed to verify the protective effect of MET on TM. Third, the experimental period was too short to prove the long-term effects of MET and the presented figures were not in the same direction. Thus, additional studies are needed in the future.”

References:

Hartwig J, Loebel M, Steiner S, Bauer S, Karadeniz Z, Roeger C, Skurk C, Scheibenbogen C, Sotzny F. 2021. Metformin attenuates ROS via FOXO3 activation in immune cells. Front Immunol 12:581799. doi:10.3389/fimmu.2021.581799

Lee JY, Lee SH, Chang JW, Song JJ, Jung HH, Im GJ. 2014. Protective effect of metformin on gentamicin-induced vestibulotoxicity in rat primary cell culture. Clin Exp Otorhinolaryngol 7:286-294. doi:10.3342/ceo.2014.7.4.286

Li B, Zhou P, Xu K, Chen T, Jiao J, Wei H, Yang X, Xu W, Wan W, Xiao J. 2020. Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/JNK signaling pathway in human osteosarcoma. Int J Biol Sci 16:74-84. doi:10.7150/ijbs.33787

Li G, Lee C, Read AT, Wang K, Ha J, Kuhn M, Navarro I, Cui J, Young K, Gorijavolu R, Sulchek T, Kopczynski C, Farsiu S, Samples J, Challa P, Ethier CR, Stamer WD. 2021. Anti-fibrotic activity of a rho-kinase inhibitor restores outflow function and intraocular pressure homeostasis. ELife 10doi:10.7554/eLife.60831

Ota S, Horigome K, Ishii T, Nakai M, Hayashi K, Kawamura T, Kishino A, Taiji M, Kimura T. 2009. Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism. Biochem Biophys Res Commun 388:311-316. doi:10.1016/j.bbrc.2009.07.164

Svitkina TM. 2020. Actin cell cortex: Structure and molecular organization. Trends Cell Biol 30:556-565. doi:10.1016/j.tcb.2020.03.005

Warkad MS, Kim CH, Kang BG, Park SH, Jung JS, Feng JH, Inci G, Kim SC, Suh HW, Lim SS, Lee JY. 2021. Metformin-induced ROS upregulation as amplified by apigenin causes profound anticancer activity while sparing normal cells. Sci Rep 11:14002. doi:10.1038/s41598-021-93270-0

Yang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, Mao M, Chen C, Huang A, Chen Y, Zhang X, Khan N, Wang L, Zhou J. 2021. Metformin induces ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J Exp Clin Cancer Res 40:206. doi:10.1186/s13046-021-02012-7

https://doi.org/10.7554/eLife.81198.sa2

Article and author information

Author details

  1. Lijuan Xu

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0361-2611

  2. Xinyao Zhang

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  3. Yin Zhao

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  4. Xiaorui Gang

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Data curation, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  5. Tao Zhou

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared

  6. Jialing Han

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  7. Yang Cao

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  8. Binyan Qi

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  9. Shuning Song

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  10. Xiaojie Wang

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  11. Yuanbo Liang

    State Key Laboratory of Ophthalmology, Optometry and Visual Science, National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    yuanboliang@126.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9685-7356

Funding

National Natural Science Foundation of China (82201177)

  • Lijuan Xu

Health Commission of Zhejiang Province (2023KY914)

  • Lijuan Xu

Key R&D Program of Zhejiang (2022C03112)

  • Yuanbo Liang

Leading Scientific and Technological Innovation Talents in Zhejiang Province (2021R52012)

  • Yuanbo Liang

National Key Research and Development Program of China (2020YFC2008200)

  • Yuanbo Liang

Zhejiang Provincial National Science Foundation of China (LQ18H120010)

  • Yuanbo Liang

Key Innovation and Guidance Program of the Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical University (YNZD 2201903)

  • Yuanbo Liang

Wenzhou Municipal Technological Innovation Program of High-level Talents (604090352/577)

  • Yuanbo Liang

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Mao Huiyan, Li Jinxin, and Zhou Mengtian, who helped with the delivery of eye drops in the mice. We also acknowledge Fang Zhouxi and Pan Liangliang for their assistance with TEM. This work was supported by the National Natural Science Foundation of China (Grant No. 82201177), Health Commission of Zhejiang Province (Grant No. 2023KY914), Key R&D Program of Zhejiang (2022C03112), Leading Scientific and Technological Innovation Talents in Zhejiang Province (2021R52012), National Key R&D Program of China (2020YFC2008200), Zhejiang Provincial National Science Foundation of China (LQ18H120010), Key Innovation and Guidance Program of the Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical University (YNZD 2201903), and the Wenzhou Municipal Technological Innovation Program of High-level Talents (No. 604090352/577).

Ethics

The Institutional Animal Care and Use Committee of the Wenzhou Medical University (wydw2022-0209).

Senior Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. Mishaela R Rubin, Columbia University Medical Center, United States

Reviewer

  1. Mishaela R Rubin, Columbia University Medical Center, United States

Publication history

  1. Received: June 19, 2022
  2. Preprint posted: July 3, 2022 (view preprint)
  3. Accepted: December 9, 2022
  4. Version of Record published: January 4, 2023 (version 1)

Copyright

© 2023, Xu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Lijuan Xu
  2. Xinyao Zhang
  3. Yin Zhao
  4. Xiaorui Gang
  5. Tao Zhou
  6. Jialing Han
  7. Yang Cao
  8. Binyan Qi
  9. Shuning Song
  10. Xiaojie Wang
  11. Yuanbo Liang
(2023)
Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals
eLife 12:e81198.
https://doi.org/10.7554/eLife.81198

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    Ignacio Bravo-Plaza, Victor G Tagua ... Miguel A Peñalva
    Research Article

    Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

    1. Cell Biology

    Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture

    Sandipan Dasgupta, Daniella Y Dayagi ... Jeffrey E Gerst
    Research Article

    Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically, <1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.