Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Abstract

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.

HTRA1, an age‐related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1

“…It cleaves and inhibits TGF beta, and several extracellular matrix proteins, including fibronectin and amyloid precursor protein (Beaufort et al., 2014; Grau et al., 2005; Oka et al., 2004). Also, when HTRA1 is overexpressed in mice, the elastic network of extracellular matrix becomes fragmented between the RPE and the choroid (Vierkotten, Muether & Fauser, 2011). …”

HTRA1 (High Temperature Requirement A Serine Peptidase 1) Gene Is Transcriptionally Regulated by Insertion/Deletion Nucleotides Located at the 3′ End of the ARMS2 (Age-related Maculopathy Susceptibility 2) Gene in Patients with Age-related Macular Degeneration

“…AMD pathogenesis and previous HTRA1 experiments related to AMD have been performed and discussed in relation to the RPE (8,16,17). However, immunostaining of HTRA1 in mouse retinas in the present study showed that the majority of transcription occurs in the photoreceptor cell layer (Fig.…”

“…A recent report using transgenic mice overexpressing human HTRA1 in the RPE showed that PCV-like capillary structures could be observed in the choroid. Vierkotten et al (17) also observed fragmentation of the elastic layer in Bruch's membrane, and Jones et al (16) reported branching of choroidal vessels, polypoidal lesions, and severe degeneration of the elastic lamina or tunica media of choroidal vessels in the same model. When HtrA1 expression was driven by the CAG promoter in a transgenic mouse model, an even more severe phenotype was induced compared with previous reports of AMD patient-like choroidal neovascularization (23).…”

“…Several cellular and molecular studies have suggested that HTRA1 plays a key role in regulating various cellular processes via the cleavage and/or binding of pivotal factors that participate in cell proliferation, migration, and cell fate (12)(13)(14)(15). Recently, up-regulation of human HTRA1 expression in the mouse retinal pigment epithelium (RPE) was shown to induce a branching network of choroidal vessels and polypoidal lesions and severe degeneration of the elastic lamina and tunica media of the choroidal vessels, similar to that observed in AMD and PCV patients (16,17). A mutation in HTRA1 has also been associated with a hereditary form of familial cerebral small vessel disease (CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy)) (18).…”

Highly Penetrant Alleles in Age-Related Macular Degeneration

“…Elastin is a principal component of Bruch's membrane, and reduced levels of functional fibulin-5 protein in the extracellular matrix may impair the normal assembly of elastin within Bruch's membrane (Stone et al 2004). Interestingly, overexpression of HTRA1, which has been associated with AMD pathogenesis (Yang et al 2010), has been shown to alter elastogenesis in Bruch's membrane through cleaving fibulin-5 (Yang et al 2010;Vierkotten et al 2011). Alternatively, interference with another function of fibulin-5 (for example, integrin-mediated cell attachment) may contribute to the pathogenic mechanism underlying fibulin-5 variants (Stone et al 2004).…”