Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery of c-Met gene.
Purpose. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)-driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities. Methods. Forty-six human corneas obtained postmortem from 23 donors with long-term diabetes (5 with diabetic retinopathy) were organ cultured and transduced with rAV-expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at approximately 10(8) plaque-forming units per cornea for 48 hours. Each control fellow cornea received control rAV (rAV expressing the beta-galactosidase gene or vector alone). After an additional 4 to 5 days of incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. The corneas were analyzed afterward by immunohistochemistry and Western blot analysis. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blot analysis, and inhibitor analysis. Results. rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular, and phosphorylated) and normalization of the patterns of select diabetic markers compared with rAV-vector-transduced control fellow corneas. Epithelial wound healing time in c-met-transduced diabetic corneas decreased twofold compared with rAV-vector-transduced corneas and became similar to normal. c-Met action apparently involved increased activation of p38 mitogen-activated protein kinase. c-Met transduction did not change tight junction protein patterns, suggesting unaltered epithelial barrier function. Conclusions. rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. c-Met gene therapy could be useful for correcting human diabetic corneal abnormalities.
Saghizadeh
Mehrnoosh
M
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, California, USA.
Kramerov
Andrei A
AA
Yu
Fu-Shin X
FS
Castro
Maria G
MG
Ljubimov
Alexander V
AV
D000256:Adenoviridae / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D048909:Diabetes Complications / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D003922:Diabetes Mellitus, Type 1 / Q000150:complications; D003924:Diabetes Mellitus, Type 2 / Q000150:complications; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D010766:Phosphorylation; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014945:Wound Healing / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met; D048051:p38 Mitogen-Activated Protein Kinases