bromochloroacetic-acid and Corneal-Neovascularization

To compare the effects of early, mid, and late subconjunctival injection of bevacizumab on corneal neovascularization (NV) and conjunctivalization in a rabbit limbal insufficiency model.. Limbal insufficiency was created surgically, and subconjunctival injections of 2.5 mg bevacizumab twice weekly for 1 month were started immediately (early group), 1 week (mid group), and 1 month after injury (late group). The corneal epithelial alterations, stromal opacity, centricity, extent, and PICS (percentage of involved corneal surface) of the corneal NV were evaluated. The expression of cytokeratins K3, K4, K12, K13, and MUC5 by the corneal surface cells was examined by immunohistochemistry.. Bevacizumab significantly inhibited corneal NV in the early and mid, but not the late, treatment groups at 4 weeks after treatment (PICS: P < 0.01 in the early group, P < 0.01 in the mid group, and P > 0.05 in the late group). Early and mid treatment produced significant inhibition of corneal alteration (P < 0.01 in the early group and P = 0.03 in the mid group) and stromal opacity (P < 0.01 in the early group and P = 0.02 in the mid group) at 4 months after treatment but not in the late group. The immunohistochemistry of cytokeratin on the corneal surface cells at 1 month after treatment was K3(+), K4(-), K12(+), K13(-), and MUC5(-) in the early group; K3(+), K4(+), K12(+), K13(+), and MUC5(-) in the mid group; and K3(+), K4(+), K12(-), K13(+), and MUC5(+) in the late treatment group.. Early and mid bevacizumab injection inhibited corneal NV, epithelial alteration, and stromal opacity in limbal insufficiency, but late treatment did not. Early treatment with bevacizumab seems to be clinically beneficial in the management of limbal injury such as chemical burn.

Topics: 3T3 Cells; Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Cell Line; Colony-Forming Units Assay; Conjunctiva; Corneal Neovascularization; Corneal Stroma; Disease Models, Animal; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Injections; Keratins; Limbus Corneae; Mice; Microscopy, Fluorescence; Mucin-5B; Phenotype; Rabbits; Time Factors; Vascular Endothelial Growth Factor A; Wound Healing

Eph cell surface receptors and their ligands, ephrins, are involved in neuronal patterning and neovascularization. Our purpose is to compare and characterize the expression of ephrinA ligands and EphA receptors to ephrinB ligands and EphB receptors in excised mouse corneal tissue, in corneal epithelial and keratocyte cell lines, and during corneal angiogenesis.. Mouse corneal epithelial cells and keratocytes were immortalized using SV40T antigen viral infection of primary cultures. The immortalized epithelial cells and keratocytes were cloned and characterized using antibodies to keratin, vimentin, integrin alpha5beta1, and alpha-smooth muscle actin. Basic fibroblast growth factor pellets were implanted to induce corneal neovascularization. The eyes of wild-type, ephrinB2(tlacZ/+), and EphB4(tlacZ/+) heterozygous mice were harvested and sectioned 7 days after pellet implantation. Confocal immunohistochemistry was performed to compare the expression of the Eph/ephrinA family (EphA1-8, ephrinA1-5) and Eph/ephrinB family (EphB1-4, EphB6 ephrinB1-3).. EphA1, EphA3, ephrinA1, ephrinA2, EphB1, EphB4, ephrinB1, and ephrinB2 were detected in wild-type mouse corneal epithelial cells and keratocytes. EphA2 was immunolocalized only in epithelial cells. Also, EphA3, ephrinA1, EphB1, EphB4, and ephrinB1 were immunolocalized to the corneal epithelium and stroma. In the vascularized corneas, ephrinB1 was immunolocalized mainly to the keratocytes around the vessels, and ephrinB2, EphB1, and EphB4 were colocalized mainly with CD31 to the vascular endothelial cells.. The characterization of ephrin ligand and Eph receptor expression during cornea angiogensis in this study suggests that the Eph/ephrin family of receptor tyrosine kinases and their ligands may play a role in the regulation of corneal angiogenesis.

Topics: Actins; Animals; Cell Line; Corneal Neovascularization; Corneal Stroma; Ephrins; Epithelium, Corneal; Fibroblasts; Fluorescent Antibody Technique, Indirect; Integrin alpha5beta1; Keratins; Ligands; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Receptors, Eph Family; Vimentin

To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency.. The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively.. Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript.. The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker.

Topics: Adult; Child; Corneal Neovascularization; DNA-Binding Proteins; Epithelium, Corneal; Female; Genes, Tumor Suppressor; Goblet Cells; Humans; Keratin-12; Keratin-3; Keratins; Limbus Corneae; Male; Middle Aged; Mucin-5B; Mucins; Phosphoproteins; RNA, Messenger; Stem Cells; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice.. Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice.. In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells.. Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

Topics: Animals; Blotting, Northern; Blotting, Western; Chondroitin Sulfate Proteoglycans; Corneal Neovascularization; Corneal Stroma; Epithelium, Corneal; Eye Proteins; Fibroblasts; Filaggrin Proteins; Gene Expression; Hyperplasia; In Situ Hybridization; Intermediate Filament Proteins; Keratan Sulfate; Keratins; Lumican; Membrane Proteins; Mice; Mice, Mutant Strains; Protein Precursors; RNA, Messenger