Abstract
Purpose: :
Variants in the chromosome 10q26 gene cluster (ARMS2/HtrA1) have conferred risk for age-related macular degeneration (AMD). HtrA1 (high temperature requirement factor A-1) acts as a serine protease and is involved in protein quality control and cell fate. Enhanced expression of HtrA1 is reported in AMD lesions. Ccl2-/-/Cx3cr1-/-.mice exhibit clinical, morphological and biochemical retinal pathology mimicking human AMD lesions. This study evaluated HtrA1 expression in retinal pigment epithelium (RPE) of the Ccl2-/-/Cx3cr1-/- mice.
Methods: :
Eight week-old Ccl2-/-/Cx3cr1-/- and age-matched wild type C57BL/6 mice were enucleated and their eyes fixed in 4% formaldehyde in phosphate buffered saline. The RPE-choroid was dissected and prepared as a flat mount preparation for immunolabeling. Expression of HtrA1 was evaluated using HtrA1-specific rabbit polyclonal antibody. Flat mounted preparations were also labeled with DAPI and the actin cytoskeletal marker phalloidin. Confocal z-series were collected to evaluate the expression level and distribution of HtrA1 in the RPE of Ccl2-/-/Cx3cr1-/- and wild type mice. Immunohistochemistry using avidin-biotin-complex immunoperoxidase was also used to assess HtrA1 expression in frozen sections of Ccl2-/-/Cx3cr1-/- and C57BL/6 mice eyes. Gene expression was examined by quantitative real time PCR (RQ-PCR) of isolated RPE and retina.
Results: :
HtrA1 protein expression in RPE-choroid flat mounted tissue from the Ccl2-/-/Cx3cr1-/- mice was 2.68-fold lower than control C57BL/6 mice. In both Ccl2-/-/Cx3cr1-/- and wild type mice, HtrA1 localized to the apical side of the RPE. HtrA1 transcript expression was decreased 1.72 ± 0.06 fold in dissected RPE cells from the AMD-mouse model as compared to C57BL/6 RPE cells. Interestingly, expression of HtrA1 mRNA in isolated neuroretina was greater than control by 2.84-fold. Immunohistochemistry also illustrated higher immunoreactivity in the Ccl2-/-/Cx3cr1-/- neuroretina.
Conclusions: :
HtrA1 protein and transcript expression is decreased in the RPE of the Ccl2-/-/Cx3cr1-/- AMD-mouse model, but increased in the retina. The discrepancy of HtrA1 expression in the mouse model RPE and human AMD may possibly result from the different expressions in the diseased RPE and neuroretinal cells, the stage of the disease, and pathological differences between mouse and human. Further investigation of HtrA1 expression intensity and localization could provide insight into its association with AMD.
Keywords: microscopy: confocal/tunneling • retinal pigment epithelium • age-related macular degeneration