ABSTRACT
A critical component of corneal scarring is the TGFß-induced differentiation of corneal keratocytes into myofibroblasts. Inhibitors of this differentiation are potentially therapeutic for corneal scarring. In this study, we tested the relative effectiveness and mechanisms of action of two electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands: cyano-3,12-dioxolean-1,9-dien-28-oic acid-methyl ester (CDDO-Me) and 15-deoxy-Δ(-12,14)-prostaglandin J(2) (15d-PGJ(2)) for inhibiting TGFß-induced myofibroblast differentiation in vitro. TGFß was used to induce myofibroblast differentiation in cultured, primary human corneal fibroblasts. CDDO-Me and 15d-PGJ(2) were added to cultures to test their ability to inhibit this process. Myofibroblast differentiation was assessed by measuring the expression of myofibroblast-specific proteins (αSMA, collagen I, and fibronectin) and mRNA (αSMA and collagen III). The role of PPARγ in the inhibition of myofibroblast differentiation by these agents was tested in genetically and pharmacologically manipulated cells. Finally, we assayed the importance of electrophilicity in the actions of these agents on TGFß-induced αSMA expression via Western blotting and immunofluorescence. Both electrophilic PPARγ ligands (CDDO-Me and 15d-PGJ(2)) potently inhibited TGFß-induced myofibroblast differentiation, but PPARγ was only partially required for inhibition of myofibroblast differentiation by either agent. Electrophilic PPARγ ligands were able to inhibit myofibroblast differentiation more potently than non-electrophilic PPARγ ligands, suggesting an important role of electrophilicity in this process. CDDO-Me and 15d-PGJ(2) are strong inhibitors of TGFß-induced corneal fibroblast to myofibroblast differentiation in vitro, suggesting this class of agents as potential novel therapies for corneal scarring warranting further study in pre-clinical animal models.
Subject(s)
Cell Transdifferentiation/drug effects , Cornea/cytology , Fibroblasts/cytology , Myofibroblasts/cytology , Oleanolic Acid/analogs & derivatives , PPAR gamma/metabolism , Prostaglandin D2/analogs & derivatives , Actins/genetics , Actins/metabolism , Biomarkers/metabolism , Blotting, Western , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Cornea/metabolism , Fibroblasts/metabolism , Fibronectins/genetics , Fibronectins/metabolism , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Humans , Ligands , Myofibroblasts/metabolism , Oleanolic Acid/pharmacology , Prostaglandin D2/pharmacology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/pharmacologyABSTRACT
OBJECTIVE: To assist ophthalmologists in treating ocular trauma patients, this study developed and validated a prognostic model to predict vision survival after open globe injury. DESIGN: Retrospective cohort review. PARTICIPANTS: Two hundred fourteen patients who sought treatment at the Wilmer Ophthalmological Institute with open globe injuries from January 1, 2001, through December 31, 2004, were part of the data set used to build the classification tree model. Then, to validate the classification tree, 51 patients were followed up with the goal to compare their actual visual outcome with the outcome predicted by the tree grown from the classification and regression tree analysis. METHODS: Binary recursive partitioning was used to construct a classification tree to predict visual outcome after open globe injury. The retrospective cohort treated for open globe injury from January 1, 2001, through December 31, 2004, was used to develop the prognostic tree and constitutes the training sample. A second independent sample of patient eyes seen from January 1, 2005, through October 15, 2005, was used to validate the prognostic tree. MAIN OUTCOME MEASURES: Two main visual outcomes were assessed: vision survival (range, 20/20-light perception) and no vision (included no light perception, enucleation, and evisceration outcomes). RESULTS: A prognostic model for open globe injury outcome was constructed using 214 open globe injuries. Of 14 predictors determined to be associated with a no vision outcome in univariate analysis, presence of a relative afferent pupillary defect and poor initial visual acuity were the most predictive of complete loss of vision; presence of lid laceration and posterior wound location also predicted poor visual outcomes. In an independent cohort of 51 eyes, the prognostic model had 85.7% sensitivity to predict no vision correctly and 91.9% specificity to predict vision survival correctly. CONCLUSIONS: The open globe injury prognostic model constructed in this study demonstrated excellent predictive accuracy and should be useful in counseling patients and making clinical decisions regarding open globe injury management.
Subject(s)
Decision Trees , Eye Injuries, Penetrating/classification , Eye Injuries, Penetrating/physiopathology , Logistic Models , Vision Disorders/physiopathology , Visual Acuity/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Prognosis , Retrospective Studies , Sensitivity and SpecificityABSTRACT
Our aims were to describe the distribution of alpha-smooth muscle actin (SMA)-containing cells in Dupuytren's tissue in vivo and to determine the effects of selected agents in regulating the expression of SMA in Dupuytren's cells in vitro. In selected hypercellular zones of Dupuytren's nodules up to 40% of the cells contained SMA, as shown by immunohistochemistry. A lower percentage (20%) of SMA-containing cells was found in regions of lower cellularity. A notable finding was that treatment in vitro of Dupuytren's cells with platelet-derived growth factor significantly reduced the content of SMA. Cells from the same patients showed a significant increase in expression of SMA in response to treatment with transforming growth factor, which confirmed recent findings. In addition, interferon-gamma, which has been previously used as a treatment for Dupuytren's disease in a clinical study, had no reproducible effect on the expression of this actin isoform. Our findings are of significance for the conservative management of contractures.
Subject(s)
Actins/metabolism , Dupuytren Contracture/metabolism , Muscle, Smooth/metabolism , Adult , Aged , Analysis of Variance , Blotting, Western , Female , Humans , Immunohistochemistry , Male , Middle AgedABSTRACT
A sensitive and precise assay of guanase was based on the conversion of 14C-guanine to uric acid in the presence of excess xanthine oxidase. The enzyme was entirely soluble in rat tissues and no inhibitors of it were detected. The most active tissues were red blood cells, lung and lactating mammary gland, with more than twice the activity of liver. The contained blood could account for the low activity in adult skeletal muscle and some other tissues. All fetal tissues examined were without activity. Activity in mammary gland rose fourfold during lactation and dropped precipitously during involution, with a secondary rise associated in time with the loss of cells from the gland. Reticulocytes present normally and after hemorrhagic anemia appeared to account for substantially all of the high guanase activity in red blood cells. The guanase level could be used to predict the degree of reticulocytosis in rats within confidence limits of +/- 0.2%. The virtual absence of guanase in human red cells was confirmed, even in bloods containing elevated reticulocyte numbers.
