Hyperglycemia-induced VEGF and ROS production in retinal cells is inhibited by the mTOR inhibitor, rapamycin

Determine the impact of the mTOR inhibitor, rapamycin, on the hyperglycemia-induced expression of vascular endothelial growth factor (VEGF) and the production of reactive oxygen species (ROS) in retinal cells. Rats made hyperglycemic for 8 weeks by streptozotocin, as well as control rats, received i.p. rapamycin (1 mg/kg) for 3 days prior to immunostaining of their retinas with anti-VEGF and anti-glial fibrillary acidic protein (GFAP) and measuring retinal protein levels of VEGF and GFAP by Western blotting. In other experiments, flow cytometry analysis of ethidium fluorescence determined intracellular ROS levels in the absence or presence of rapamycin (1 μM) under normoglycemic (5.5 mM) and hyperglycemic (25 mM) conditions in a rat retinal Müller cell line (TR-MUL5) and primary human retinal microvascular endothelial cells (HRMECs). In the diabetic retina, VEGF was elevated and colocalized with the glial marker, GFAP, whose level was also elevated. Treatment with rapamycin inhibited the diabetes-induced VEGF and GFAP increases. We also found that raising extracellular glucose from 5.5 mM to 25 mM resulted in significant rapamycin-sensitive increases in the ROS levels of TR-MUL5 cells and HRMECs. In rat retina, rapamycin attenuates the diabetes-induced VEGF overexpression, and in cultured Müller cells and HRMECs, inhibits the hyperglycemia-induced boost ROS.


Induction of diabetes.
Diabetic and non-diabetic rats were produced as described previously 27 . Diabetes was induced by administering a single 60 mg/kg intraperitoneal injection of streptozotocin in 10 mM sodium citrate buffer, pH 4.5, to each rat after an overnight fast. As controls, nondiabetic animals received an injection of citrate buffer only. Animals with blood glucose levels higher than 250 mg/dL at 24 h after injections were considered diabetic. All experiments were conducted 8 weeks after the induction of diabetes. The blood glucose was checked again at the time of the experiments to ensure that the rats showed hyperglycemia. The blood glucose level at the time of sacrifice was 384.8 ± 23.9 mg/dL. Intraperitoneal injection of rapamycin. Rapamycin (1 mg/kg) (Tokyo Chemical Industry, Tokyo, Japan) was injected intraperitoneally once a day for 3 days in STZ-induced diabetic and normoglycemic rats. Animals received general anesthesia as previously described, and perfused fixation was performed 24 h after the intraperitoneal injections.
Immunohistochemistry of retinal slices. All procedures were performed as described previously 27 .
Protein levels of VEGF and GFAP in retinas by western blotting. All procedures were performed as described previously 27,28 . Retinas were excised from the eyes and homogenized in lysis buffer containing 1 mM phenyl methanesulfonyl fluoride, 10 μM pepstatin A, 10 μM leupeptin, 10 μM aprotinin, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 5% sodium deoxy cholate, 50 mM Tris-HCl (pH 7.6), and 150 mM sodium chloride. The suspension was centrifuged, and the total protein concentration in the supernatant was determined using the Lowry method (DC Protein Assay Reagent, Bio-Rad, Hercules, CA, USA). Samples were separated on a 10-12% SDS-polyacrylamide gel and blotted onto PVDF membranes. The membranes were then blocked with 5% skim milk in Tris-buffered saline, pH 7.4, with 0.1% Tween 20 (TBS-T) followed by an overnight incubation at 4 °C with a rabbit polyclonal anti-VEGF (1:1000, sc-507, Santa Cruz Biotechnology) or GFAP (1:1000, 04-1031, Merck Millipore). Tubulin (α-tubulin, 1:1000; Merck Millipore) was used as an internal control. The membranes were washed in TBS-T followed by incubation with a peroxidase-conjugated goat anti-rabbit (W401B) or mouse IgG (W402B, 1:2500, Promega, Madison, WI, USA) secondary antibody for 2 h at 37 °C. The protein bands were visualized following the addition of an ECL Plus Western blotting detection system (GE Healthcare Life Sciences, Little Chalfont, UK). Protein band densities were measured with a luminescent image analyzer (LAS-3000, Fujifilm, Tokyo, Japan). Relative protein levels were quantified using the embedded software (Multi Gauge version 3.0) and standardized according to α-tubulin protein levels. and HRMECs at passages 4 and 6 were used in this study. These cells were maintained in DMEM that contained 5.5 mM glucose. Before reaching confluence, the media was changed to the control media (5.5 mM glucose in DMEM) or the high glucose media (25 mM glucose in DMEM, D6429) to compare ROS levels, as described later. Detailed procedures and treatment protocols for each experiment are described in subsequent sections.

Immunostaining of cultured Müller cells.
To determine the effect of the mTOR inhibitor rapamycin on the expression of VEGF and GFAP in the TR-MUL5 cells under high glucose condition, the cells that were incubated in high glucose (25 mM) media with and without rapamycin were examined by immunocytochemistry. After fixation by 4% formaldehyde, the cells were incubated with primary antibodies of mouse monoclonal anti-VEGF (

Flow cytometry analyses of ROS formation in müller cells and HRMECs.
We examined the generation of ROS in cultured Müller cells (TR-MUL5) and HRMECs by treating them with hydroethidine and measuring ethidium fluorescence by flow cytometry (EC800 cell analyzer, SONY, Tokyo, Japan). After a few passages of TR-MUL5 and HRMECs, the media was changed to the control media (5.5 mM glucose in DMEM) or the high glucose media (25 mM glucose in DMEM, D6429) with 10% fetal bovine serum (FBS). The cells were incubated overnight at 37 °C with or without rapamycin (1 μM) under physiological (5.5 mM) and high-glucose (25 mM) conditions for 48 h. The cells were cultured in a medium lacking 10% FBS and were serum-deprived overnight before the following FACS assay. Cells were harvested via trypsinization, centrifuged at 1000×g for 5 min, re-suspended in phenol red-free DMEM, and incubated with hydroethidine, a fluorogenic probe, for 20 min at 37 °C. Cell densities were adjusted to 2.0 × 10 5 cells/mL. Hydroethidine is oxidized by ROS to form ethidium, a fluorescent product, which is then retained intracellularly to allow a semi-quantitative estimation of intracellular ROS levels 31,32 . The intracellular levels of ROS were measured using an EC800 cell analyzer at 488 nm excitation and 590-610 nm emission wavelengths. The acquisition and analysis software programs on the EC800 was used to acquire and quantify the fluorescence intensities. This assay was performed to determine the levels of ROS generated by the cultured cells in control and high glucose media in the presence or absence of rapamycin (1 μM). In addition, the cultured Müller cells (TR-MUL5) were also incubated overnight with or without VEGF (5 ng/mL) and rapamycin under physiological (5.5 mM) and high-glucose (25 mM) conditions to determine ROS production by VEGF and the effects of rapamycin on this ROS production. The ethidium fluorescence in TR-MUL5 cells exposed to VEGF and/or rapamycin under physiological (5.5 mM) and high-glucose (25 mM) conditions was also measured by flow cytometry.

Results
Immunohistochemistry of retinal slices. Compared with the nondiabetic control rats, immunoreactivity to VEGF was increased in the STZ-induced diabetic rats. As shown in Fig. 1, VEGF immunoreactivity was seemed to be higher in the diabetic rat retinas than in the controls. Injection of rapamycin into the nondiabetic rats seemed to decrease VEGF immunoreactivity. In the diabetic retina, VEGF expression seemed to be intensified mainly in these internal retinal layers. Rapamycin likely decreased the intensity in diabetic rats.
Immunoreactivity to GFAP seemed to be higher in the diabetic rat retina than in the control retina. The observed longitudinal pattern of immunoreactivity to GFAP in the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), and the outer nuclear layer (ONL) suggested that these GFAP expression may involve Müller cells. Double staining with VEGF and GFAP demonstrated that the expression of VEGF was co-localized with GFAP expression especially in the diabetic rat retinas. Rapamycin seemed to inhibit the increases in GFAP expression in the diabetic retina. www.nature.com/scientificreports/

Immunostaining of cultured Müller cells. Representative photomicrographs of immunostaining with
anti-VEGF (green) and anti-GFAP (red) in the TR-MUL5 cells are shown in Fig. 2. Immunoreactivities to VEGF and GFAP in high glucose condition seemed to be depressed by the mTOR inhibitor rapamycin.  Protein levels of GFAP in the diabetic retina were significantly higher (28.1%) than in the control retina (P = 0.0004). The increases were significantly suppressed by rapamycin (P < 0.0001) (Fig. 4, n = 3 each).  (25 mM) conditions. Flow cytometry analyses showed significant increase in intracellular levels of ROS in TR-MUL5 under the high-glucose condition, and these increases were significantly suppressed by rapamycin. This figure also shows the effects of rapamycin on VEGF in ROS production of TR-MUL5 under physiological (5.5 mM) and high-glucose (25 mM) conditions. We performed this assay to determine the effect of rapamycin on the ROS production of cultured Müller cells (TR-MUL5) exposed to VEGF. Flow cytometry analyses showed significant increases in the intracellular levels of ROS in TR-MUL5 cells exposed to VEGF, and these increases were significantly suppressed by rapamycin (P < 0.0018, the Bonferroni correction). Figure 6 shows the intracellular ROS levels in HRMECs exposed to rapamycin under physiological (5.5 mM) and high-glucose (25 mM) conditions. Significant increases in the ROS levels in HRMECs under high-glucose condition were observed, and these increases were significantly suppressed by rapamycin (P < 0.0083, the Bonferroni correction).

Discussion
A key finding of this study is that the diabetes-induced increase in VEGF expression in the diabetic rat retina is inhibited by the mTOR inhibitor, rapamycin. This enzymatic inhibitor also blocked the up-regulation of GFAP by glial cells of the diabetic retina. An additional novel observation is that rapamycin inhibits the hyperglycemiainduced increase in ROS in cultured Müller cells as well as HERMCs. We also found that under hyperglycemic conditions, the VEGF-induced increase in Müller cell ROS was also suppressed by rapamycin.
The increase in VEGF levels in the diabetic retina have been previously reported 19,27,[33][34][35] , and this response was suppressed by rapamycin. Generally, mTOR coordinates protein synthesis, mitochondrial energy production 36 . Figure 3. Protein levels of VEGF in retinas determined with Western blotting. VEGF expression was significantly higher in retinas obtained from STZ-induced diabetic rats than from nondiabetic controls. The increased VEGF expression was suppressed by treatment with the mTOR inhibitor rapamycin (P < 0.05, n = 3 each). www.nature.com/scientificreports/ Figure 4. Protein levels of GFAP in retinas determined with Western blotting. GFAP expression was significantly higher in retinas obtained from STZ-induced diabetic rats than from nondiabetic controls. The increased GFAP expression was suppressed by rapamycin (P < 0.05, n = 3 each).

Figure 5.
ROS levels in cultured Müller cells (TR-MUL5) exposed to rapamycin and/or VEGF under physiological (5.5 mM) and high-glucose (25 mM) conditions. Flow cytometry analyses showed significant increases in intracellular ROS levels in TR-MUL5 under a high-glucose condition, and these increases were significantly suppressed by rapamycin. In addition, flow cytometry analyses showed significant increases in intracellular levels of ROS in TR-MUL5 exposed to VEGF, and these increases were significantly suppressed by rapamycin (P < 0.0018, the Bonferroni correction).  5) and primary HRMECs (Fig. 6). This indicated that rapamycin seemed to decrease ROS levels by inhibiting the mTOR pathway in retinal Müller cells; however, we did not assess its effects on non-Müller rat retinal cells. Our results are consistent with co-localization being at least in part within the Müller cells, and it is not excluded from the present study that some non-Müller cells may also co-express VEGF and GFAP. In the retina, Müller cells and vascular endothelial cells might express mTOR. ROS production, oxidative stress, and cellular death can be altered by several signaling pathways triggered by the hyperglycemia that causes diabetic retinopathy 37 . Our data indicated that VEGF-induced increase of the ROS levels in Müller cells under high-glucose conditions and this increase was suppressed by rapamycin. We also analyzed the intracellular ROS levels in primary HRMECs which have the potential to cause retinal vascularization observed in human diabetic retinopathy. Our findings indicate that rapamycin might be an effective alternative drug for the treatment of diabetic macular edema.
We also showed that GFAP, a glial marker, increased in the diabetic retina. Our findings showed that rapamycin can reduce the increase in GFAP expression in the diabetic retina, suggesting that mTOR inhibition may inhibit glial activation and/or proliferation. In non-diabetic rats, the effect of rapamycin on GFAP levels was observed as well. Rapamycin might inhibit the activity of retinal glia even also under the physiological condition. In diabetic retinopathy, neurodegeneration starts even at an early stage of the disorder where reactive gliosis plays a crucial role 17,38 . It is known that there are two types of mTOR, mTORC1 (mainly retinal ganglion cells) and mTORC2 (mainly glial cells) in the retina [39][40][41] . The effects of rapamycin in this study seem to be caused mainly by mTORC2 inhibition in the diabetic rat retina.
A limitation of this study is that although the chief focus was on oft-studied rat models of diabetes, it will be of importance for future analyses to extend analyses to the retinas of animals and humans exhibiting anatomical and functional manifestations of diabetic retinopathy. Another possible limitation is that although mTOR is reported to increase in the retinas of rats with experimental diabetes 20 , this remains to be confirmed in retinas of rats made hyperglycemic by STZ for 8 weeks. Thirdly, although FACS was used to quantify ROS in cultured retinal Müller cells and HRMECs, it remains to be demonstrated in future studies that rapamycin suppresses excessive ROS production in vivo within the diabetic retina.
In conclusion, our findings from in vivo and in vitro experiments showed that the mTOR inhibitor, rapamycin, blocks the up-regulation of VEGF and GFAP in the diabetic rat retina and also inhibits the hyperglycemiainduced increase in ROS in cultured Müller cells and HERMCs. These findings suggest that rapamycin may be a potential candidate drug for ameliorating retinal complications in diabetes.
Received: 24 June 2020; Accepted: 7 January 2021 Figure 6. ROS levels in primary human retinal microvascular endothelial cells (HRMECs) exposed to rapamycin under physiological (5.5 mM) and high-glucose (25 mM) conditions. Flow cytometry analyses showed significant increases in intracellular ROS levels in HRMECs under a high-glucose condition, and these increases were significantly suppressed by rapamycin (P < 0.0083, the Bonferroni correction).