TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy

Abstract

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFβ and pro-apoptotic BIGH3 (TGFβ-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFβ1 and TGFβ2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68⁺ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFβ induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 μg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFβ induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

Figure 1

Immunocytochemistry localization of BIGH3 protein in cultured HRP. Primary HRP were cultured in EBM-2 supplemented with 10% FBS and stained with BIGH3 antibody (red, a) or rabbit IgG (e; negative control) followed by DAPI (nuclei; blue, b and f), phalloidin (actin; green, c and g). In d (a–c merged) shows cytosolic and extracellular locations of BIGH3 protein. Images are shown at × 20 magnification.

Figure 2

TGFβ increased BIGH3 expression in HRP. (a, b) HRP were cultured with increasing concentrations of TGFβ1 or 2 added to conditioned media for 24 h. BIGH3 mRNA expression was measured by qPCR after TGFβ treatment. One-way ANOVA showed significant treatment effects by TGFβ 1 and 2 (F(4,14)=8.083; P<0.01)], and (F(4,14)=4.975; P<0.05). Dunnett's multiple comparison to test differences between groups was significant (*P<0.05, **P<0.01) between BIGH3 mRNA expression and TGFβ1 in all treatment groups (0 ng/ml TGFβ used as control group). Dunnett's test only showed 10 ng/ml of TGFβ2 to have a significant difference from the control group. (c, d) HRP were cultured with either TGFβ1 (c) or TGFβ2 (d) at 1, 2, 5, or 10 ng/ml for 24 h and refreshed media were collected after a 24 h period and probed for BIGH3 protein by western blot. Lane 1, 0.1 μg recombinant BIGH3; TGFβ1 or 2: lane 2, 0 ng/ml; lane 3, 1 ng/ml; lane 4, 2 ng/ml; lane 5, 5 ng/ml; lane 6, 10 ng/ml. (e, f) Densitometry results from c and d, respectively.

Figure 3

TGFβ-mediated apoptosis mitigated by BIGH3 antibodies. HRP were cultured in EMB-2 for 24 h, media was refreshed with 5 ng/ml of TGFβ1 or TGFβ2, with and without rabbit anti-BIGH3 antiserum (BIGH3 Ab). BIGH3 Ab significantly reduced TGFβ-induced apoptosis (Tukey's multiple comparison test, *P<0.05). One-way ANOVA was significant F(5,27)=5.695; P<0.01.

Figure 4

BIGH3 induced HRP apoptosis. (a) Dose-dependent increase in apoptosis: HRP were cultured in media containing recombinant BIGH3 protein at increasing concentrations. TUNEL assay was performed following 24 h treatment period, and a dose-dependent relationship existed between BIGH3 amount and percent apoptosis. Results from one-way ANOVA were significant (F(4,18)=9.154; P<0.001). A Dunnett's multiple comparison test was performed using 0 μg/ml as the control (*P<0.05, **P<0.01). (b) Time-dependent increase in apoptosis: cultured HRP were treated with 3 μg/ml of BIGH3 protein for 72 h. TUNEL assay was performed every 24 h, and a time-dependent relationship was observed. One-way ANOVA was significant (F(5,11)=17.54; P<0.01) and Dunnett's test was performed using recombinant BIGH3 vehicle (PBS) as control (*P<0.05). Camptothecin used a positive control (72 h; topoisomerase I inhibitor). (c) Cell type dependency in apoptosis: HRP (open circle), rhesus monkey RECs (closed triangle) and human RPTECs (closed square) were cultured and treated with increasing concentrations of BIGH3 protein. Cell Death was confirmed by TUNEL assay following 24 h treatment period. HRP were most sensitive to low concentration (1–5 μg/ml) of BIGH3 treatment which induced a 25% increase in apoptosis (or 6% increase in apoptosis per unit increase in BIGH3 concentration—μg/ml). RhRECs were less sensitive to BIGH3 treatment (2–10 μg/ml) which induced a 22% increase in cell apoptosis (or 2.5% increase in apoptosis per unit increase in BIGH3 concentration—μg/ml). RPTEC were the least sensitive to BIGH3 treatment (at a higher concentration of 10–30 μg/ml) which induced a 16% increase in cell apoptosis (or 0.8% increase in apoptosis per unit increase in BIGH3 concentration—μg/ml).

Figure 5

Distribution of BIGH3 and macrophage marker, CD68 in the retina of a 68-year old woman with 7-year history of Type II diabetes mellitus who died of renal and congestive heart failure. (a) H&E-stained section showing hyaline thickening of a vessel wall (between arrows). (b) Periodic acid schiff stain showing arteriolosclerosis with early luminal narrowing (arrow). (c) Immunohistochemical localization of BIGH3. The most prominent expression was found in the retinal vessels (arrows) and occasionally in the choroidal vessels (asterisk). Counter stain here and in d was Azure B, which stains melanin metachromatically blue/green allowing the brown reaction product to be clearly distinguished from the retinal pigmented epithelium and choroid melanosomes. (d) Adsorbed control performed by co-incubating the primary antibody with purified recombinant BIGH3 protein. Immunospecific staining was abolished. *Retinal arteriole. (e) Distribution of CD68 expressing macrophages (arrows). The macrophages were often associated with the retinal arterioles and capillaries (asterisk). Each of the panels represents perifoveal/mid-peripheral retina of the right globe photographed at × 200. Arrowhead in each panel marks the outer limiting membrane.