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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

Autophagy is evident in diabetic human retinas. (a) Immunohistochemistry for LC3B in human retinal sections: non-diabetic (non-DM), diabetic without clinical retinopathy (DM) and diabetic with clinical retinopathy (DR). DAPI (blue) was used to visualise the nuclei. Scale bar, 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Punctate staining of LC3B (green) was present in both groups of diabetic retinas but was minimal in non-diabetic retinas. There was no obvious difference between the two diabetic groups. (b) Western blots for ATG-5 (white bars), Beclin-1 (grey bars) and LC3B (black bars) were performed on total retinal protein extracts from individual human retinas and quantified by densitometry (mean ± SD, n = 3 or 4, *p < 0.05 vs non-DM)
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Fig1: Autophagy is evident in diabetic human retinas. (a) Immunohistochemistry for LC3B in human retinal sections: non-diabetic (non-DM), diabetic without clinical retinopathy (DM) and diabetic with clinical retinopathy (DR). DAPI (blue) was used to visualise the nuclei. Scale bar, 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Punctate staining of LC3B (green) was present in both groups of diabetic retinas but was minimal in non-diabetic retinas. There was no obvious difference between the two diabetic groups. (b) Western blots for ATG-5 (white bars), Beclin-1 (grey bars) and LC3B (black bars) were performed on total retinal protein extracts from individual human retinas and quantified by densitometry (mean ± SD, n = 3 or 4, *p < 0.05 vs non-DM)

Mentions: LC3B immunohistochemistry was performed in retinas from individuals with type 2 diabetes with and without diabetic retinopathy, and from non-diabetic individuals. In diabetic retinas, punctate staining (indicating autophagosomes) was observed in the ganglion cell layer and inner nuclear layer, but in non-diabetic retinas, punctate staining was absent (Fig. 1a). Retinal protein lysates were analysed (western blotting) for LC3B and two other autophagy markers, ATG-5 and Beclin-1. LC3B and ATG-5 were higher in diabetic vs non-diabetic individuals, but retinopathy status had no effect; Beclin-1 levels tended to be higher in diabetic retinas (Fig. 1b). Overall, autophagy was increased in the diabetic retina; the similarity between those with and without retinopathy may reflect pre-clinical injury in people who appear disease-free.Fig. 1


Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
Autophagy is evident in diabetic human retinas. (a) Immunohistochemistry for LC3B in human retinal sections: non-diabetic (non-DM), diabetic without clinical retinopathy (DM) and diabetic with clinical retinopathy (DR). DAPI (blue) was used to visualise the nuclei. Scale bar, 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Punctate staining of LC3B (green) was present in both groups of diabetic retinas but was minimal in non-diabetic retinas. There was no obvious difference between the two diabetic groups. (b) Western blots for ATG-5 (white bars), Beclin-1 (grey bars) and LC3B (black bars) were performed on total retinal protein extracts from individual human retinas and quantified by densitometry (mean ± SD, n = 3 or 4, *p < 0.05 vs non-DM)
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Related In: Results  -  Collection

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Fig1: Autophagy is evident in diabetic human retinas. (a) Immunohistochemistry for LC3B in human retinal sections: non-diabetic (non-DM), diabetic without clinical retinopathy (DM) and diabetic with clinical retinopathy (DR). DAPI (blue) was used to visualise the nuclei. Scale bar, 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Punctate staining of LC3B (green) was present in both groups of diabetic retinas but was minimal in non-diabetic retinas. There was no obvious difference between the two diabetic groups. (b) Western blots for ATG-5 (white bars), Beclin-1 (grey bars) and LC3B (black bars) were performed on total retinal protein extracts from individual human retinas and quantified by densitometry (mean ± SD, n = 3 or 4, *p < 0.05 vs non-DM)
Mentions: LC3B immunohistochemistry was performed in retinas from individuals with type 2 diabetes with and without diabetic retinopathy, and from non-diabetic individuals. In diabetic retinas, punctate staining (indicating autophagosomes) was observed in the ganglion cell layer and inner nuclear layer, but in non-diabetic retinas, punctate staining was absent (Fig. 1a). Retinal protein lysates were analysed (western blotting) for LC3B and two other autophagy markers, ATG-5 and Beclin-1. LC3B and ATG-5 were higher in diabetic vs non-diabetic individuals, but retinopathy status had no effect; Beclin-1 levels tended to be higher in diabetic retinas (Fig. 1b). Overall, autophagy was increased in the diabetic retina; the similarity between those with and without retinopathy may reflect pre-clinical injury in people who appear disease-free.Fig. 1

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