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Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery of c-Met gene.

ABSTRACT: 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.

SUBMITTER: Saghizadeh M

PROVIDER: S-EPMC2846188 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWe recently reported the clinical safety profile of RJX, a well-defined intravenous GMP-grade pharmaceutical formulation of anti-oxidant and anti-inflammatory vitamins as active ingredients, in a Phase 1 study in healthy volunteers (ClinicalTrials.gov Identifier: NCT03680105) (Uckun et al., Front. Pharmacol. 11, 594321. 10.3389/fphar.2020.594321). The primary objective of the present study was to examine the effects of GMP-grade RJX on wound and burn injury healing in diabetic rats.MethodsIn the present study, a rat model of T2DM was used that employs HFD in combination with a single injection of STZ intraperitoneally (i.p) at a moderate dose level (45 mg/kg). Anesthetized diabetic rats underwent full-thickness skin excision on the back or were subjected to burn injury via a heated brass probe and then started on treatments with normal saline (NS = vehicle) or RJX administered via intraperitoneal injections for three weeks.FindingsNotably, diabetic rats treated with the 1.25 mL/kg or 2.5 mL/kg RJX (DM+RJX groups) rapidly healed their wounds as fast as non-diabetic control rats. Inflammatory cell infiltration in the dermis along with fibrin and cell debris on the epithelial layer persisted for up to 14 days in the DM+NS group but not in RJX-treated groups. The histopathological score of wound healing on days 7 and 14 was better in diabetic rats treated with RJX than diabetic rats treated with NS and comparable to the scores for non-diabetic healthy rats consistent with an accelerated healing process. The residual wound area of RJX-treated rats was significantly smaller than that of NS-treated diabetic rats at each evaluation time point (P<0.001). The accelerating effect of RJX on diabetic wound healing was dose-dependent. We obtained similar results in the burn injury model. Our results demonstrate that RJX - at a dose level >10-fold lower than its clinical maximum tolerated dose (MTD) - accelerates the healing of excision wounds as well burn injury in diabetic rats.

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Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery of c-Met gene.

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