Retinal HtrA1 upregulates inflammatory factors in response to hypoxia in vivo

Abstract

Purpose : Elevated HTRA1 is significantly associated with retinopathy of prematurity (ROP) in human disease and animal models. Mechanisms for this association are not clear. We hypothesize that HTRA1 modulates ocular inflammatory gene expression during hypoxia.

Methods : HtrA1 and inflammatory factors with predicted roles in ROP disease were measured using RT-PCR in murine retinal or retinal pigment epithelial (RPE) cells. ΔΔCt values were compared among P14 transgenic HtrA1 overexpression (HtrA1^TG) pups and wildtype (WT) C57Bl6J littermate controls under normoxic or hypoxic oxygen induced retinopathy (OIR) conditions (N=3/group). Following humane euthanasia and enucleation, retinal tissue was isolated using microdissection. RPE cells were isolated from choroid via mechanical dissociation in RNAprotect solution for RNA extraction. HtrA1, IL-1ß, TNFα, IL6, and Iba1 cDNA was amplified in triplicate, and Ct values normalized to ß2M. Statistical significance was assessed using one-way ANOVA followed by Bonferroni-corrected two tailed t-tests for multiple comparisons.

Results : Baseline HtrA1 expression in normoxia-reared HtrA1^TG pups was 3.3-fold and 19-fold higher in retinal and RPE tissues, respectively (p<0.0001) compared with WT littermates. Under OIR conditions, WT retinal HtrA1 levels decreased significantly (2-fold; p<0.001), whereas RPE levels did not change. In contrast, OIR conditions in HtrA1^TG pups increased HtrA1 expression significantly in both retina (1.5-fold; p<0.005) and RPE (2-fold; p=0.005). Compared to normoxic conditions, OIR increased HtrA1^TG retinal expression of IL-1ß (4.5 fold; p<0.001), TNFα (23 fold; p<0.0001), and IL6 (4.3-fold; p<0.005). In WT retinas, a more moderate hypoxia-induced increase of IL-1ß (3 fold; p<0.005) and TNFα (12-fold; p<0.0001), but not IL6 were observed. We found no statistically significant expression changes for any inflammatory gene within the RPE.

Conclusions : We demonstrated that inflammatory gene expression was upregulated in hypoxic P14 retinae, further amplified by HtrA1 overexpression. In contrast to previous reports, hypoxia did not increase inflammatory gene expression in RPE cells. However, our study isolated RPE cells from the underlying choroid to prevent contamination from macrophages/monocytes within the choroidal vasculature. Our data suggest that HtrA1-mediated inflammatory activity specific to the retina rather than the RPE can exacerbate ROP.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.