| Purpose:The cornea is physiologically transparent and its transparency depends on many factors including avascularity. Corneal neovascularization may be evoked under some pathological condition, such as inflammatory disorders, corneal graft rejection, infection keratitis, contact lens-related hypoxia, alkali burns, stromal ulceration, and limbal stem cell deficiency. Although neovasculature is benefit for recovering the injured cornea, neovascularization in the corneal tissue induced unwanted opacification, resulting in significant reduction in visual function.Furthermore, corneal neovascularization is the main risk factor for rejection after keratoplastyBeing one of anti-angiogenic factors, angiostatin is an internal fragment (kringle1-4) of plasminogen and expressed in cultured corneal epithelial cells and corneal epithelium, but down-regulated after wounding.Excitingly, accelerating evidences reveal that delivery of recombinant virus containing angiostatin fragment or local administration of angiostatin can suppress efficiently the development of vasculatures in cornea after wounding, suggesting that angiostatin should be one candidate to treat corneal vascularization. Certainly, throughout understanding of the mechanism underlying this potential is helpful for clinic translation. Method:Being a kind of important biological macromolecules, proteins undertake the majority of life activities. Proteomics has achieved a great advance recently. Compared to traditional research on a single protein, proteomics is a new field of research in the post-genomic era, which aims at the expression and functional study of proteins in cells, tissues and organisms. So in this study, we administrate angiostatin on the alkali burn induced rabbit corneal neovascularization model. Proteins of corneal were separated by two-dimensional gel electrophoresis, and proteins that up-regulated and down-regulated their expressions in rabbit cornea responding to angiostatin were detected by means of mass spectrum analysis.Process:Experimental Animals and GroupingThis study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No.85-23, revised1996) and was approved by the local ethics committee for animal’s research. Thirty two New Zealand white rabbits weighing from2.5kg to3.5kg and without eye surface disorders were purchased from Experimental Animal’s Center of Southern Medical University, China. Animals were housed individually in the cages and free access to commercial chow and tap water. After being acclimated surroundings for one week, the animals were randomly divided into three groups:normal control (n=8), control (n=12) and experimental group (n=12).Alkali-Induced Corneal NeovascularizationAfter animals were anesthetized topically with0.5%tetracaine, alkali injuries to the control and experimental eyes were created by1.5min exposure of the central cornea to a10mm diameter disk of filter paper soaked in1.5mol/L sodium hydroxide followed by rinsing with50ml sterile saline, and tobramycin solution and tobramycin ophthalmic ointment. The experimental eyes received locally30μg/ml angiostatin three times per day until sampling, whereas the control eyes were not treated. Observation and Quantification of Corneal NeovascularizationThe eyes in both control and experimental animals were observed using a slit lamp (YZ5T,66Vision Tech Co., Ltd, Suzhou, China) every other day. The area of corneal neovascularization was determined using the following formula: AC=C/12×3.1416×[r2-(r-1)2], where C represents the clock hours of corneal neovascularization, r indicates the radius of the animal’s cornea, and L means the length of the longest new vessel grew toward the opacus center of cornea with continuous slight curvature. The length of vessel was measured with a reticule from the limbus to the tip of the vessel [10]. In present experimentation, the measured radius of the rabbit cornea was7mm.Sample PreparationOn postoperative week3, the corneas from each group were dissociated, and stored immediately in5ml microtubes at-80℃. Before protein extraction, the samples were grinded throughout to fine powder, and then, homogenated with lysis buffer. The total proteins were collected by centrifugation, and quantized using Bradford method (Bio-Rad, Hercules, CA, USA) according to the manufacture’s protocol. In brief,2μl total proteins form each sample was added into18μl distilled water, and then, mixed with200μl dye from the Bradford reagent. After being incubated for5min at room temperature, absorbances of the proteins at595nm optic light were read by the fluorophotometer (Model680Bio-Rad), to submit to calculate the protein concentration according to the linearized absorbance curve of bovine serum albumin (BSA), which was used as standard reference.Two-Dimensional Gel ElectrophoresisSamples containing120μg proteins were separated by two-dimensional gel electrophoresis (2-DE). The first-dimension isoelectric focusing (IEF) was performed on immobilized pH gradient strips (24cm; pH4-7, nonlinear, purchased from GE Healthcare) using an Ettan IPGphor system (GE Healthcare). Isoelectric focusing was performed using the following step voltage focusing protocol:30v10-12h;500v1h; OOOv1h;8000v10h. After the first dimensional IEF, the IPG strips were quilibrated in a sodium dodecyl sulfate (SDS) equilibration buffer containing1%ithiothreitol (DTT) for15min. The IPG gel strips were then removed to another equilibration buffer containing2.5%iodoacetamide and equilibrated for a further15min. The equilibrated IPG strips were then placed onto a12.5%SDS-PAGE. The second-dimension electrophoresis was performed using the Ettan DALTsix electrophoresis Unit (GE Healthcare). The conditions are2-2.5W/gel40min then17W/gel until the run is completed. At the end of each run, the gels were sliver stained with EMBL in following the manufacture’s protocol (GE Healthcare, Milwaukee, WI, USA). The stained gels were scanned with an ImageScanner (PowerLook1100scanner, UMAX) and were analyzed with ImageMaster2D Elite software.Image analysis and select differential proteinsThe gels after2-DE were scanned into computer. We analysed each gel by a software named ImageMaster2D. Then we chose proteins which have statistical significance, with t test, P value less than0.05.MALDI-TOF/TOF analysisPlugs containing spots with significant and reproducible changes were excised from the gel. The proteins were identified with a MALDI-TOF/TOF tandem mass spectrometer ABI4700proteomics analyzer (Applied Biosystems, USA) according to following parameters:UV wavelength=355nm; repetition rate=200Hz; accelerated voltage=20000V; optimal resolution for the quality=1500Da; mass range700-3200Da.The mass spectrometer was corrected using an inner standard, which was identified as peak of instinct digestion of typsin. All mass spectrums of samples were obtained under a recommend module.Database SearchingUsing Mascot distiller software, the base peaks were filtered to identify the signals. Mascot software (Matrixscience) was used to search the matched proteins according to following criteria:1):The acquisition mass range was set at800to4,000Da;2);No restriction regarding molecular weight (Mr)or Isoelectric point (pI);3) mass measurement accuracy of±50ppm;4) up to two missed tryptic cleavages;5) taxonomy:restricted to mammalian sequences;6)MH+monoisotopic masses;7) carbamidomethyl cysteine as fixed modification, methionine oxidation as variable modification.Result1. The modle of neovasculation in cornea of rabbit was setup successfully.2. Local administration of angiostatin suppresses the neovascularization area of alkali-induced rabbit corneal neovascularization on postoperative day16, the neovascularization area in alkali burned rabbit cornea was46.77±8.98mm2, whereas it was statistically declined to37.62±9.65mm2by locally administrated with30μg/ml angiostatin. Using SPSS13.0software, the difference of neovascularization area in experimental and control animals, which was presented as mean and standard error, was analyzed with t test. P value less than0.05was considered as significant.3. Among normal, control and experimental proteins, thirteen spots were found to be significantly changed in volumes by values greater than1.5folds in the experimental animals compared to those in control.4. Thirteen protein spots above-mentioned were recognized and can be classified as two categories. The first category indicated that proteins down-regulated their expressions after alkali burn, but up-regulated responding for local administration of angiostatin, including serum albumin precursor, HSPA8protein, pyruvate kinase, bataB3-crystallin, retinol binding protein1and a unknown protein. In contrary, some proteins who up-regulated their expressions in alkali burned rabbit cornea, but down-regulated due to local administration of angiostatin were classified as category two, including reticulocalbin3,(EF-hand calcium binding domain), keratin14, actin-11, Ig lambda chain, immunoglobulin kappa-chain, and haptolobin.ConclusionPast decades have witnessed the rapid development of angiostatin used as an efficient inhibitor to overcome the neovascularization both in various carcinomas and other disorders, including corneal neovascularization introduced by gene fragment delivery or local administration. And, significant incline by locally administrated with angiostatin on area of neovascularization induced by alkali burn in rabbit reported here provided novel evidence that angiostatin can efficiently release the corneal neovascularization that was thought to attributing its capability to down-regulate the proliferation and migration of the vascular endothelial cells, and induce these cells to apoptosis. Using proteomic approach, we found here that local administration of angiostain suppresses the alkali induced overexpressions of defined proteins, including reticulocalbin3, keratin14and haptoglobin, which has been considered as participators for neovascularture formation. And, this treatment recovers the alkali induced lowed expressions of proteins as basis for corneal transparency, such as crystallins and retinol binding protein. Additionally, local administration of angiostatin has capability to up-regulate the expressions of proteins that involved in corneal epithelial wound healing, energy metabolism and protein synthesis, including albumin precursor, pyruvate kinase, and HSPA8protein. Certainly, the dynamic changes in protein expressions based on different time points after alkali burn in rabbit cornea should be explored in further, in order to throughout definition of anti-angiogenic efficiency of angiostatin for clinical translation. |
PDF Full Text Request