Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy
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Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
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.
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.
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.
KeywordsApoptosis Autophagy Diabetic retinopathy ER stress LC3B Modified LDL Oxidative stress Pericytes
Autophagy-related homologue 5
Eukaryotic initiation factor 2α
Green fluorescent protein
Highly oxidised glycated LDL
Human retinal capillary pericyte
c-Jun NH(2)-terminal kinase
Poly ADP ribose polymerase
Protein kinase R‑like endoplasmic reticulum kinase
Retinal pigment epithelium
Reactive oxygen species
Small interfering RNA
Unfolded protein response
Vascular endothelial growth factor
Diabetic retinopathy remains a major cause of visual impairment in the working-age population . Pericytes are critical in maintaining retinal vascular integrity ; their loss is considered an initiating event of diabetic retinopathy [3, 4, 5, 6] but the mechanisms of pericyte loss are poorly understood, hindering development of effective therapies.
Autophagy, a catabolic process by which cells degrade and recycle their own constituents through a lysosomal mechanism, acts as a cellular sensor of intra- and extracellular stresses [7, 8]. In diabetes, misfolded proteins accumulate in the endoplasmic reticulum (ER) leading to ER stress. ER stress activates the unfolded protein response (UPR), restoring protein homeostasis and promoting cell survival. Autophagy is an alternative pathway to the UPR. Rapamycin, an inducer of autophagy, inhibits angiogenic sprouting and vascular endothelial growth factor (VEGF) production in a co-culture model of retinal pigment epithelial (RPE) and endothelial cells [9, 10, 11], and in diabetic rats it suppresses retinal oxidative stress and VEGF expression  and prevents age-related retinopathy . Targeting autophagy may therefore have therapeutic potential; however, in certain circumstances, autophagy may activate apoptotic death , and depending on context, stress-induced autophagy may promote survival or death of a given cell species .
Diabetic retinopathy is generally viewed as a consequence of hyperglycaemia, but in previous studies we showed that extravasated modified LDL also plays a critical role [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. The effects of extravasated lipoproteins in atherosclerosis are well-established ; we have demonstrated analogous effects in the retina once the blood–retinal barrier (BRB) integrity is compromised, or bypassed, as in an animal model we described recently . Initial leakage may be mild and transient but, as extravasated LDL accumulates, vicious cycles of damage may be established. While oxidised lipoprotein-induced autophagy is implicated in atherogenesis , little is known regarding the retina. Previously, we showed that in human retinal capillary pericytes (HRCPs) and retinal pigment epithelium (RPE), ER stress that had been induced by highly oxidised glycated human LDL is mitigated by the UPR, but that in the presence of continued severe stresses ER homeostasis could not be preserved, resulting in apoptosis [22, 23]. In this study, we aim to determine the underlying mechanisms whereby autophagy and apoptosis contribute to pericyte death.
The study was approved by the Institutional Review Board at the University of Oklahoma Health Sciences Centre (OUHSC) and the Ethics Committee at the Queen’s University of Belfast, and was conducted according to the principles of the Declaration of Helsinki. Animal experiments were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina and by the Queen’s University Belfast Ethical Review Committee for Animal Research. All the animal experiments were randomised and blind to group assignment and outcome assessment.
Immunohistochemistry of human retinas
Human retinas were obtained post-mortem from the National Disease Research Interchange (NDRI; Philadelphia, PA, USA) as described [23, 24]. Retinas were from age-matched individuals categorised as follows: non-diabetic, diabetic without clinical diabetic retinopathy; diabetic with retinopathy (n = 3 or 4 per group). The antibody used for immuno-histochemical detection of autophagy marker LC3B (catalogue No. 3868, 1:100 dilution; Cell Signaling Technology, Danvers, MA, USA) was also used in western blots (below): according to the manufacturer, it recognises human and murine LC3BI and LC3BII, and may exhibit some cross-reactivity with LC3A. Absence of non-specific tissue binding by secondary antibodies was confirmed. See Methods in electronic supplementary material (ESM) for details of retinal sample preparation and immunohistochemistry.
LDL preparation, modification and characterisation
Lipoproteins were prepared as described [23, 28]. Briefly, native LDL (N-LDL) was prepared by sequential ultracentrifugation of freshly pooled plasma from healthy human volunteers. Highly oxidised glycated LDL (HOG-LDL) was prepared by glycating N-LDL, then oxidising with CuCl2. See ESM Methods for details.
Genetically modified mouse model of hyperlipidaemia
Genetically modified C57B16 male mice (Genentech, South San Francisco, CA, USA) with double knockout of the genes encoding the LDL receptor (Ldlr −/−) and apolipoprotein B mRNA-editing catalytic polypeptide (converts ApoB100 to ApoB48) (Apobec1 −/−) were used to model hypercholesterolaemia (vs wild-type [WT] controls) . When mice reached 7 weeks of age, diabetes was induced by streptozotocin (STZ) as described , yielding groups with and without diabetes and with and without hypercholesterolaemia. All mice were maintained under a 12 h light–12 h dark cycle (07:00–19:00 hours) and constant temperature (25°C) throughout the study, with food and water given ad libitum. Retinas were studied 40 weeks after diabetes induction: see ESM Methods for details.
Diabetes induction and LDL intravitreal injection in rats
Diabetes was induced by STZ in adult (8–10 weeks) male Sprague–Dawley rats weighing 280–330 g. After 8 weeks, human HOG-LDL or N-LDL (5 μl, 0.5 or 2.0 g protein/l in PBS, to yield 50 or 200 mg/l final intravitreal concentration), or PBS alone, was injected intravitreally. After 7 days, retinas were harvested for western blot. The rats were maintained under a 12 h light–12 h dark cycle (07:00–19:00 hours) and constant temperature (25°C) throughout the study, with food and water given ad libitum. See ESM Methods for details.
HRCP cell culture
HRCPs (Cambrex, Walkersville, MD, USA) were cultured in EBM-2 medium. Cells (passages 3–9) at 85% confluence were treated with either N-LDL or HOG-LDL as indicated. Where appropriate, cells were pre-treated with pharmacological reagents prior to lipoprotein exposure. See ESM Methods for details.
Cell viability assay
HRCPs were studied in 96-well plates (1 × 104 cells/well). Cell viability was measured using a cell counting assay (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA), per the manufacturer’s protocol.
Human retinas or pericytes were homogenised with a complete lysis buffer (Roche, Indianapolis, IN, USA). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL, USA). Protein (30 μg) was resolved by SDS-PAGE and then blotted with antibodies against autophagy-related homologue 5 (ATG-5), Beclin-1, LC3B (detects both LC3B-I and LC3B-II; some cross-reactivity with LC3A), phosphorylated c-Jun NH(2)-terminal kinase (p-JNK), total JNK, cleaved poly ADP ribose polymerase (PARP), activated caspase-3, C/EBP-homologous protein (CHOP), β-actin (1:3000) (all from Cell Signaling Technology); and phosphorylated‑protein kinase R‑like ER kinase (p‑PERK), 78 kDa glucose-regulated protein (GRP78) and phosphorylated eukaryotic initiation factor 2α (eIF2α) (all from Abcam, Cambridge, MA, USA). All antibodies were diluted 1:1000 unless indicated otherwise. Antibody to β-actin was used as a loading control. All cell culture experiments were repeated three times independently and quantified by densitometry.
TUNEL assay for apoptosis
HRCPs were seeded and grown to 85% confluence on glass coverslips, and treated as indicated. Apoptosis was assessed (in situ cell death detection kit; Roche, Indianapolis, IN, USA) as per manufacturer’s instructions. Immunofluorescence was visualised under a fluorescence microscope (Nikon, Tokyo, Japan).
Measurement of intracellular reactive oxygen species
Reactive oxygen species (ROS) were measured with chloromethyl derivative of H2DCFDA (CM-H2DCFDA) (Life Technologies, Invitrogen, Carlsbad, CA, USA) as previously described . Briefly, cells were seeded in 96-well plates (1 × 104 cells/well). When they reached 80% confluence, cells were washed and incubated with 20 μmol/l DCFDA at 37°C for 20 min, then exposed to experimental conditions. Fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 525 nm (VICTOR3 microplate reader; PerkinElmer, Waltham, MA, USA).
Data are expressed as means ± SD. Statistical significance was determined by Student’s t test or one-way ANOVA followed by post hoc Dunnett’s test as appropriate (Prism 5 software; Graphpad, La Jolla, CA, USA). A p value of ≤ 0.05 was considered significant.
Autophagy in human diabetic retina
Autophagy in diabetic and hypercholesterolaemic mouse retina
LC3B staining was significantly higher in the two diabetic groups vs the non-diabetic group, with hyperlipidaemic diabetic mice showing the greatest intensity, localised predominantly in the ganglion cell and inner nuclear layers (ESM Fig. 1). Again, retinal autophagy was increased in the presence of diabetes, and more so in the added presence of long-standing hypercholesterolaemia.
HOG-LDL induces autophagy in pericytes
JNK mediates HOG-LDL-induced ER stress and autophagy
Jun amino-terminal kinases have been implicated in stress-induced autophagy [33, 34]. In cultured HRCPs, HOG-LDL vs N-LDL significantly increased JNK phosphorylation (Fig. 2d), to an extent equivalent to that induced by tunicamycin, an ER stress inducer. This response was obliterated by pre-treatment with sodium phenylbutyrate, an ER stress inhibitor, confirming that JNK mediates HOG-LDL-induced ER stress in pericytes.
To determine the role of JNK in autophagy, JNK phosphorylation inhibitor SP60012 was employed. JNK pathway inhibition was confirmed by western blot (ESM Fig. 3a). SP60012 pre-treatment attenuated HOG-LDL-induced autophagy, as demonstrated by decreased expression of LC3BII, ATG-5 and Beclin-1 (Fig. 2e). JNK knockdown using small interfering RNA (siRNA) had a similar effect, reducing both phosphorylated and total JNK (ESM Fig. 3b) as well as HOG-LDL-induced autophagy (Fig. 2f). The data support an essential role for JNK activation in the mediation of HOG-LDL-induced autophagy in HRCPs.
A dual role for autophagy in HOG-LDL-induced pericyte death
Comparison of the dose–response relationships for HOG-LDL-induced oxidative stress, ER stress, JNK activation and CHOP expression
CHOP, not JNK, is responsible for HOG-LDL-induced apoptosis
Extravascular HOG-LDL dose-dependently induced ER stress, autophagy and apoptosis in diabetic rat retinas
To maintain normal cellular function, autophagy may be upregulated in response to environmental stress. In the retina, all cell types rely on one or more aspects of autophagy to maintain structure and/or function . Retinal autophagy was first observed (in rats) by Remé et al in 1977 , occurring primarily in the outer retina, where later it was shown to exhibit circadian variation . More recently, Piano et al demonstrated upregulation of autophagy in retinal rods as an early feature of diabetic retinopathy (i.e. after 4–12 weeks diabetes) in STZ-induced diabetic mice . In general, cellular homeostasis relies on regulated interplay between basal and stress-induced autophagic pathways . Previously we showed that both extravasated modified lipoproteins and markers of ER stress were present in human diabetic retinas in proportion to the severity of retinopathy . In the present study, the retinal findings from humans and genetically hypercholesterolaemic mice, with and without diabetes, are generally consistent with the findings described above: intra-retinal formation of autophagosomes (punctate LC3 staining) was increased by diabetes and further increased (in mice) by prolonged hypercholesterolaemia. In a new diabetic rat model using intravitreal LDL injection, we found additional supportive evidence: exposure of the retina to HOG-LDL but not N-LDL stimulated an autophagic response. Taken together, the data are consistent with autophagy being implicated in lipoprotein-mediated retinal injury and, specifically, from the cell culture work, in pericyte injury in diabetic retinopathy.
From the present data, we propose that the interplay between autophagy and apoptosis is critical for pericyte survival. The balance between survival and death depends on the level of stress—minor stress may be countered by autophagy but severe stress leads to cell death. This concept is in concert with the findings of Piano et al regarding effects of early diabetic retinopathy on retinal neural cells . In the present work, the survival–death balance is seen in the observed dose-dependent effects of HOG-LDL on pericyte oxidative stress, ER stress, apoptosis and autophagy, with consistent findings following intravitreal LDL injections in rats, summarised in Fig. 7. HOG-LDL induced various molecular responses that were dose-dependent over different concentration ranges. Oxidative stress and ER stress were dose-dependent up to 200 mg/l, autophagy up to 50 mg/l and apoptosis from 50 mg/l to 200 mg/l. When cells experienced relatively mild stress (<50 mg/l), autophagy was induced, promoting cell survival, but under more severe stress (100 or 200 mg/l), autophagy was no longer protective but instead contributed to disruption of cellular homeostasis and death. Thus, pericytes utilise autophagy as a cytoprotective mechanism unless, or until, a critical stress threshold is exceeded. At that point, neither the UPR nor autophagy can protect the cells; instead, apoptotic mechanisms are activated and autophagy shifts from a protective to a lethal role. The definition of 50 mg/l as the threshold for this shift is clearly inexact, but nevertheless relevant in the retina where LDL is normally excluded by the BRB: any degree of BRB leakage would likely lead to accumulation and transition through this value. The pathophysiological relevance of the concentrations of HOG-LDL (0–200 mg/l) used in this study has been described and justified previously [21, 23], and we believe them to be relevant in vivo.
A dual role for autophagy has been proposed in other studies. In rheumatoid arthritis, autophagy in synovial fibroblasts may promote cell survival or death, depending on the level of stress . In the liver, autophagy is essential for maintenance of hepatocyte mitochondria and control of oxidative stress, thus preventing carcinogenesis, but once hepatocarcinoma is established, autophagy can promote the disease . In diabetic retinopathy, apparently contradictory roles for autophagy can inhibit or promote retinal vascular injury, depending on context (e.g. severity of retinopathy, extent of LDL leakage, extent of modification). In early retinopathy, when BRB leakage is mild, the retina is exposed only to small quantities of modified LDL  and autophagy may enhance cell survival. As retinopathy progresses and BRB leakage and lipoprotein leakage/modification become more severe, neither UPR nor autophagy can maintain ER homeostasis and autophagic death ensues. This dual action may complicate the development of treatments for diabetic retinopathy that aim to modulate autophagy.
The JNK pathway has been implicated in a range of cellular stress responses [7, 33, 43]. In this study, we demonstrated that JNK phosphorylation was essential to autophagy induced by HOG-LDL and ER stress. This is consistent with data implicating PERK–eIF2a and IRE1–JNK signalling pathways in autophagy [7, 44, 45]. Apart from autophagy, JNK activation is implicated in ER stress-induced apoptosis . However, in the present study, we showed that JNK ‘knockdown’ did not affect HOG-LDL (200 mg/l)-induced apoptosis, and enabled lower concentrations of HOG-LDL (50 mg/l) to induce apoptosis. This suggests that JNK activation was not implicated in HOG-LDL-induced apoptosis, but at low levels of stress it may promote autophagy and thus protect cells against apoptosis. It remains unclear whether or how activation of JNK through ER stress affects upstream components of the autophagy pathway (e.g. mechanistic target of rapamycin; mTOR) . Further studies regarding the detailed pathway of HOG-LDL-induced autophagy, including the relative roles of LC3B and LC3A, which may not be clearly distinguished in the present study, are needed: possibly, differential effects of the two isoforms could be important.
In conclusion, we present further evidence that autophagy is present in human diabetic retinas, and a role for modified lipoproteins is supported by in vivo findings in diabetic mouse and rat retina and by in vitro studies of HRCPs. We show that ER stress-mediated autophagy may play a dual role in pericyte loss induced by modified LDL. At low levels of exposure autophagy has a pro-survival effect, but as stresses become severe it promotes cell death. This dual function has implications for the development of any future autophagy-based therapies, which might only be applicable early in disease evolution when intra-retinal stresses remain mild.
The NDRI provided valued assistance in obtaining human retinal tissues. We thank M. Leyva and T. Friedel for expert technical and logistic support.
Compliance with ethical standards
This work was supported by the Oklahoma Center for the Advancement of Science and Technology (HR08-067), by a Research Grant from the American Diabetes Association (7-12-CT46), and by the National Institutes of Health (USA) COBRE Program of the National Center for Research Resources (P20 RR 024215).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
DF, SY, MW, SMH, ARC and MD conducted experiments, researched data and wrote the manuscript. JYY and JC researched data and wrote the manuscript. TJL conceived and conducted the study, researched data and wrote the manuscript. TJL is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All the authors have approved publication.
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