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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.


CHOP, but not JNK, is essential for HOG-LDL-induced apoptosis. HRCPs were transfected with si-CHOP or si-JNK for 36 h, then exposed to N-LDL (N; 200 mg/l) or HOG-LDL (HOG; 50, 200 mg/l) for 12 h. At 200 mg/l HOG-LDL, si-CHOP decreased TUNEL-positive cells (a). At 200 mg/l, si-CHOP decreased HOG-LDL-induced expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) (b). At 50 mg/l HOG-LDL, si-JNK increased expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) but had no effect at 200 mg/l (c). si-CHOP did not alter expression of p-JNK (white bars) or LC3BII (black bars) at 50 or 200 mg/l HOG-LDL (d). Data are means ± SD, n = 3; *p < 0.05, **p < 0.01
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Fig5: CHOP, but not JNK, is essential for HOG-LDL-induced apoptosis. HRCPs were transfected with si-CHOP or si-JNK for 36 h, then exposed to N-LDL (N; 200 mg/l) or HOG-LDL (HOG; 50, 200 mg/l) for 12 h. At 200 mg/l HOG-LDL, si-CHOP decreased TUNEL-positive cells (a). At 200 mg/l, si-CHOP decreased HOG-LDL-induced expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) (b). At 50 mg/l HOG-LDL, si-JNK increased expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) but had no effect at 200 mg/l (c). si-CHOP did not alter expression of p-JNK (white bars) or LC3BII (black bars) at 50 or 200 mg/l HOG-LDL (d). Data are means ± SD, n = 3; *p < 0.05, **p < 0.01

Mentions: Both CHOP and JNK have been implicated in ER stress-induced apoptosis [35]. To determine their relative roles in HOG-LDL-induced apoptosis, we employed siRNA against CHOP (si-CHOP) or JNK (si-JNK), then measured apoptosis (TUNEL assay, western blots). si-CHOP significantly reduced TUNEL-positive staining (Fig. 5a, ESM Fig. 4b), cleaved PARP and activated caspase-3 (Fig. 5b), indicating a role for CHOP in apoptosis. However, in pericytes exposed to 50 mg/l HOG-LDL, si-JNK increased protein levels of CHOP, cleaved PARP and activated caspase-3 (Fig. 5c), responses that promote apoptosis. In contrast, in the presence of 200 mg/l HOG-LDL, si-JNK had no effect. This is consistent with JNK knockdown inhibiting autophagy, thus blocking the protective effects of autophagy at lower levels of cell stress. Finally, we showed that si-CHOP did not change expression of p-JNK or LC3BII in pericytes exposed to HOG-LDL at 50 or 200 mg/l (Fig. 5d), indicating that autophagy induced by HOG-LDL, in contrast to apoptosis, was CHOP-independent.Fig. 5


Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
CHOP, but not JNK, is essential for HOG-LDL-induced apoptosis. HRCPs were transfected with si-CHOP or si-JNK for 36 h, then exposed to N-LDL (N; 200 mg/l) or HOG-LDL (HOG; 50, 200 mg/l) for 12 h. At 200 mg/l HOG-LDL, si-CHOP decreased TUNEL-positive cells (a). At 200 mg/l, si-CHOP decreased HOG-LDL-induced expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) (b). At 50 mg/l HOG-LDL, si-JNK increased expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) but had no effect at 200 mg/l (c). si-CHOP did not alter expression of p-JNK (white bars) or LC3BII (black bars) at 50 or 200 mg/l HOG-LDL (d). Data are means ± SD, n = 3; *p < 0.05, **p < 0.01
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Fig5: CHOP, but not JNK, is essential for HOG-LDL-induced apoptosis. HRCPs were transfected with si-CHOP or si-JNK for 36 h, then exposed to N-LDL (N; 200 mg/l) or HOG-LDL (HOG; 50, 200 mg/l) for 12 h. At 200 mg/l HOG-LDL, si-CHOP decreased TUNEL-positive cells (a). At 200 mg/l, si-CHOP decreased HOG-LDL-induced expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) (b). At 50 mg/l HOG-LDL, si-JNK increased expression of CHOP (white bars), cleaved PARP (grey bars) and activated caspase-3 (black bars) but had no effect at 200 mg/l (c). si-CHOP did not alter expression of p-JNK (white bars) or LC3BII (black bars) at 50 or 200 mg/l HOG-LDL (d). Data are means ± SD, n = 3; *p < 0.05, **p < 0.01
Mentions: Both CHOP and JNK have been implicated in ER stress-induced apoptosis [35]. To determine their relative roles in HOG-LDL-induced apoptosis, we employed siRNA against CHOP (si-CHOP) or JNK (si-JNK), then measured apoptosis (TUNEL assay, western blots). si-CHOP significantly reduced TUNEL-positive staining (Fig. 5a, ESM Fig. 4b), cleaved PARP and activated caspase-3 (Fig. 5b), indicating a role for CHOP in apoptosis. However, in pericytes exposed to 50 mg/l HOG-LDL, si-JNK increased protein levels of CHOP, cleaved PARP and activated caspase-3 (Fig. 5c), responses that promote apoptosis. In contrast, in the presence of 200 mg/l HOG-LDL, si-JNK had no effect. This is consistent with JNK knockdown inhibiting autophagy, thus blocking the protective effects of autophagy at lower levels of cell stress. Finally, we showed that si-CHOP did not change expression of p-JNK or LC3BII in pericytes exposed to HOG-LDL at 50 or 200 mg/l (Fig. 5d), indicating that autophagy induced by HOG-LDL, in contrast to apoptosis, was CHOP-independent.Fig. 5

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.