Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes.
Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1-2 x 10(8) plaque forming units of rAV per cornea were added to cultures for 48 h with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6-10 days additional incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol. Sildenafil significantly increased epithelial transduction efficiency, apparently by stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin alpha(3)beta(1), nidogen-1, nidogen-2, and laminin gamma2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic corneas. The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.
Saghizadeh
Mehrnoosh
M
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Kramerov
Andrei A
AA
Yaghoobzadeh
Yousha
Y
Hu
Jinwei
J
Ljubimova
Julia Y
JY
Black
Keith L
KL
Castro
Maria G
MG
Ljubimov
Alexander V
AV
D000256:Adenoviridae / Q000472:pathogenicity* / Q000502:physiology; D000257:Adenoviridae Infections / Q000150:complications*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D056646:Cathepsin F / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D006224:Cricetinae; D003412:Cricetulus; D003920:Diabetes Mellitus / Q000209:etiology*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D014161:Transduction, Genetic / Q000379:methods; D014945:Wound Healing / Q000235:genetics
D005136:Eye Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D010447:Peptide Hydrolases; C534090:CTSF protein, human; D056646:Cathepsin F