Overexpression of HTRA1 in mice compromises the extracellular matrix integrity in Bruch’s membrane and choroidal/retinal vasculature

Abstract

Purpose : Genome-wide association studies implicate dysregulation of ECM homeostasis as a major pathogenic mechanism in age-related macular degeneration (AMD). High risk alleles at the ARMS2/HTRA1 locus confer the highest genetic risks for AMD and are associated with elevated high-temperature requirement A1 (HTRA1) expression. To investigate the role of the ECM protease HTRA1 in ocular pathology, mice overexpressing HTRA1 were characterized.

Methods : RNAScope was performed to assess ocular HTRA1 gene expression in C57BL/6 mice. A doxycycline (dox)-inducible transgenic mouse line overexpressing HTRA1 systemically (HTRA1 iTg) was utilized for the studies. An MSD assay was used to measure HTRA1 protein levels. Fibulin-5 and ApoE levels were quantified by Western blot. Assessment of structural changes were performed using ICGA/SLO, smooth muscle actin (SMA) staining of RPE/choroid flatmounts, and TEM. Retinal function was evaluated by ERG.

Results : HTRA1 mRNA expression by RNAScope was localized primarily to the inner nuclear layer of the retina in native C57BL/6 mice. Serum HTRA1 concentration was increased by 15-fold in HTRA1 iTg mice, compared to non-transgenic (Tg) controls, and were further elevated with dox treatment. Overexpression of HTRA1 in iTg mice treated with dox for 5 days, reduced the levels of fibulin-5 and ApoE by approximately 50%. Increased HTRA1 protease activity caused a reduction in vascular SMA staining in RPE/choroid flatmounts as well as Bruch’s membrane (BM) thickening with loosening or separation of collagen fibrils by TEM. HTRA1 iTg mice retinal vessels displayed mild dilation and tortuosity on ICGA compared to their non-Tg controls. PCV phenotype was not observed in iTg mice. ERG a, b and c-wave amplitudes did not differ significantly between iTg and non-Tg groups with or without dox treatment.

Conclusions : HTRA1 mRNA is expressed in mouse retina. Increased HTRA1 protease activity reduced HTRA1 substrate fibulin-5 and ApoE levels in HTRA1 iTg mice. Polymorphisms in fibulin-5 and ApoE are linked to increased risk of AMD. By affecting multiple substrates that play a critical role in ECM homeostasis and lipid metabolism, over-expression of HTRA1 causes BM thickening and choroidal vessel abnormalities, both of which are characteristic features of AMD. Inhibition of HTRA1 activity may protect ECM integrity and is a potential AMD therapeutic strategy.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.