HFD/STZ Type 2 diabetes (T2D)-induced DR animal model
All experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University and Shanxi Medical University Committee on Animal Care. In order to establish Type 2 diabetes (T2D) induced DR model, adult C57BL/6J mice were fed high fat diet containing 60% energy from fat (D12492; Open Source Diets) combined with streptozotocin (STZ) (Sigma Chemical, St. Louis, MO) [14]. The mice were fed HFD for 12 weeks to induce obesity, characterized by abnormal glucose tolerance and insulin resistance. The age-matched nondiabetic mice were fed standard diet (SD-diet). Then, mice were subjected to STZ (50 mg/kg body weight, once) at 12 weeks of HFD [14] followed with 8 more weeks of HFD to establish DR model (Supplemental Figure 1A). In addition, mice fed standard diet as control were given an injection of equivalent volume of citrate buffer. Fasting blood glucose level was examined using a RocheAccu-Chek™ blood glucose monitoring systems at the 12 weeks of HFD. Mice were considered diabetic when blood glucose exceeded 12 mmol/L. The globular form of CTRP3 (gCTRP3, 0.5μg/g/d) or double-distilled water was administered to HFD/STZ-T2D mice and the age-matched non-diabetic mice via peritoneally implanted osmotic pumps during the last 2 weeks of HFD. Meanwhile, to inhibit AMPK activation, 100 µl (20 mg/mL) Compound C (Sigma Chemical, St. Louis, MO, USA) was intraperitoneally injected into diabetic mice daily during the last 2 weeks.
Glucose and Insulin Tolerance Assay
To assess glucose tolerance, mice were intraperitoneally injected with D-glucose (1.5 g/kg) after fasting. The venous blood was collected 30 min before (time 0) and after injection at 0, 15, 30, 60 and 120 min. The blood glucose was measured using a Roche Accu-Chek™ glucose monitoring systems per manufacturer’s instruction. To assess insulin tolerance, a single dose of Novolin R regular insulin (0.5unit/kg) was intraperitoneally administered to the mice after fasting for 4 hours with free access to water. The blood glucose level was measured as described above.
Capillary permeability assay in vivo
To observe capillary permeability in vivo, the T2D-DR mice and the age-matched non-diabetic mice were anesthetized. Evans Blue (EB) dye (25 mg/kg, 0.5% in PBS) was injected. After the mice were euthanized, mouse ear tissue was collected with an 8 mm skin punch and fixed in 10% formamide at 56°C for 48 hours. The quantification of Evans blue in the tissues was determined by the optical density assessment on Image J (NIH) [15]. To assess CTRP3’s role in ear capillary permeability, gCTRP3 (0.5 μg/g/d) or vehicle was administered via peritoneal implanted ALZET osmotic pumps (Cupertino, CA) for 2 weeks to T2D-DR mice and the age-matched nondiabetic mice.
Retinal capillary leakage assay in vivo
Retinal capillary leakage assay was analyzed by a previously reported methods with modification. [15-18] Briefly, mixture of tetramethylrhodamine isothiocyanate conjugate Concanavalin A (TRITC-ConcanavalinA) (indicating vascularity, 25 μg/g, Cat#C860, Thermo Fisher Scientific, MA) and FITC-dextran (indicating vascular leakage, 50 μg/g, Cat#46945, Sigma-Aldrich, MO) were injected from the superior vena cava. Then, the mice were euthanized in 15 minutes and retina tissues were obtained. The retina were flatly mounted and used for examining the retinal vascular pattern. Images were obtained with a Nikon EclipseE800 confocal microscopy (Nikon). 5 images for each eye (full view) were taken. The mean intensity of TRITC-ConcanavalinA and FITC-dextran was determined by Image J software (NIH).
Cell culture and treatments
Human retinal microvascular endothelial cells (HRMECs) (Cat# cAP-0010, Angio-Proteomie, Boston, MA) were plated on six-well plates and cultured at 37°C in 5% CO2 incubator. Upon reaching 100% confluence, HRMECs were randomized to receive one of the following treatments: 1) Normal glucose normal lipids (NGNL, 5.5 mM D-glucose/19.5mM L-glucose) receiving vehicle or gCTRP3 (0.3, 1, and 3μg/ml, Cat# 00082-01; Aviscera Bioscience, Santa Clara, CA) for 24 hours; 2) high glucose/high lipids (HGHL, 25 mM D-glucose/250 μM palmitates) [19] receiving vehicle or gCTRP3 treatment (0.3, 1, and 3μg/ml) for 24 hours; 25mM L-glucose plus fatty acid-free BSA was used as an osmotic control [20].
iBRB permeability assay in vitro
The permeability of the endothelial membrane was assessed by passage of FITC-dextran through the HRMEC monolayer [21]. To measure endothelial barrier function in iBRB, in vitro transwell endothelial permeability assay (Sigma-Aldrich, MA) was performed. Briefly, after HRMEC monolayer was built, culture medium was pre-treated with gCTRP3 (3μg/ml) for 15 minutes, then followed by NGNL/HGHL challenge for 24 hours. FITC-dextran (20 μg/ml) was added to upper chamber for 20 minutes. The fluorescence signal in lower chamber was determined at 450 nm using Bio-Rad 450 microplate reader (Bio-Rad, Hercules, CA).
To investigate continuous real-time monitoring of the HRMEC monolayer migration and cell-cell junctions in vitro, we conducted xCELLigence electrical conductivity assays [22, 23]. HRMECs (3.0x104 cells) were seeded onto a 16-well E-plate. After equilibration, plates were inserted into the xCELLigence station, and the base-line impedance was measured to ensure that all wells and connections were working within acceptable limits. Cell index curves were determined by the xCELLigence RTCA System (Roche, RTCA DP Station) per manufacturer instruction.
Western blot analysis
HRMECs or tissue were lysed by lysis buffer (Cell Signaling). Total protein (50 μg) was loaded on Bio-Rad 4-20% gel system, transferred to PVDF membrane, immunoblotted with one of the following primary antibodies: Serine473 phosphorylated Akt (Cat#4060, Cell Signaling), total Akt (Cat#4691,Cell Signaling), Threonine172 phosphorylated AMPK (Cat#2535, Cell Signaling), total AMPK (Cat#5832,Cell Signaling), Acetyl-CoA Carboxylase(Cat #3662, Cell Signaling), Phospho-Acetyl-CoA Carboxylase (Cat #11818, Cell Signaling), Claudin-1(Cat#13995, Cell Signaling), zo-1 (Cat#13663, Cell Signaling), Claudin-5 (Cat#35-2500, Thermo-Fisher Scientific), Occludin (Cat#71-1500, Thermo-Fisher Scientific)), and ß-Actin (Cat#SC-4778, Sigma). The secondary antibody was obtained from Cell Signaling Company. The membrane was exposed to Super-Signal Reagent (Pierce, Illinois, USA), and imaged on a Bio-Rad ChemiDoc Touch station (Bio-Rad).
Cellular Immuno-fluorescence staining
Cells were fixed with 1% paraformaldehyde for 10 minutes at room temperature. The cells were incubated with primary antibodies against anti-Claudin5 (Cat#35-2500, Thermo-Fisher Scientific) and Occludin (Cat#71-150, Thermo-Fisher Scientific). After overnight incubation at 4°C, the cells were incubated with fluorescein-labeled secondary antibody. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Micrographs of all immunostains were acquired by Olympus BX51 Fluorescence Microscope and Olympus DP72 image system. Quantification of fluorescence intensity at the cell boundary was performed with Image J (NIH).
Measurement of serum and retinal globular domain CTRP3 (gCTRP3)
Animal retina and serum were collected and stored at -80°C. Frozen retinas were homogenized, centrifuged for 10 min at 13,000 g at 4°C, and the supernatants were collected. The serum was centrifuged for 15 min at 3,000 g at 4°C and the supernatants were collected. gCTRP3 was measured using a commercial ELISA kit (Cat: SK00082-03-100, Aviscera Bioscience, Santa Clara, CA) per the manufacturer’s instruction. The minimum detectable level of CTRP3 is 16 ng/ml (intra-assay coefficient of variability (CV): 4-6%; interassay CV: 8–10%).
Tube formation assay
To examine the impact of CTRP3 on the tube formation of HRMECs, we used a u-slide angiogenesis system (ibidi). Briefly, the plate was coated with Matrigel (growth factor reduced, BD Biosciences). Upon gel solidification, HRMECs (1x104 cells) were seeded onto pre-coated Matrigel. Then, gCTRP3 (0.3 μg/ml, 1 μg/ml, and 3μg/ml) was added and then the insert was removed. After overnight incubation, the capillary-like structures of HRMECs were photographed with a microscope and analyzed via Image J software (NIH). For conditioned medium experiments, endothelial cell basal medium was replaced with NG medium or HGHL medium.
Small interfering RNA transfection
HRMECs were transfected by siPORTER siRNA transfection kit (Cat#AM4503, Ambion) per manufacturer's protocol with siRNA duplex against AMPK-α1 and universal negative control (Santa Cruz). Briefly, HRMECs were plated on six-well plates. After HRMECs reached 80% confluence, siRNA was applied to each well (final concentration 50 nM). Protein suppression was evaluated by Western blot 72 hours later.
Statistical Analysis
Data were analyzed with GraphPad Prism-7 statistic software (La Jolla, CA). All values are presented as the mean±SD of independent experiments. For analysis of differences between two groups, the unpaired Student t-test was performed. For multiple groups, one-way ANOVA was conducted across all investigated groups. Tukey post hoc tests confirmed where statistically significant differences existed between groups. P values less than or equal to 0.05 were considered statistically significant.