Human iPSC-based disease modeling studies identify a common mechanistic defect and potential therapies for AMD and related macular dystrophies

Abstract

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) primarily affect the retinal pigment epithelium (RPE) in the eye. A hallmark of AMD/MDs that drives later-stage pathologies is drusen. Drusen are sub-RPE lipid-protein-rich extracellular deposits, but how drusen forms and accumulates is not known. We utilized human induced pluripotent stem cell (iPSC)-derived RPE from patients with AMD and three distinct MDs to demonstrate that reduced activity of RPE-secreted matrix metalloproteinase 2 (MMP2) contributes to drusen in multiple maculopathies in a genotype-agnostic manner by instigating sterile inflammation and impaired lipid homeostasis via damage-associated molecular pattern molecule (DAMP)-mediated activation of receptor for advanced glycation end-products (RAGE) and increased secretory phospholipase 2-IIA (sPLA2-IIA) levels. Therapeutically, RPE-specific MMP2 supplementation, RAGE-antagonistic peptide, and a small molecule inhibitor of sPLA2-IIA ameliorated drusen accumulation in AMD/MD iPSC-RPE. Ultimately, this study defines a causal role of the MMP2-DAMP-RAGE-sPLA2-IIA axis in AMD/MDs.

Trial registration: ClinicalTrials.gov NCT01782989.

Keywords: ADRD; DHRD; Doyne honeycomb macular dystrophy; SFD; Sorsby’s fundus dystrophy; age-related macular degeneration; autosomal dominant radial drusen; drusen; macular dystrophy; matrix metalloproteinase 2; retinal pigment epithelium; sterile inflammation; tissue inhibitor of metalloproteinase 3.

Figure 1.. Increased TIMP3 levels and decreased MMP2 activity precede drusen in SFD iRPE cultures.

(a, b) Western blot images (a) and quantification (b) of TIMP3 levels in the ECM underlying control versus SFD iRPE at days 14, 30 and 90 of culture. Data is presented normalized to control sample. * p < 0.05, ** p< 0.01. n = 3–5 biological replicates. (c, d) Gelatin zymography images (c) and quantification (d) of total active MMP2 levels in the RPE-CM collected from the basal chamber of control versus SFD iRPE cells at days 14, 30 and 90 in culture. Data is represented as normalized to control. * p< 0.01, *** p<0.005. n = 3 biological replicates. (e) TEM images showing absence of drusen in day 30 SFD iRPE culture (top panel) but presence of drusen (marked by white arrow, bottom panel) in day 90 SFD iRPE culture. Scale bar = 500 nm. (f) Confocal microscopy images (top panel) and quantification of the count and area of co-localized APOE (green) and Nile Red (red)-positive drusen beneath day 90 control and SFD iRPE cells. Scale bar = 50 μm. Note the absence of DAPI-positive cells is because RPE was removed prior to immunocytochemical analysis for drusen proteins on transwell membranes. ** p< 0.01. n = 3 biological replicates represented by three distinct colored data points. (g) Schematic depicting the longitudinal changes in the levels of ECM TIMP3, MMP2 activity and formation of drusen at varying timepoints (Day 14, 30 and 90) in control versus SFD iRPE cultures. See also Figure S1, S2 and Table S1.

Figure 2.. Increased TIMP3 and decreased MMP2 activity precede drusen in DHRD and AMD iRPE cultures.

(a, b) Western blot images (a) and quantification (b) of TIMP3 levels in the ECM underlying control versus DHRD and control versus AMD iRPE cells in day 30 cultures. TIMP3 levels were calculated relative to total protein. * p < 0.05. n = 3–6 biological replicates. (c, d) Gelatin zymography images (c) and quantitative analyses (d) of the levels of total active MMP2 in the RPE-CM collected from the basal chamber of control versus DHRD and control versus AMD iRPE cells at ~30 days of culture. Data is represented as normalized to control. * p < 0.05, *** p<0.005. n = 3 biological replicates. (e) TEM image of day 30 DHRD iRPE culture showing absence of drusen. Scale bar = 200 nm. (f) Confocal microscopy images showing lack of co-localized APOE (green) and Nile Red (red) drusen in day 30 control and AMD iRPE cultures. Note: DAPI-positive cells are absent as RPE monolayer was removed prior to immunocytochemistry for drusen proteins on transwell membrane. Scale bar = 50 μm. (g) TEM image showing drusen-like deposits in day 90 DHRD iRPE culture. Scale bar = 200 nm. (h) Confocal microscopy images (top panel) and quantification (bottom panel) showing count and area of APOE (green) and Nile Red (red) co-localized drusen deposits beneath control versus AMD iRPE after ~90 days in culture. Note the absence of DAPI as the RPE monolayer was removed from transwell membrane prior to immunocytochemical analyses of drusen proteins. Scale bar = 50 μm. *** p<0.005, n = 3 biological replicates represented by the three distinct colored data points. (i) Schematic showing the time course of ECM-TIMP3 accumulation, reduced MMP2 activity and presence of drusen in DHRD and AMD iRPE cultures. See also Figure S1, S2 and Table S1.

Figure 3.. Pharmacological inhibition of MMP2 activity promotes pro-maculopathy changes in control iRPE cultures.

(a, b) Western blot images (a) and corresponding quantification (b) showing levels of HMGB1 and sPLA2-IIA in basally-secreted RPE-CM of untreated versus MMP2-I1-treated (5 μM; daily for 6 days) iRPE cultures at day 3 (HMGB1) and day 6 (sPLA2-IIA) of treatment. Data is presented relative to total protein in the RPE-CM and was normalized to the untreated condition. * p<0.05. n = 6 biological replicates. (c, d) Western blot image (c) and corresponding quantification (d) showing RAGE levels in MMP2-I1-treated (5 μM; daily for 6 days) iRPE cells compared to untreated iRPE cells at day 6 of treatment. * p<0.05. n = 4 biological replicates. (e) Quantitative real time PCR analyses showing expression of complement pathway genes, C3 and CFB, in MMP2-I1-treated (5 μM; daily for 6 days) iRPE cells compared to untreated iRPE cells at day 6 of treatment. * p<0.05. n = 3 biological replicates. (f) TER measurement of untreated and MMP2-I1-treated (5 μM; daily for 6 days) cultures at baseline (day 0; prior to starting the treatment) and day 6 of MMP2-I1 treatment. The dashed lines represent the reported threshold of in vivo RPE TER. ** p<0.01. n = 3 biological replicates. (g) TEM image of an RPE section from MMP2-I1-treated iRPE culture showing lipid deposits (white arrowheads) at day 14 of 5 μM daily treatment. Scale bar = 500 nm. (h, i) Light microscopy images (h) and quantification (i) of Oil Red O positive neutral lipids in untreated versus MMP2-I1-treated (5 μM; daily for 14 days) iRPE cells. *** p<0.005. n = 3 biological replicates. Scale bar = 25 μm. (j) TEM images of RPE sections from MMP2-I1-treated iRPE culture showing presence of drusen deposits (white arrowheads) at day 14 of 5 μM daily treatment. Scale bar = 500 nm. (k) Confocal images showing co-localization of sPLA2-IIA (green) and APOE (red) in drusen beneath untreated versus MMP2-I1-treated (5 μM; daily for 6 days) RPE cultures at day 6 of treatment. Scale bar = 50 μm. Note: The absence of DAPI-positive cells is because the RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. (l) Quantitative analyses of the count and area of co-localized sPLA2-IIA (green) and APOE (red) positive drusen deposits in untreated versus MMP2-I1-treated (5 μM; daily for 6 days) iRPE cultures at day 6 of treatment. Data is presented normalized to count and area of untreated iRPE cultures. *** p<0.005. n = 3 biological replicates represented by three distinct colored data points. (m) Fluorescence microscopy images of Calcein-AM-stained live cells in untreated versus MMP2-I1-treated (5 μM; daily for 6 days) RPE cultures at baseline (day 0; prior to starting the treatment; top panel) and day 6 (bottom panel) of treatment. Scale bar = 50 μm. See also Figure S2 and S3.

Figure 4.. MMP2 and RAGE mediate pro-maculopathy cellular changes in Dox-treated iRPE.

(a, b) Western blot images (a) and quantification (b) showing levels of complement C3 in basally secreted RPE-CM of untreated versus Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and RAP-supplemented (5 μM; daily for 6 days) iRPE cultures. Data is presented normalized to Dox-treated cultures. * p<0.05, ** p<0.01. n = 4–6 biological replicates. (c) TER measurement of Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and RAP-supplemented (5 μM; daily for 6 days) iRPE cultures at baseline (day 0) and day 6 of treatments. *** p<0.005. n = 3–5 biological replicates. (d, e) Immunofluorescence images (d) and quantification (e) showing amount (count, area) of co-localized HMGB1 (green), Nile Red (red) and APOE (blue) drusen beneath Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and RAP-supplemented (5 μM; daily for 6 days) iRPE cultures at day 6 of treatment. Note: The absence of DAPI-positive cells is due to removal of RPE monolayer prior to staining for drusen proteins on transwell membrane. Data is presented normalized to Dox-treated iRPE cultures. Scale bar = 50 μm. * p≤0.05; ** p≤0.01. n = 3 biological replicates. (f) Fluorescence images showing Calcein-AM-stained live cells at baseline (top panel) and day 6 (bottom panel) in Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and RAP-supplemented (5 μM; daily for 6 days) iRPE cultures. Scale bar = 50 μm. (g, h) Western blot images (g) and quantification (h) showing levels of HMGB1 and sPLA2-IIA in basally secreted RPE-CM of Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and MMP2-supplemented (150 nM; basally supplemented daily for 6 days) iRPE cultures at day 3 (HMGB1) and day 6 (sPLA2-IIA) of treatment respectively. Data is presented normalized to Dox-treated cultures. * p<0.05. n = 3 biological replicates. (i) TER measurement of Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and MMP2-supplemented (150 nM; basally supplemented daily for 6 days) iRPE cultures at baseline (day 0, before the start of treatment) and day 6 of treatment. The dashed lines mark the in vivo threshold of TER for RPE cells. * p<0.05. n = 3 biological replicates. (j, k) Immunofluorescence images (j) and quantification (k) showing amount (count, area) of co-localized TIMP3 (green), Nile Red (red) and APOE (blue) drusen beneath Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and MMP2-supplemented (150 nM; basally supplemented daily for 6 days) iRPE cultures at day 6 of treatment. Data is presented normalized to Dox-treated iRPE cultures. Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm. *** p≤0.005. n = 3 biological replicates shown by three distinct colored data points. (l) Fluorescence microscopy images of Calcein-AM-stained live cells in Dox-treated (30 μM; daily for 6 days) versus Dox-treated (30 μM; daily for 6 days) and MMP2-supplemented (150 nM; basally supplemented daily for 6 days) iRPE cultures at baseline (day 0, before the start of treatment; top panel) and day 6 (bottom panel) of treatments. Scale bar = 50 μm. See also Figure S2 and S3.

Figure 5.. MMP2 supplementation decreases drusen in SFD, DHRD and AMD iRPE cultures.

(a, a’) Confocal images (a) and quantitative analyses (a’) showing levels (count, area) of co-localized TIMP3 (green) and APOE (red) sub-RPE drusen deposits (on transwell membrane after removing RPE cells) in unsupplemented versus MMP2-supplemented (150 nM; basally supplemented for 18h) in aged (>90 days in culture) SFD iRPE cultures. Data is presented normalized to unsupplemented SFD iRPE cultures. Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm. * p<0.05; ** p<0.01. n = 3 biological replicates shown by three distinct colored data points. (b, b’) Confocal images (b) and quantitative analyses (b’) showing amount (count, area) of co-localized TIMP3 (green) and APOE (red) drusen deposits (on transwell membrane after removing RPE cells) in day 90 DHRD iRPE cultures that were either unsupplemented or MMP2-supplemented (150 nM; basally supplemented for 18 h). Data is presented normalized to unsupplemented DHRD iRPE cultures Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm. *** p≤0.005. n = 3 biological replicates shown by three distinct colored data points. (c, c’) Confocal images (c) and quantification (c’) of the count and area of co-localized sPLA2-IIA (green)/APOE (red) drusen (on transwell membrane after removing RPE cells) in >90-day cultures of unsupplemented versus MMP2-supplemented (150 nM; basally supplemented for 18 h) AMD iRPE cultures. Data is presented normalized to unsupplemented AMD iRPE cultures. Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm ** p<0.01. n = 3 biological replicates is shown by three distinct colored data points. See also Figure S4.

Figure 6.. RAGE antagonist peptide (RAP) and a sPLA2-IIA inhibitor decrease drusen accumulation in SFD, DHRD and AMD iRPE cultures.

(a-d) Confocal images (a-c) and quantification (d) showing amount (count, area) of co-localized APOE (green) and sPLA2-IIA (red) drusen in >90-day unsupplemented versus RAP-supplemented (5 μM; daily for 14 days) SFD (a, d), DHRD (b, d) and AMD (c, d) iRPE cultures. Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm. ** p<0.01 and *** p≤0.005. n = 3 biological replicates for each disease shown by distinct colored data points. (e-h) Confocal images (e-g) and quantification (h) of >90-day cultures showing amount (count, area) of co-localized APOE (green) and Nile Red (red) or APOE (green) and sPLA2-IIA (red) drusen in unsupplemented versus sPLA2-IIA inhibitor-supplemented (5 μM; daily for 7 days) SFD (e, h), DHRD (f, h) and AMD (g, h) iRPE. Note: The absence of DAPI-positive cells is due to the fact that RPE monolayer was removed prior to immunostaining for drusen proteins on transwell membrane. Scale bar = 50 μm. * p<0.05; ** p<0.01 and *** p≤0.005. n = 3 biological replicates for each disease shown by distinct colored data points. See also Figure S4 and S5.

Figure 7.. Histopathological analyses of AMD donor eyes and SFD mouse model is concordant with a pathogenic role of MMP2-DAMP-RAGE-sPLA2-IIA axis in AMD/MDs.

(a-c) Confocal images of donor tissue sections from AMD patients (top panel) and normal adults (bottom panel) showing co-localization of HMGB1 (green) and APOE (red) (a), sPLA2-IIA (green) and APOE (red) (b), and (ω‐[2‐carboxyethyl]pyrrole) CEP protein adducts (green) and APOE (red) (c) in drusen. Scale bars = 50 μm. BrM and RPE nuclei are denoted by a white arrow and arrowhead respectively. (d) Confocal images showing co-localization of HMGB1 (green) and APOE (red) (top panel), sPLA2-IIA (green) and APOE (red) (middle panel), and CEP (green) with APOE (bottom panel) positive drusen underlying >90-day AMD iRPE cultures. Scale bars = 50 μm. Note: BrM and RPE nuclei are denoted by a white arrow and arrowhead respectively. (e, f) Confocal images (e) and quantification (f) of in situ zymography (ISZ) measuring gelatinase activity in the RPE-BrM of wild-type (WT) versus Timp3^KI-mut mice. BrM is labeled with COL4 antibody (red) and ISZ is shown in green. DAPI-stained nuclei are shown in blue. Scale bars = 50 μm. ** p≤0.01. n = 4 biological replicates are shown by four distinct colored data points. Note: BrM and RPE nuclei are denoted by a white arrow and arrowhead respectively. (g) Confocal and brightfield superimposed images post-immunostaining for PLA2G2A (sPLA2-IIA) of tissue sections from in Timp3^KI-mut mice and an AMD donor section. BrM is also labeled with COL4 antibody (red) in Timp3^KI-mut mice section. Note: BrM and RPE nuclei are denoted by a white arrow and arrowhead respectively. (h) Light microscopic images from 2 independent experiments showing vascular networks in iPSC-choriocapillaris (iCC) supplemented apically with RPE-CM derived from basal chamber of either untreated or MMP2-I1-treated (5 μM; daily for 6 days) control iRPE cultures. Note that both cultures received untreated control RPE-CM daily for 5–6 days until the formation of prominent vascular networks. Thereafter, one of the parallel cultures was switched to daily feeding of the MMP2-I1-treated RPE-CM cultures and the other cultures continued receiving untreated control RPE-CM until the end of the experiment at day 12–14. n = 2 independent experiments. See also Figure S5 and Table S2.