Angiographic features of transgenic mice with increased expression of human serine protease HTRA1 in retinal pigment epithelium

Abstract

Purpose: Polypoidal choroidal vasculopathy (PCV) is characterized by a branching vascular network (BVN) of choroid that terminates in polypoidal dilations. We have previously reported the generation of the first PCV model by transgenically expressing human HTRA1 (hHTRA1(+)), a multifunctional serine protease, in mouse RPE. The purpose of this study was to perform a comprehensive examination of the PCV phenotypes (e.g., lesion type and distribution) of hHTRA1(+) mice by a variety of in vivo imaging techniques.

Methods: We generated improved hHTRA1(+) mice with a more consistent phenotype. Transgenic mice were examined by indocyanine green angiography (ICGA), fluorescein angiography, funduscopy, and spectral-domain optical coherence tomography. In particular, we performed ICGA by tail vein injection of ICG to obtain high-quality ICGA comparable to human studies in terms of the three phases (early, middle, and late) of angiography.

Results: The polyps can be detected in the early "fill-in" phase of ICGA, and most lesions become visible in the middle phase and are more distinct in the late phase with the fading of surrounding vessels. In addition to the two key features of PCV (polypoidal dilations and BVNs), hHTRA1(+) mice exhibit other features of PCV (i.e., late geographic hyperfluorescence, pigment epithelial detachment, and hyperfluorescent plaque). Polypoidal lesions appear as reddish orange nodules on funduscopy.

Conclusions: Transgenic hHTRA1(+) mice exhibit a rich spectrum of "clinical" features that closely mimic human PCV. This animal model will serve as an invaluable tool for future mechanistic and translational studies of PCV and other forms of choroidal vasculopathies.

Keywords: HTRA1; indocyanine green angiography; late geographic hyperfluorescence; pigment epithelial detachment; polypoidal choroidal vasculopathy.

Figure 1

Expression of human HTRA1 in mouse RPE. (A) Western blot analysis of human HTRA1 expression in RPE and choroid of Tg44 mice. The Western blot shows the relative levels of human HTRA1 signal from 4 μg RPE and choroid lysate of Tg44 mice (n = 3) and 15 μg lysate of human RPE (n = 3). (His)₆-tagged recombinant human HTRA1 protein (approximately 52 kDa) was used as the standard (in nanograms). The myc–(His)₆-tagged transgenic HTRA1, (His)₆-tagged recombinant HTRA1, and native HTRA1 (from human RPE and choroid) ran at 55, 52, and 50 kDa, respectively. (B) The pixel values of the recombinant protein bands in (A) in arbitrary units (AU) were plotted against the protein amounts to construct a standard curve. The expression levels of human HTRA1 in Tg44 mice and human RPE were determined according to their AU and the standard curve. (C) Comparison of human HTRA1 protein levels in Tg44 mice and human RPE (n = 3). Data are expressed as means (SEMs). ***P < 0.001. (D) Retinal sections from WT and Tg44 mice immunostained with a mouse anti-human HTRA1 antibody (green) or the isotype control mouse IgG2b together with a rabbit anti-bovine RPE65 antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Transgenic HTRA1 was detected at the basal side of RPE (white arrows), Bruch's membrane (white arrowheads), and choroid (yellow arrows) of hHTRA1⁺ mice. BM, Bruch's membrane; Cho, choroid; ROS, rod outer segment. Scale bar: 10 μm.

Figure 2

Angiographic features of Tg44 mice on time-course IV-ICGA. The early, middle, and late phases of ICGA were recorded for WT control (A) and Tg44 mice (B–E). The Tg44 mice developed a variety of phenotypes related to PCV, polyp dilations ([B, C], blue arrows), BVNs ([B–D], red circles on the left), LGHs ([C, D], red circles), and hyperfluorescent plaques ([E], red circle).

Figure 3

The PCV lesion numbers increased during a 10-month period in Tg44 mice. The numbers of small (≤100 μm) and large (>100 μm) lesions per eye were counted at age 1 (n = 40), 2 (n = 50), 3 (n = 76), 4 (n = 46), 5 (n = 36), and 10 (n = 41) months. Data are expressed as means (SEMs).

Figure 4

Funduscopic features of PCV lesions. Funduscopic examination was performed in WT control and Tg44 mice. In Tg44 mice, reddish orange nodules, which correspond to PCV lesion structures, are indicated ([B], white arrowhead; [C], red box). Hemorrhagic ([B], white stars) and serous ([C], white asterisks) PEDs, RPE degeneration ([C], yellow arrow), and yellowish hard exudates were observed near the lesion site ([B], green arrow).

Figure 5

Tomographic features of PCV lesions. Spectral-domain optical coherence tomography was used to examine WT controls (A), cluster polyps (B), and PEDs (C) in Tg44 mice. (B) The blue arrow indicates the start of the lesion. The red bracket indicates an RPE bump, which corresponds to the middle polyp (red arrow). The blue bracket indicates RPE degeneration between the middle and right polyps. (C) The red bracket indicates a PED located in a PCV lesion area (red arrow). Shown at the bottom is a magnified (×3) region of the PED. Detachment of RPE can be clearly seen. Cho, choroid; ELM, external limiting membrane; GCL, ganglion cell layer; INL, inner nuclear layer; IS/OS, the transition zone between the inner and outer segments (also referred to in human clinical literature as the inner segment ellipsoid); ONL, outer nuclear layer.

Figure 6

A representative 4-month-old Tg44 mouse was examined by ICGA, FA, funduscopy, and SD-OCT. The red box indicates an LGH-type lesion on ICGA and funduscopy. The OCT scan line is indicated by a green horizontal line on the ICGA image. Two blue arrows mark the boundary of the LGH lesion. A yellow arrow points to a notch of the LGH, which corresponds to the beginning of a choroidal hyperreflective structure on OCT. A red bracket on OCT indicates the location of a polyp (purple arrow on ICGA) inside the sub-RPE space. Note that the junction of the IS/OS (the transition zone between the inner and outer segments) and the RPE layers are present, suggesting no significant RPE atrophy.