| Angiogenesis is the process of neovascularization from pre-existing blood vessels. Angiogenesis is required for a variety of physiological processes such as embryonic development, wound healing, and tissue as well as organ regeneration. Abnormal growth of new blood vessels can lead to the progression of many eye diseases related to the neovascularization, such as corneal infection, proliferative vitroretinopathy, exudative age-related macular degeneration and so on. The switch to the pathogenic angiogenic phenotype requires both up-regulation of angiogenic stimulators and down-regulation of angiogenesis inhibitors abnormally, which then leads to the processes of quiescent endothelia entering into the cell cycle, migrating, degrading the underlying basement membrane, and forming a lumen. Vascular endothelial growth factor (VEGF), as the key element in this complex angiogenic response and the strong vascular permeable factor, has been reported to be an endothelium-specific mitogen, and then plays an important role in the neovascularization.The kringle domain is a protein structure comprising about 80 amino acids with conserved triple disulfide bonds that appears to function as an independent folding unit. Kringle domains are found in many proteins with a surprisingly diverse array of functions such as growth factors, proteases, or coagulation factors (hepatocyte growth factor, plasminogen, prothrombin, and urokinase, etc.). They are thought to play an important role in protein-protein interactions that provide specificity to and regulation of their parent proteins. Moreover, many kringle domains have been identified as inhibitors of angiogenesis. Based on these findings, kringles have been suggested to constitute the first example of a conserved architecture that specifically inhibits blood vessel growth. A considerable numble of kringles have been confirmed to be able tosuppress the angiogenesis, including of plasminogen kringle 5 (K5).Lipoprotein is a spherical large molecular complex that is composed of lipid in plasm and special proteins, the former is called as apolipoprotein. Lipoprotein(a), [Lp(a)] includes apolipoprotein B100 (apo B) and apolipoprotein (a) [apo(a)]. It consists of tandemly repeated kringle domains that closely resemble plasminogen kringle 4 (termed KIV), followed by sequences that are homologous to the kringle 5 (termed KV) and protease domains of plasminogen. Although the physiological and biochemical roles of apo(a) kringle domains remain to be elucidated, there exists some evidence suggesting that they can inhibit angiogenesis. However, its mechanism of action and the exact region that serves this special function was not clearly elucidated.This study was performed to explore the the exact anti-angiogenic region in the amino acid sequence of human apo(a), and to evaluate the role of this synthetic peptide on VEGF-induced angiogenesis of mouse cornea in vivo. The characterization of the structure and biological activity of the amino acid sequence of apo(a) KV was analyzed, applying the bioinformatic methods, which include sequence alignment, analysis of antigenicity, surface accessibility and hydrophilicity, and then a peptide, YTMNPRKLFDY, was selected. The peptide was synthesized with a high efficiency solid-phase method. Then the mouse corneal micropocket model was applied to test the effect of this synthetic peptide on the VEGF-induced mouse corneal neovascularization.Section 1 Sequence analysis and peptide selection by bioinformaticsObjective: Analyzing the amino acid sequence of apo(a), applying the bioinfomatic methods to predict the active anti-angiogenic region in the apo(a). Method: The amino acid sequence of apo(a) KV was inquired by opening the homepage of National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/. and entering the NRDB protein sequence secondary database. Then this sequence was submitted through the Standard Protein-Protein BLAST (BLASTp) in the same BLAST section of the NCBInet. The characterizationof the structure and biological activity of the amino acid sequence of apo(a) KV was analyzed, throμgh the analysis of antigenicity, surface accessibility, hydrophilicity and amino acid composition and then a peptide, was selected, using the related programmes in the Lasergene software and the BioEdit software respecitively. Results: The amino acid sequence of human apo(a) KV (4227-4327) acquired from the NRDB protein sequence secondary database was reported as follows: DCMF GNGKGYRGKK ATTVTGTPCQ EWAAQEPHRH STFIPGTNKW AGLEKNYCRN PDGDINGPWC YTMNPRKLFD YCDIPLCASS SFDCGKPQVE PKKCPGS. Analysis by BLAST programme showed that apo(a) KV and plasminogen K5 have 85% of the same amino acids, and 91% of the resemble ones (Expect = 1e-51). It is speculated that there might be some active anti-angiogenic regions in the conserved sequenence of apo(a). According to the results of the sequence alignment above, combined with the spacial configuration, the sequence of apo(a) was divided into 4 parts, Pept 1, Pept 2, Pept 3 and Pept 4, respectively. Antigenicity analysis showed that there were antigenic peaks in part of Pept 1 and Pept 3, the whole of Pept 4. Surface accessibility analysis showed that there were peaks appeared in middle Pept 1, foreside of Pept 2 and the whole of Pept 4. Hydrophilicity analysis showed that both of Pept 3 and Pept 4 had a hydrophilicity hollow. The percentage of hydrophobic amino acids (AILFWV) in Pept 1 to Pept 4 were 15.33 %, 29.10%, 24.16% 和 18.00%, respectively. Conclusion: Among all peptide-segments in the sequence of apo(a), Pept 4 had the largest proportion of conserved amino acid residues, and also had better biologic acitivity, including antigenicity, surface accessibility and hydrophilicity, than the other three ones. Therefore, it is presumed that Pept 4, which has the sequence of YTMNPRKLFDY, may be related to the anti-angiogenic acitivity of apo(a) KV.Section 2 Establishment of a mouse corneal micropocket modelPurpose: The purpose of this part of research was to establish a model of non-inflammatious angiogenesis in the mouse cornea, with VEGF as the direct and only stimulus. Method: 12% PolyHEMA in ethanol and sucralfate solution wasmixed together in the same volumes, and micropeilets (0.35 * 0.35 mm) were made. Subsequently, 160 ng VEGF was added to each pellet. All procedures were perfonned under sterile condition. A stromal linear keratotomy was nicked parallel to the insertion of the lateral rectus muscle in the eye, then an intrastromal micropocket was dissected. The pellets with or without VEGF were implanted into the corneal stroma pocket in each eye. Every eye was observed after surgery everyday until the subsequent vascular response appeared, and then it was photographed every other day with the maximal vessel length (VL) extending from the limbal vasculature toward the pellet and the contiguous circumferential zone of neovascularization (CN) were measured. The area of neovascular response was calculated using the formula: Area (mm~2)=0.2 × π × VL (mm) × CN (mm). Histologic analysis of mouse corneas were also conducted at every observing time. Result: Biomicroscopic observation revealed that in the group of corneas with pellet containing VEGF, the healing process of the corneal epithelium and the stroma to be mildly edematous within 24h after surgery. Limbal vessels began to develop toward the implant within a previously avascular cornea on postoperative day 3. The neovascularization was induced by VEGF pellet in the corneal stroma and reached the peak at day 7. The neovascular response was intense, well localized and reproducible. While in the group of corneas with pellet not containing VEGF have not been seen any neovascularization within 2w after operation. Conclusion: The neovascularization in this mouse corneal micropocket model is induced directly by VEGF, excluding the inflammtion response of the operation. Therefore, this noninflammatory model of corneal neovascularization is especially advantageous because it is reproducible, economical, and facilitates investigation of anti-angiogenic drμgs.Section 3 Study on the effects of the synthetic peptide on the mouse corneal neovascularization induced by VEGFPurpose: The peptide selected by bioinformatic methods was synthesized. Applying the mouse corneal micropoket model to determine whether this peptidecould inhibit VEGF-induced angiogenesis. Methods: The peptide was synthesized automatically according to its sequence of amino acid residues, and was purified by high performance liquid chromatography (HPLC). 12% PolyHEMA in ethanol and sucralfate solution was mixed together in the same volumes, and micropellets (0.35 * 0.35 mm) were made. Subsequently, 160 ng VEGF with 0μg, 0.5μg, 1μg, and 1.5μg of the peptide, and only 1μg of the peptide were added to each pellet respectively. All procedures were performed under sterile condition. Choosing the right eye of each C57B1/6 mouse as the expermental eye, then 50 eyes were randomly divided into 5 groups of 10 eyes per group. A stromal linear keratotomy was nicked parallel to the insertion of the lateral rectus muscle in the eye, then an intrastromal micropocket was dissected. Different pellets were implanted into the corneal stroma pocket in each group. At day 7 after the surgery, the vascular response in mouse corneas was photographed and the the maximal vessel length (VL) extending from the limbal vasculature toward the pellet and the contiguous circumferential zone of neovascularization (CN) were measured. The area of neovascular response was calculated using the formula: Area (mm~2) = 0.2 × π × VL (mm) × CN (mm).Histologic analysis of mouse corneas were also conducted. Results: YTMNPRKLFDY, a small peptide containing only 11 amino acid residues, was synthesized in the form of white powder (purity > 95%). The doses of 1.0 μg and 1.5 μg synthetic peptide showed significant inhibition of the vessel length, the clock hours and the area of VEGF-induced mouse corneal neovascularization compared with the control group on day 7 after the operation (P <0.01; P <0.01). The dose of 1.5 μg showed no more potent inhibitory effect than 1.0 μg (P >0.05). The dose of 0.5 μg did not demonstrate any significant inhibition of neovascularization compared with the control group (P >0.05). While in the group of corneas with pellet containing only 1.0 μg peptide were not seen any neovascularization within 2 w after the operation. Conclusion: The peptide, YTMNPRKLFDY, appears to inhibit VEGF-induced angiogenesis in a mouse corneal micropocket assay in vivo, therefore, this amino acid sequence may locate at the active anti-angiogenic region of apo(a) KV.
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