Dulbecco's modified Eagle's medium (DMEM) is purchased from PAN Biotech and Foetal Bovine Serum (FBS) from Invitrogen Life Technologies (Gaithersburg, MD). Human recombinant leptin, Bradykinin, Phenylmethylsulfonyal fluoride (PMSF), Picoll, 5Fluorouracil (5FU), 5-Chloromethylflouorescein diacetate (CMFDA), and all the primary antibodies are purchased from Abcam, USA. Propidium Iodide (PI), L-NAME, L-Arginine, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), recombinant VEGF were from Sigma Chemical Co (St. Louis, MO). L-NIO is purchased from Enzo life sciences and Wortmannin from Santa Cruz Biotechnologies. Collagen type-1 purchased from Pan Biotech GmbH Am Gewerbeperk. DAF- FM (4 amino-5-methylamino-2'7'difluoroscein) and Calcium green1/AM were from Molecular probes, Eugene, Oregon, USA. Protease inhibitor tablets from Roche Diagnostics. All the primary antibodies are purchased from Abcam, and all the secondary antibodies and DAB systems are purchased from Bangalore Gene, India. All the other chemicals are at least of the reagent grade and are obtained commercially.
The T24 bladder carcinoma cell line formerly designated as ECV 304, stably transfected with eNOS-GFP constructs, is a kind gift from Dr. Vijay Shah, Mayo Clinic, Rochester, USA, and was used for immunofluorescence analysis. Another immortalized endothelial hybrid cell line EA.hy926 is a kind gift from Dr. C.J.S. Edgell, University of North Carolina, Chapel Hill. The cells are cultured in DMEM medium supplemented with 10 % FBS (v/v) and 1 % penicillin/streptomycin (w/v) and maintained at 37 °C in a humidified CO2 incubator. Bovine aortic endothelial cells (BAECs) are isolated from the bovine aorta. The aorta is collected from a government-authorized slaughterhouse. BAECs are separated according to protocols described elsewhere . The isolated cells are confirmed as endothelial cells by using the antibody against endothelial marker eNOS. Primary endothelial cells are used till passage 6.
Fertilized White Leghorn chicken (Gallus Gallus) eggs weighing 50 ± gms are obtained from the Poultry research station, Nandanam, Chennai, Tamil Nadu, India. The eggs are incubated at 37–38ºC, at a relative humidity of 70 to 80% using an egg incubator. All experiments are performed based on the Hens Egg Test- Chorioallantoic Membrane (HET-CAM) method [8, 9].
Scratch monolayer wound healing assay
Scratch wound healing assay is performed using EA.hy926 cells as described elsewhere . In brief, a monolayer scratch is made on EA.hy926 cells and incubated with 0, 1, 5, 10, 25, and 50 nM concentrations of leptin for 6 hours. Images are recorded every 2 hours of interval. To confirm leptin's temporal and after wash effect after 6 hours, the wounded area is washed with PBS and continued incubation up to 10 hours without leptin. The rate of healing is calculated from the images. The experiment is repeated with 5 nM concentration of leptin, and the effect is compared with 3 mM concentration of 5- Fluorouracil. Throughout the healing experiments, the cell-free area of the wound is recorded using a Nikon Camera attached with a Bright field microscope at 4X magnification. It is subsequently analyzed using Image J software to calculate the percentage and rate of wound healing from the initial and final wounded areas.
Endothelial ring formation assay
Ring formation assay is performed as described elsewhere . In brief, EA.hy926 cells are incubated with 0, 1, 5, 10, 25, and 50 nM concentrations of leptin for 6 hours. Images are recorded every 2 hours of the interval using a Nikon Camera attached with a Bright Field Phase Contrast microscope at 20X magnification. The rings formed by two or more endothelial cells are considered ring structures and are counted from every field. The rate of endothelial ring formation every 2 hours is calculated. The endothelial ring structure's stability under leptin is confirmed from real-time image analysis and compared with the L-NIO (0.01µM) effect. After the incubation, each coverslip is mounted on a customized live-cell chamber on a microscope stage and is fixed after focusing on a single ring structure. The cells are monitored for 30 minutes continuously. Live-cell images are recorded at 0, 10, and 30 minutes of incubation at 20X magnification using a Nikon Camera attached with a Bright Field Phase Contrast microscope.
Measurement of Nitric Oxide
The level of NO released from ECs after leptin incubation is measured by employing three methods. (A) DAF-FM method – The experiment was performed using EA.hy926 cells, as mentioned earlier . Cells are treated with L-NIO (0.1µM) for 30 minutes before the incubation with 0, 5, 10, and 25 nM concentrations of leptin for 4 hours. After PBS washing, the cells are treated with 1µM DAF-FM in DMEM along with 1 mM of L-Arginine for another 30 minutes. The supernatant of 100 µl in volume is collected from each group. Using a varian cary eclipse UV-vis fluorescence spectrophotometer, the OD is measured at an excitation/emission of 495/515nm wavelength. (B) Griess assay – Griess assay is performed as mentioned earlier , repeating the same experimental procedure described above. After the incubation with L-NIO and Leptin, cells were treated with 2X HBS and 1mM L-Arginine for another 30 minutes. The supernatant of 100 µl in volume is collected from each group and mixed with an equal volume of PBS (50 µl) and Griess assay reagent A and B. After 10 minutes of incubation, the OD value at 540 nm is measured using a Varian Cary 4000 UV-Vis Spectrophotometer. The nitrite concentration is determined from a standard curve generated with Sodium Nitrate (NaNO2). The early NO-releasing capacity of 5 and 10 nM concentration of leptin for 3, 6, and 240 minutes of incubation is measured repeating the same protocol mentioned above. A comparative study is carried out using a known inducer of NO such as VEGF (10 ng) and an inhibitor, Bradykinin (1µM). (C) NO measurement using NO electrode - Direct measurement of NO immediately after the release from ECs was performed using an Apollo 4000 Free radical analyzer (at 37ºC, pH 7.4), an optically isolated multi-channel free radical analyzer with a NO selective membrane as described elsewhere . BAEC cells are incubated with 0, 5, and 10 nM concentrations of Leptin for 6 and 12 minutes. After the incubation, the cells are incubated with 1 mM L-Arginine for another 30 minutes. The real-time acquisition of NO release is measured at 100 nAº voltage at 37ºC and is recorded through a single–board computer. During the 9th minutes of the experiment, 5 and 10 nM concentration of Leptin is added separately, and the entire set-up is allowed to run for 30 minutes and compared with the control value for the same incubation period.
Analysis of eNOS-GFP localization pattern using eNOS-GFP stably transfected endothelial cells
As mentioned earlier, the localization pattern of eNOS in eNOS - GFP stably transfected ECs is performed . ECV-304 eNOS GFP tagged cells are incubated with 0, 5, and 10 nM concentrations of leptin for 1st and 4th hours separately. After fixing and permeabilizing, the cells are incubated with 2 μL of DAPI (stock-1 mg/ml) for 10 minutes and are mounted with 70% glyceraldehyde. Images are recorded at 60X magnification using a DP71 color camera attached with an Olympus1X71 Epifluorescence microscope at 515 nm wavelength. The same procedure is followed for 0, 5, and 10 nM concentrations of Leptin for 3, 6, 9, and 12 minutes. The cells are categorized into the plasma or perinuclear membrane brightness based on the localization eNOS.
Fluorescence imaging analysis of the intracellular calcium in endothelial cells using Calcium Green Probe
Intracellular calcium level is measured as described earlier . EA.hy926 cells were incubated with 0, 5, and 10 nM concentrations of leptin for 6 minutes, followed by incubation with 5 µM Calcium green1/AM for 30 minutes. Additional incubation with PBS is given for another 15 minutes and after washing with PBS for 3 times to complete hydrolysis of any intact ester linkages in the intracellular calcium green 1/AM. Coverslips were loaded in a live cell chamber and placed onto the stage of an Olympus 1X71 Epifluorescence microscope. Fluorescence images of the cells are recorded using a DP71 camera attached to the microscope, and the fluorescence intensity of the cells is calculated using Adobe Photoshop version 6.
Immunofluorescence analysis is performed as described elsewhere to evaluate the phosphorylation pattern of eNOS and co-localization of eNOS and caveolin-1 . ECV-304 eNOS GFP tagged cells are incubated with 0 and 5 nM concentrations of leptin for 6 and 9 minutes. Followed by fixing and permeabilization, the cells are blocked with 0.5% of BSA for 10 minutes. Overnight incubation is done with primary antibodies of phospho Ser-1177 and with Caveolin-1 rabbit polyclonal primary antibody (1:1000). TRITC labeled secondary antibody (1:2000) is used for the detection, and the nuclear staining is performed with DAPI. Images are recorded at 60X magnification using a DP71 color camera attached with an Olympus 1X71 Epifluorescence microscope with the required filter.
Western blot method
(A) Direct western blot - Direct western blot method is performed as described earlier to analyze the protein level expression of phospho Serine 1177 and eNOS . ECV-304 eNOS GFP tagged cells are treated with 0 and 5 nM concentrations of leptin for 6 and 9 minutes. Following the protein separation and wet blotting, the membrane is probed for anti-eNOS (rabbit polyclonal, dilution of 1:1000), phospho eNOS (Ser-117, dilution 1:1000), and detected using HRP labeled secondary Ab of 1:2000 dilutions. The blots are developed using TMB/H2O2 substrate. Images of the membrane are recorded using BIO-RAD (USA) system. (B) Western blot analysis after ultracentrifugation – This experiment is performed to identify the localization of Caveolin-1 and eNOS in endothelial cell subcellular fraction after leptin incubation. The procedure is adopted from the protocol described elsewhere . In brief, ECV-304 eNOS GFP tagged confluent cells are incubated with 0 and 5 nM concentrations of Leptin for 6 minutes, and after the ultracentrifugation, all the 10 fractions are carefully collected into sterile vials. The western blot analysis for Caveolin-1and eNOS is performed using rabbit polyclonal antibodies (1:1000 dilution) as described above.
Egg yolk angiogenesis assay
Egg yolk (area vasculosa) angiogenesis assay is performed as described below. In brief, 2 to 3 mL of albumin is withdrawn on the 3rd day of incubation, and a square window is opened. During the 4th day of incubation, sterile filter paper disks soaked with 0 and 5 nM leptin, L-NAME (nitro arginine methyl ester) (1 µM) and leptin +L-NAME are then placed on the egg yolks and incubated for another 8 hours at 37°C in an egg incubator. Images are taken at 4X magnification using a Nikon Cool Pix camera (Olympus India Pvt Ltd, New Delhi, India) adapted to a stereomicroscope at 0, 4, and 8 hours incubation. Quantification of angiogenesis is performed using AngioQuant Toolbox, MATLAB 6.5 (R13) software. Histological analysis is performed as described elsewhere [8, 9].
All the experiments are performed in triplicate or more (n≥3) until otherwise specified. Data are presented as mean ± SEM. Data analyzed using Student t-test as appropriate. Data with p-values equal or less than p=<0.001 is selected as a criterion for showing a statistically significant difference.