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Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy

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ABSTRACT

Aims/hypothesis: Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.

Methods: Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.

Results: Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.

Conclusions: Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Electronic supplementary material: The online version of this article (doi:10.1007/s00125-016-4058-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

No MeSH data available.


Related in: MedlinePlus

HOG-LDL induces JNK phosphorylation-dependent autophagy in HRCPs. (a) HRCPs were transfected with GFP–LC3B plasmid for 36 h, then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 24 h, with or without CQ (10 μmol/l) pre-treatment for 1 h. Untreated cells served as control (Ctrl) in this and the following figs. Autophagy was quantified by counting GFP–LC3B puncta within cells (n = 5 experiments; *p < 0.05, **p < 0.01). (b) HRCPs were directly exposed to LDL with or without CQ and analysed by western blot for LC3BII. White bars, control; black bars, CQ (mean ± SD, n = 3; *p < 0.05). (c) HRCPs were exposed to N-LDL (200 mg/l) or HOG-LDL (25–200 mg/l) for 12 h and analysed by western blot. ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (d) HRCPs were treated with tunicamycin (TM; 2 μmol/l), N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h or pre-treated with 4-phenylbutyric acid (PBA; 10 mg/l) before exposure to HOG-LDL. Western blot analysis was carried out and total JNK (white bars) and p-JNK (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (e, f) HRCPs were pre-treated with p-JNK inhibitor SP600125 (10 μmol/l) for 1 h (e), or transfected with siRNA against JNK (si-JNK) or with si-Ctrl for 36 h (f), then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h. Western blotting was carried out and levels of ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) were quantified (mean ± SD, n = 3; **p < 0.01)
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Fig2: HOG-LDL induces JNK phosphorylation-dependent autophagy in HRCPs. (a) HRCPs were transfected with GFP–LC3B plasmid for 36 h, then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 24 h, with or without CQ (10 μmol/l) pre-treatment for 1 h. Untreated cells served as control (Ctrl) in this and the following figs. Autophagy was quantified by counting GFP–LC3B puncta within cells (n = 5 experiments; *p < 0.05, **p < 0.01). (b) HRCPs were directly exposed to LDL with or without CQ and analysed by western blot for LC3BII. White bars, control; black bars, CQ (mean ± SD, n = 3; *p < 0.05). (c) HRCPs were exposed to N-LDL (200 mg/l) or HOG-LDL (25–200 mg/l) for 12 h and analysed by western blot. ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (d) HRCPs were treated with tunicamycin (TM; 2 μmol/l), N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h or pre-treated with 4-phenylbutyric acid (PBA; 10 mg/l) before exposure to HOG-LDL. Western blot analysis was carried out and total JNK (white bars) and p-JNK (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (e, f) HRCPs were pre-treated with p-JNK inhibitor SP600125 (10 μmol/l) for 1 h (e), or transfected with siRNA against JNK (si-JNK) or with si-Ctrl for 36 h (f), then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h. Western blotting was carried out and levels of ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) were quantified (mean ± SD, n = 3; **p < 0.01)

Mentions: To determine whether modified LDL elicits autophagy in vitro, cultured HRCPs were transfected with a green fluorescent protein (GFP)-labelled LC3 plasmid, then exposed to HOG-LDL vs N-LDL. HOG-LDL elicited a punctate intracellular GFP–LC3B distribution, characteristic of autophagy [31], which was not observed in response to N-LDL (Fig. 2a and ESM Fig. 2). The effect was further enhanced by chloroquine (CQ), an autophagosome–lysosome fusion blocker [32], confirming that HOG-LDL enhances autophagic flux. In concert, western blots of HRCP lysates showed increased levels of LC3BII (the lipidated form present in autophagosomes) in response to HOG-LDL vs N-LDL, and a further increase in response to HOG-LDL + CQ (Fig. 2b). HOG-LDL increased protein expression of ATG-5, Beclin-1 and LC3BII in a dose-dependent manner over the concentration range 0–50 mg/l, but caused no further increase at concentrations from 50 to 200 mg/L (Fig. 2c). Concentrations > 200 mg/l were not tested due to cellular toxicity.Fig. 2


Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
HOG-LDL induces JNK phosphorylation-dependent autophagy in HRCPs. (a) HRCPs were transfected with GFP–LC3B plasmid for 36 h, then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 24 h, with or without CQ (10 μmol/l) pre-treatment for 1 h. Untreated cells served as control (Ctrl) in this and the following figs. Autophagy was quantified by counting GFP–LC3B puncta within cells (n = 5 experiments; *p < 0.05, **p < 0.01). (b) HRCPs were directly exposed to LDL with or without CQ and analysed by western blot for LC3BII. White bars, control; black bars, CQ (mean ± SD, n = 3; *p < 0.05). (c) HRCPs were exposed to N-LDL (200 mg/l) or HOG-LDL (25–200 mg/l) for 12 h and analysed by western blot. ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (d) HRCPs were treated with tunicamycin (TM; 2 μmol/l), N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h or pre-treated with 4-phenylbutyric acid (PBA; 10 mg/l) before exposure to HOG-LDL. Western blot analysis was carried out and total JNK (white bars) and p-JNK (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (e, f) HRCPs were pre-treated with p-JNK inhibitor SP600125 (10 μmol/l) for 1 h (e), or transfected with siRNA against JNK (si-JNK) or with si-Ctrl for 36 h (f), then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h. Western blotting was carried out and levels of ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) were quantified (mean ± SD, n = 3; **p < 0.01)
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Fig2: HOG-LDL induces JNK phosphorylation-dependent autophagy in HRCPs. (a) HRCPs were transfected with GFP–LC3B plasmid for 36 h, then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 24 h, with or without CQ (10 μmol/l) pre-treatment for 1 h. Untreated cells served as control (Ctrl) in this and the following figs. Autophagy was quantified by counting GFP–LC3B puncta within cells (n = 5 experiments; *p < 0.05, **p < 0.01). (b) HRCPs were directly exposed to LDL with or without CQ and analysed by western blot for LC3BII. White bars, control; black bars, CQ (mean ± SD, n = 3; *p < 0.05). (c) HRCPs were exposed to N-LDL (200 mg/l) or HOG-LDL (25–200 mg/l) for 12 h and analysed by western blot. ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (d) HRCPs were treated with tunicamycin (TM; 2 μmol/l), N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h or pre-treated with 4-phenylbutyric acid (PBA; 10 mg/l) before exposure to HOG-LDL. Western blot analysis was carried out and total JNK (white bars) and p-JNK (black bars) levels were quantified (mean ± SD, n = 3; **p < 0.01). (e, f) HRCPs were pre-treated with p-JNK inhibitor SP600125 (10 μmol/l) for 1 h (e), or transfected with siRNA against JNK (si-JNK) or with si-Ctrl for 36 h (f), then exposed to N-LDL (200 mg/l) or HOG-LDL (200 mg/l) for 12 h. Western blotting was carried out and levels of ATG-5 (white bars), Beclin-1 (grey bars) and LC3BII (black bars) were quantified (mean ± SD, n = 3; **p < 0.01)
Mentions: To determine whether modified LDL elicits autophagy in vitro, cultured HRCPs were transfected with a green fluorescent protein (GFP)-labelled LC3 plasmid, then exposed to HOG-LDL vs N-LDL. HOG-LDL elicited a punctate intracellular GFP–LC3B distribution, characteristic of autophagy [31], which was not observed in response to N-LDL (Fig. 2a and ESM Fig. 2). The effect was further enhanced by chloroquine (CQ), an autophagosome–lysosome fusion blocker [32], confirming that HOG-LDL enhances autophagic flux. In concert, western blots of HRCP lysates showed increased levels of LC3BII (the lipidated form present in autophagosomes) in response to HOG-LDL vs N-LDL, and a further increase in response to HOG-LDL + CQ (Fig. 2b). HOG-LDL increased protein expression of ATG-5, Beclin-1 and LC3BII in a dose-dependent manner over the concentration range 0–50 mg/l, but caused no further increase at concentrations from 50 to 200 mg/L (Fig. 2c). Concentrations > 200 mg/l were not tested due to cellular toxicity.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Aims/hypothesis: Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.

Methods: Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein&ndash;LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5&nbsp;mmol/l) and/or caspase inhibitor Z-VAD-fmk (100&nbsp;&mu;mol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200&nbsp;mg protein/l intravitreal concentration) and, after 7&nbsp;days, retinas were analysed for ER stress, autophagy and apoptosis.

Results: Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50&nbsp;mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100&ndash;200&nbsp;mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25&ndash;200&nbsp;mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50&nbsp;mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200&nbsp;mg/l elicited greater ER stress and apoptosis.

Conclusions: Autophagy has a dual role in diabetic retinopathy: under mild stress (50&nbsp;mg/l HOG-LDL) it is protective; under more severe stress (200&nbsp;mg/l HOG-LDL) it promotes cell death.

Electronic supplementary material: The online version of this article (doi:10.1007/s00125-016-4058-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

No MeSH data available.


Related in: MedlinePlus