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

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–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

Dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy. (a) Summary of dose-dependent effects of HOG-LDL on oxidative stress, ER stress, autophagy and apoptosis in HRCPs. (b) Contrasting roles of autophagy in moderate vs severe HOG-LDL-induced cell stress. (c) Study hypothesis: in diabetic retina, modified LDL-triggered ER stress may be overcome by UPR or autophagy (as a pro-survival, compensatory pathway) but if cellular stress becomes more severe, both UPR and autophagy may shift to promote apoptotic and autophagic cell death
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Fig7: Dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy. (a) Summary of dose-dependent effects of HOG-LDL on oxidative stress, ER stress, autophagy and apoptosis in HRCPs. (b) Contrasting roles of autophagy in moderate vs severe HOG-LDL-induced cell stress. (c) Study hypothesis: in diabetic retina, modified LDL-triggered ER stress may be overcome by UPR or autophagy (as a pro-survival, compensatory pathway) but if cellular stress becomes more severe, both UPR and autophagy may shift to promote apoptotic and autophagic cell death

Mentions: Therapies targeting autophagy are of increasing interest and may be effective in retinal disease [9–11] but little is known regarding diabetic retinopathy. In the current study, we showed for the first time that autophagy markers (by immunohistochemistry and western blot) were increased in human retinas in the presence of diabetes, with or without concomitant retinopathy, and that in cell culture, exposure of pericytes to modified lipoproteins stimulated autophagy. These findings are consistent with our overall hypothesis that ectopic (extravasated) modified lipoproteins, when present in the diabetic retina, mediate responses (some defensive, some injurious) even before clinical diabetic retinopathy is evident [21, 23]. More interesting, our in vitro and in vivo data both suggest that autophagy may play a dual role: protecting against cell death under moderate stress, but contributing to it under severe stress (Fig. 7).Fig. 7


Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
Dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy. (a) Summary of dose-dependent effects of HOG-LDL on oxidative stress, ER stress, autophagy and apoptosis in HRCPs. (b) Contrasting roles of autophagy in moderate vs severe HOG-LDL-induced cell stress. (c) Study hypothesis: in diabetic retina, modified LDL-triggered ER stress may be overcome by UPR or autophagy (as a pro-survival, compensatory pathway) but if cellular stress becomes more severe, both UPR and autophagy may shift to promote apoptotic and autophagic cell death
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig7: Dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy. (a) Summary of dose-dependent effects of HOG-LDL on oxidative stress, ER stress, autophagy and apoptosis in HRCPs. (b) Contrasting roles of autophagy in moderate vs severe HOG-LDL-induced cell stress. (c) Study hypothesis: in diabetic retina, modified LDL-triggered ER stress may be overcome by UPR or autophagy (as a pro-survival, compensatory pathway) but if cellular stress becomes more severe, both UPR and autophagy may shift to promote apoptotic and autophagic cell death
Mentions: Therapies targeting autophagy are of increasing interest and may be effective in retinal disease [9–11] but little is known regarding diabetic retinopathy. In the current study, we showed for the first time that autophagy markers (by immunohistochemistry and western blot) were increased in human retinas in the presence of diabetes, with or without concomitant retinopathy, and that in cell culture, exposure of pericytes to modified lipoproteins stimulated autophagy. These findings are consistent with our overall hypothesis that ectopic (extravasated) modified lipoproteins, when present in the diabetic retina, mediate responses (some defensive, some injurious) even before clinical diabetic retinopathy is evident [21, 23]. More interesting, our in vitro and in vivo data both suggest that autophagy may play a dual role: protecting against cell death under moderate stress, but contributing to it under severe stress (Fig. 7).Fig. 7

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–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