This exploratory grant application (R21) has been prepared by a new investigator to obtain the preliminary data to prepare a competitive R01 application. Treatments that specifically target the fibrotic process in diabetes are not available primarily because few of the molecular mechanisms of fibrogenesis have been described sufficiently. A critical step in the development of fibrosis is the formation of myofibroblasts. The flavonoid apigenin disrupts the myofibroblast phenotype, the primary collagen-producing cell type in fibrotic tissues by selectively blocking basal and TGF-?-stimulated expression of a1(l) collagen and a smooth muscle actin, genes that are characteristically up-regulated in myofibroblasts. Our preliminary data indicate that apigenin blocks H2O2 production and stimulates expression of catalase mRNA. We have also observed that the attenuation of the TGF- ? -stimulated increase of a smooth muscle actin mRNA by apigenin can be partially overcome by inhibition of the Ca2+-activated K+ channel. Furthermore, inhibition of the Na+/Ca2+ exchanger or the Na+/K+/2CI- cotransporter attenuates expression of a1(l) collagen and a smooth muscle actin. These findings suggest that cation metabolism and H2O2 regulate myofibroblast differentiation. The objective of this application is to investigate the effects of flavonoids on myofibroblast function. The long-range goal is to identify efficacious interventions for the management of fibrosis. The central hypothesis of the proposed research is that specific flavonoids disrupt the maintenance of the myofibroblast phenotype by blocking production of reactive oxygen species and by blocking non-enzymatic N-linked glycation. We will test our hypothesis through the following specific aims:
Aim I. To characterize the signaling events induced by glucose that stimulate expression of a1(l) collagen and a smooth muscle actin. We hypothesize that reducing sugars such as glucose induce expression of a1(l) collagen and a smooth muscle actin mRNAs through a mechanism that initiates with non-enzymatic glycation.
Aim II. To identify the mechanism by which apigenin and other structurally related flavonoids disrupt maintenance of the myofibroblast phenotype. We hypothesize that apigenin, luteolin, and chrysoeriol modulate expression of specific genes that are required to maintain the myofibroblast phenotype and block glycation. The rationale for this study is that understanding the mechanisms for flavonoid modulation of fibrogenesis allows the development of novel therapeutic interventions that specifically target the fibrogenic pathway. We expect that, by the completion of this project, we will have characterized the signal transduction pathway that maintains the myofibroblast phenotype and the effects of apigenin on this pathway. The results from the research proposed will be significant because they will identify critical physiological and cellular processes involved in fibrogenesis. ? ?