RESUMO
Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.
Assuntos
Retinopatia Diabética/enzimologia , Retinopatia Diabética/prevenção & controle , Epóxido Hidrolases/antagonistas & inibidores , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Progressão da Doença , Ácidos Docosa-Hexaenoicos/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Ependimogliais , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/patologia , Presenilina-1/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/metabolismo , Retina/patologia , Solubilidade , Corpo Vítreo/metabolismoRESUMO
Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31 P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.