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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

HOG-LDL dose–responses for ROS generation, ER stress, JNK activation and CHOP expression. Cellular ROS level are shown following exposure to various doses of HOG-LDL (mean ± SD, n = 3; **p < 0.01 vs N-LDL) (a). Western blots are shown for p-PERK (white bars), GRP78 (grey bars) and p-eIF2α (black bars) (b), p-JNK and total JNK (c), and CHOP (d). Data are means ± SD, n = 3; **p < 0.01. Ctrl, control
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Fig4: HOG-LDL dose–responses for ROS generation, ER stress, JNK activation and CHOP expression. Cellular ROS level are shown following exposure to various doses of HOG-LDL (mean ± SD, n = 3; **p < 0.01 vs N-LDL) (a). Western blots are shown for p-PERK (white bars), GRP78 (grey bars) and p-eIF2α (black bars) (b), p-JNK and total JNK (c), and CHOP (d). Data are means ± SD, n = 3; **p < 0.01. Ctrl, control

Mentions: To elucidate underlying signalling pathways and mechanisms, we compared dose–response relationships of oxidative stress (ROS), ER stress (chaperone: GRP78; sensors: p-PERK, p-eIF2α), JNK activation and CHOP expression in cultured HRCPs exposed to HOG-LDL. HOG-LDL increased levels of ROS, GRP78, p-PERK and p-eIF2α dose-dependently (25–200 mg/l) (Fig. 4a, b). JNK activation increased over the 25–50 mg/l HOG-LDL range, then remained constant up to 200 mg/l (Fig. 4c), similar to autophagy (Fig. 2c). HOG-LDL did not increase CHOP expression until its concentration reached 100 mg/l (Fig. 4d), consistent with effects on apoptosis (Fig. 3d). Together, the data suggest that low-dose HOG-LDL (up to 50 mg/ml) induces mild oxidative and ER stress, triggering a protective action of autophagy via JNK; at higher concentrations (100–200 mg/l), HOG-LDL further increases stresses leading to autophagic and apoptotic death.Fig. 4


Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
HOG-LDL dose–responses for ROS generation, ER stress, JNK activation and CHOP expression. Cellular ROS level are shown following exposure to various doses of HOG-LDL (mean ± SD, n = 3; **p < 0.01 vs N-LDL) (a). Western blots are shown for p-PERK (white bars), GRP78 (grey bars) and p-eIF2α (black bars) (b), p-JNK and total JNK (c), and CHOP (d). Data are means ± SD, n = 3; **p < 0.01. Ctrl, control
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Related In: Results  -  Collection

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Fig4: HOG-LDL dose–responses for ROS generation, ER stress, JNK activation and CHOP expression. Cellular ROS level are shown following exposure to various doses of HOG-LDL (mean ± SD, n = 3; **p < 0.01 vs N-LDL) (a). Western blots are shown for p-PERK (white bars), GRP78 (grey bars) and p-eIF2α (black bars) (b), p-JNK and total JNK (c), and CHOP (d). Data are means ± SD, n = 3; **p < 0.01. Ctrl, control
Mentions: To elucidate underlying signalling pathways and mechanisms, we compared dose–response relationships of oxidative stress (ROS), ER stress (chaperone: GRP78; sensors: p-PERK, p-eIF2α), JNK activation and CHOP expression in cultured HRCPs exposed to HOG-LDL. HOG-LDL increased levels of ROS, GRP78, p-PERK and p-eIF2α dose-dependently (25–200 mg/l) (Fig. 4a, b). JNK activation increased over the 25–50 mg/l HOG-LDL range, then remained constant up to 200 mg/l (Fig. 4c), similar to autophagy (Fig. 2c). HOG-LDL did not increase CHOP expression until its concentration reached 100 mg/l (Fig. 4d), consistent with effects on apoptosis (Fig. 3d). Together, the data suggest that low-dose HOG-LDL (up to 50 mg/ml) induces mild oxidative and ER stress, triggering a protective action of autophagy via JNK; at higher concentrations (100–200 mg/l), HOG-LDL further increases stresses leading to autophagic and apoptotic death.Fig. 4

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