Materials
Fertilized white leghorn chicken eggs are purchased from Tamil Nadu Poultry Research Station, Madras Veterinary University, Nandanam, Tamil Nadu, India. Gelatine sponges are purchased from Jhonson & Jhonson Pvt Lmtd, India. Paraffin film, wax, TRIzol reagent, agarose, and EtBr are purchased from Sigma, Aldrich, USA. High molecular weight Heparin purchased from CALBIOCHEM, USA (Product No. 375054). ImProm-11™ Reverse Transcriptase kit and GoTaq Green Master Mix PCR amplification kit were from Promega, USA, Oligo (dt) of length 18-meres from Eurofins, mwg operon, Germany, Random hexamers from MP Biomedicals, USA. All primers are purchased from Bioserve, India. DNA ladders purchased from Invitrogen, USA. DAB system purchased from Bangalore Genei, India. Bradford reagent and FITC (Goat ant-rabbit IgG were from Bangalore Genei, India. SDS-PAGE Standards marker purchased from BIO-RAD, CANADA. Rabbit polyclonal FGF2 and MMP2 are a kind gift from Dr. Li Haiqing, MD, Ph.D., Technology transfer specialist, National Cancer Institute, Rockville, USA. Unless otherwise specified, all other common reagents and chemicals are purchased from Sigma, Aldrich, USA.
Methods
Selection of template for homology modeling
The experimental crystal structures of major chicken-specific pro-angiogenic growth factors such as FGF2, MMP2, MMP9, NOS3, VEGF A, and VEGF C are not available in Protein Data Bank (PDB). Hence, we try to validate the crystal structures of mentioned chicken-specific pro-angiogenic growth factors for the time. The UniProt Knowledgebase (UniProtKB) sequence of these proteins is obtained with accession number and is followed as FGF2-P48800, MMP2-Q90611, MMP9-A0A3Q2U2B5, NOS3-H9CZQ2, VEGF A - P67964, and VEGF C- E1C7C6, respectively [12]. For building an appropriate query sequence and sequence identity models, the protein IDs were up-loaded in the webserver of SWISS-MODEL [13]. A Global Model Consistency Estimation (GMQE) and Qualitative Model Energy Analysis (QMEAN) have been chosen to obtain the most accurate 3D structure for each protein [14, 15]. The modeled structures are visualized by using chimera [16].
Structure validation of modeled protein
The quality of the modeled 3D structures created through SWISS-MODEL was verified using PROCHECK. The PDB file formats of the modeled structure of all the proteins were downloaded and submitted to the SAVS server to obtain both the Ramachandran plot and the Ramachandran plot statistics. The Ramachandran plot is an experimental structure. The statistics of the Ramachandran plot include details on the total number of residues of amino acids present at the favorable, permitted, and disallowed regions [17].
Molecular Docking Analysis
Protein preparation
The homology modeled 3D structure of the target proteins, such as FGF2, MMP2, MMP9, NOS3, VEGF A, VEGF C, are downloaded from SWISS-MODEL in its PDB format. The modeled protein structures were defined as receptors and were prepared by the computation of Gasteiger charges, with the polar hydrogens and merging of the nonpolar hydrogens using AutoDock Tools 1.5.6. The structures are then saved for input into PyRx in the PDBQT file format.
Ligand Preparation
The structure of Heparin was downloaded from the PubChem Compound Database in the Spatial Data File (SDF) format and converted into the PDB file format using the Online Smile Translator. First, the ligand structure was stored for input to AutoDock vina-PyRx as a pdf file format, and then it was saved as input into PyRx in the PDBQT file format.
Molecular docking
Molecular docking analysis is performed using the Autodock module available in PyRx Version 0.8 [18, 19]. The size of the docking grid is expanded to fit the entire protein within the grid with dimensions. Genetic Algorithm (GA) is used for screening the highest suitable blind docking conformers. During the docking period, the ligands are considered flexible and the protein as rigid. The configuration file for grid parameters is created by using the PyRx Auto Grid engine. The framework is used to know or predict the amino acids associated with the ligand at the active protein site. The maximum conformers have considered each compound to predict the best conformers in genetic algorithms through molecular docking. The best binding energy (most negative) is considered to note the ligand with maximum binding affinity. From each compound, the best conformer is selected for the interaction analysis. The selected docked complex of proteins and ligands were retrieved in PDB format and examined in PyMOL.
The CAM vascular bed model
Fertilized white leghorn chicken eggs weighing 50 ± 2 grams were incubated at 37°C in a humidified atmosphere (>60% relative humidity) based on Hen's Egg Test- Chorioallantoic Membrane (HET-CAM) protocol. On the third day of post-incubation, 2 to 3 mL of albumin is withdrawn using a 21-gauge needle through the sizeable blunt edge of the eggs to minimize the adhesion of the shell membrane with the CAM. A square window of 1 cm² in size is opened in the eggshell and was sealed with paraffin film to prevent dehydration. On day 9, gelatinase sponges of 1 mm³ in size are placed on the top of the growing CAM under a sterile condition. The sponges are soaked with ten μl of 50, 100, and 150 μM concentrations of Heparin. Control CAM is incubated with ten μl of 1X PBS. The window was closed with transparent adhesive tape, and the eggs are returned for further incubation till day 12 (72 hours of incubation), at which the vascularization potential of CAM reaches its maximum. The experiment groups are divided into 4 of, each containing 40 numbers of eggs. Group1 represents the control, groups 2, 3, and 4 correspond to 10 μL volume of 50, 100, and 150 μM concentrations of Heparin. CAMs were photographed at 0, 24, 48, and 72 hours of incubation using a Cannon digital camera of 12X50 MegaPixel (Power Shot A95). The images are analyzed with Angioquant Toolbox, MATLAB 6.5 software to identify the blood vessels [4].
Semi-Quantitative Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
According to the manufacturer protocol, total RNA is isolated from the tested CAMs (10 numbers each) using TRIzol reagent (100 mg/1 mL). The quantity and the purity of the isolated RNA are checked using a UV-visible spectrophotometer. Synthesize of cDNA of 20 μl in volume was done using ImProm-11™ Reverse Transcriptase kit with Oligo (dt) of length 18-meres and random hexamers. PCR amplification is performed using a Go Taq Green Master Mix kit, and the changes in the level of mRNA expression of FGF2, MMP2, MMP9, NOS, VEGF A, VEGF C, and GAPDH are evaluated using 100 Pico moles of chicken specific primers. The relative expression level of each mRNA transcript is normalized with control. PCR products were subjected to electrophoresis on 1.5 % agarose gel containing 0.5 µg/ml EtBr and are photographed using a Canon Digital Camera of 12X5.0 MegaPixel resolution (Power Shot A95). The base pair products are compared against a DNA ladder of 10 base pairs. The relative density of the bands per experiment is calculated using a Scion Image release α 4.0 3.2 software. Specific primer sequences and the PCR reaction set up were given in Table 1 and 2, respectively [10].
Gelatin zymography
CAM tissues were homogenized (100 mg/mL) using Tris buffer (0.5 M Tris–HCl (pH-6.8), 10 % SDS, glycerol and 0.01 % bromophenol blue) and centrifuged at 12,000 rpm/4°C/10 minutes. The protein concentration from the supernatant is determined using the Bradford reagent, and the gelatinase activity of the extracted samples is examined on a 10% SDS-PAGE electrophoresis containing 1.0 mg/mL of gelatine. Protein samples of 25 μg/40 μL in concentration and 20 μL of pertained SDS-PAGE Standards marker are loaded in the system. After electrophoresis, the gels were washed with 2.5% of Triton-X-100 and incubated with the digestion buffer (50 mM Tris HCl-pH-7.5,100 mM CaCl2, 1 μM ZnCl2, 1 % Triton X-100, 0.02 % NaN3-100 mL) for 16 to 18 hours at 37 °C with gentle agitation. Gels are stained with staining solution (0.05 % (w/v, Coomassie blue in 50 % methanol and 10 % acetic acid) for 1 hour and de-stained with methanol/acetic acid mixture. The gels are scanned, and the images are recorded using a BIO-RAD Calibrated Densitometer Software (GS 800, USA). The bands' density is calculated with PD Quest Advances Software and normalized with control value [11].
Immunohistochemistry
CAM tissues incubated with ten μL of 100 μM Heparin (10 μM in thickness) are deparaffinized and dehydrated and were allowed to undergo antigen retrieval process using Sodium Citrate (10 mM-pH 6.0) in a microwave oven for 20 minutes and washed with DDH2O for 3X5 minutes in 1X PBS (pH 7.3). Standard Goat Serum Blocking Solution (2 % goat serum,1 % BSA, 0.1 % cold fish skin gelatin, 0.1 % Triton X-100, 0.05 % Tween- 20, 0.05 % Sodium Azide, 0.01 M PBS (pH 7.2) of 50 to 75 μL in volume was added immediately on the sections and are incubated for 1 hour in a humidified chamber. After washing (IX PBS), the primary antibodies of FGF2, MMP2, and MMP9 were applied on the sections after diluted in a blocking serum. After overnight incubation, the sections are rinsed with 1X PBS with 0.05 % of Tween-20. Diluted FITC (Goat anti-rabbit IgG) and HRP (both Goat anti-rabbit and Goat anti-mouse IgG) secondary antibodies (1:40 dilution) are applied for 1 hour according to the manufacturer's instruction. For HRP conjugated secondary antibodies DAB system was used for color development. The slides were finally counterstained with Mayer's hematoxylin and mounted with 90 % of glycerol. For HRP conjugated system, the images are recorded using a light microscope. For FITC conjugation, the images are taken using BX51 Olympus Fluorescence Microscope at a wavelength of 515 nm with ASI FISH View 5.5 software at 40X magnification [10].
Scanning electron microscopic (SEM) analysis
The tested area of the CAM (with 100 μM Heparin) is dissected after 72 hours of incubation and washed with 1X PBS. After drying at room temperature, the unfolded air-dried membranes are glued onto stubs with carbon spattered with gold (10 min, 14-17 Ma, and 0.07 m bar). The sections are observed under a Hitachi S-3400N Variable Pressure Scanning Electron Microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 15-30 kV, and the images are recorded at 100X magnification [20].
Data analysis and statistics
All the experiments were performed in triplicate (n=3) unless otherwise specified. Data are presented as mean ± SEM and are analyzed by One-Way ANOVA analysis of variance test, Student's t-test, and Turkey post hoc tests as appropriate by Sigma Stat 2.0. Values of p = < 0.001 and p = 0.001 were considered as statistically significant.