Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Abstract

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDL-induced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to 'highly-oxidised glycated' LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

Figures

Fig. 1

Decreased HRCP viability is induced by HOG-LDL vs N-LDL and is partially mitigated by Poly-I, NAC, 4-PBA and CsA. HRCP were pre-treated (1 h) with or without Poly-I (50 µg/ml), NAC (100 µmol/l), 4-PBA (0.5 mmol/l) or CsA (2 µmol/l) for 1 h, then treated with N-LDL or HOG-LDL (200 mg/l) for 24 h. Cell viability was measured by CCK-8 assay. HOG-LDL, but not N-LDL, decreased viability. This effect was partially blocked by each of the four agents. In comparison, pretreatment with the same four agents did not alter cell viability following exposure to N-LDL. Data, as percentage of untreated control (SFM), are expressed as mean ± SD; n=3; *p<0.05 vs HOG-LDL control; ††p<0.01 vs N-LDL control

Fig. 2

HOG-LDL induces oxidative stress and nitrosative stress in HRCP. (a) Time course of intracellular ROS levels in HRCP. After incubation in SFM for 18 h, cells were exposed to N-LDL (grey line) or HOG-LDL (black line) (200 mg/l). Significantly higher levels of ROS were produced in response to HOG-LDL vs N-LDL. Values are mean ± SD; n=3; ††p<0.01; †††p<0.001. (b) Cells were treated with N-LDL or HOG-LDL (200 mg/l) for up to 24 h and western blot experiments performed on total protein extracts. (c) Histogram of time course for levels of GPX-1, (d) SOD-2 and (e) 3-NT in HRCP after treatment as above (b) (white bars, N-LDL; black bars, HOG-LDL). Data are expressed as fold change vs 0 h N-LDL, and are mean ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL at 0 h. (f) Poly-I and NAC, but not 4-PBA or CsA, inhibit HOG-LDL-activated oxidative and nitrosative stress in HRCP. Cells were treated with N-LDLor HOG-LDL (200 mg/l) for 12 h following pre-incubation (1 h) with or without inhibitors, and protein levels as labelled were detected by western blot performed on total protein extracts. (g) The graphs show fold changes for GPX-1, (h) SOD-2 and (i) 3-NT, expressed as mean ± SD; n=3; *p<0.05 vs HOG-LDL control; ††p<0.01 vs N-LDL control

Fig. 3

HOG-LDL activates ER stress in HRCP. (a) Time course of ER stress markers GRP78, p-eIF2α and CHOP in HRCP. Cells were treated as above (Fig. 2b). (b) Quantification of blot for GRP78, (c) p-eIF2α and (d) CHOP, expressed as means ± SD; n=3; *p<0.05, **p<0.01 and ***p<0.001 vs HOG-LDL at 0 h. White bars, N-LDL; black bars, HOG-LDL. (e) Poly-I, NAC and 4-PBA, but not CsA, inhibit HOG-LDL-activated ER stress in HRCP. Cells were treated for 12 h as above (Fig. 2f), and protein levels detected by western blot and quantified for (f) GRP78, (g) p-eIF2α and (h) CHOP. Values are mean ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL control; ††p<0.01 and †††p<0.001 vs N-LDL control. (i) Immunocytochemistry images showing nuclear translocation of ATF6 in HRCP. Cells were treated with N-LDL or HOG-LDL (200 mg/l) for 12 h. Data are representative of three separate experiments and show that HOG-LDL, but not N-LDL induced ATF6 translocation from cytoplasm to nucleus. (j, k) mRNA expression in HRCP. Real-time PCR documented mRNA expression for CHOP (j) and sXBP-1 (k) in HRCP treated for 12 h with N-LDL or HOG-LDL (200 mg/l) following pre-incubation (1 h) with or without Poly-I, NAC, 4-PBA and CsA. Relative mRNA levels were normalised to 18 s mRNA. Values are means ± SD; n=3; **p<0.01 vs HOG-LDL control; ††p<0.01 vs N-LDL control

Fig. 4

HOG-LDL decreases mitochondrial membrane potential. (a) Mitochondrial membrane potential detection in HRCP. Cells were treated for 12 h as above (Fig. 3j, k). Mitochondrial membrane potential analyses were performed by flow cytometry with DiOC6(3), a fluorescent dye used to measure membrane potential. The percentage of total events associated with low membrane potential corresponds to low fluorescence events, which are shown as horizontal bars (denoted ‘M1’) in the histograms. Data are representative of three independent experiments. (b) Time course of CYT-C levels in HRCP. Cells were treated as above (Fig. 2b) and western blot experiments performed on total protein extracts, with β-actin used as loading control. The quantification of blots is expressed as mean ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL at 0 h. (c) Poly-I, NAC, 4-PBA and CsA inhibit HOG-LDL-induced CYT-C upregulation in HRCP. Cells were treated for 12 h as above (Fig. 2f) and CYT-C levels detected by western blot, with quantification expressed as mean ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL control; ††p<0.01 vs N-LDL control. White bars, N-LDL; black bars, HOG-LDL

Fig. 5

HOG-LDL induces apoptosis in HRCP. (a) TUNEL staining in HRCP. Apoptotic cells were observed by TUNEL assay when HRCP were exposed to HOG-LDL (200 mg/l) vs N-LDL (200 mg/l) for 24 h. Apoptosis significantly increased after HOG-LDL, but not after N-LDL treatment. Images are representative of three independent experiments. (b) Time course of activated caspase-3, cleaved PARP, BAX and BCL-2 in HRCP. Cells were treated as above (Fig. 2b) and western blot experiments performed on total protein extracts, with β-actin used as loading control. (c) Quantification of findings for activated caspase-3, (d) cleaved PARP and (e) BAX and BCL-2, expressed as means ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL 0 h. White bars, N-LDL; black bars, HOG-LDL. (f) Poly-I, NAC, 4-PBA and CsA inhibit HOG-LDL-induced apoptosis in HRCP. Cells were treated for 24 h as shown and protein levels as indicated detected by western blot. (g) Quantification of findings for activated caspase-3, (h) cleaved PARP and (i) BAX: BCL-2 ratio, expressed as means ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL control; ††p<0.01 vs N-LDL control

Fig. 6

HOG-LDL induces autophagy in HRCP. (a) Time course of conversion of LC3-I to LC3-II, and of ATG5 and beclin-1 abundance in HRCP. Cells were treated as above (Fig. 2b) and western blot performed on total protein extracts, with β-actin used as loading control. (b) Quantification of findings for LC3-I to LC3-II, (c) ATG5 and (d) beclin-1, expressed as means ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL 0 h. White bars, N-LDL; black bars, HOG-LDL. (e) Poly-I, NAC, 4-PBA and CsA inhibit HOG-LDL-induced autophagy in HRCP. Cells were treated for 24 h as shown, and protein conversion and abundance as indicated detected by western blot. (f) Quantification of findings for LC3-II/LC3-I ratio, (g) ATG5 and (h) beclin-1, expressed as means ± SD; n=3; *p<0.05 and **p<0.01 vs HOG-LDL control; ††p<0.01 vs N-LDL control