Hyperglycaemic memory affects the neurovascular unit of the retina in a diabetic mouse model
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The aim of this study was to evaluate damage to the neurovascular unit in a mouse model of hyperglycaemic memory.
A streptozotocin-induced mouse model of diabetes (C57BL/6J background) received insulin-releasing pellets and pancreatic islet-cell transplantation. Damage to the neurovascular unit was studied by quantitative retinal morphometry for microvascular changes and microarray analysis, with subsequent functional annotation clustering, for changes of the retinal genome.
Sustained microvascular damage was confirmed by persistent loss of pericytes in the retinal vasculature (PC/mm2): compared with healthy controls (1981 ± 404 PC/mm2), the pericyte coverage of the retinal vasculature was significantly reduced in diabetic mice (1571 ± 383 PC/mm2, p < 0.001) and transplanted mice (1606 ± 268 PC/mm2, p < 0.001). Genes meeting the criteria for hyperglycaemic memory were attributed to the cytoskeletal and nuclear cell compartments of the neurovascular unit. The most prominent regulated genes in the cytoskeletal compartment were Ddx51, Fgd4, Pdlim7, Utp23, Cep57, Csrp3, Eml5, Fhl3, Map1a, Mapk1ip1, Mnda, Neil2, Parp2, Myl12b, Dynll1, Stag3 and Sntg2, and in the nuclear compartment were Ddx51, Utp23, Mnda, Kmt2e, Nr6a1, Parp2, Cdk8, Srsf1 and Zfp326.
We demonstrated that changes in gene expression and microvascular damage persist after euglycaemic re-entry, indicating memory.
The datasets generated during and/or analysed during the current study are available in the GEO repository, GSE87433, www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=idmbysgctluxviv&acc=GSE87433.
KeywordsAnimal Microarray Microvascular disease Mouse Retinopathy
- 6 W/12 W
6 weeks/12 weeks
Diabetes Control and Complications Trial
Diabetic control group
Normal control group
Hyperglycaemic memory is part of the pathogenesis of diabetic retinopathy [1, 2]. Perpetuation of oxidative stress, irreversible accumulation of AGEs and hyperglycaemia-induced epigenetic changes are possible underlying mechanisms.
In diabetic dogs, retinopathy develops during euglycaemia after initial hyperglycaemia. In the human retina, the effect of a less-well-controlled period is perpetuated into the period of euglycaemic control. In the Diabetes Control and Complications Trial (DCCT), progression to diabetic retinopathy was increased in the conventional treatment group. In the follow-up study, this effect persisted even after equalising HbA1c levels.
Cell culture experiments revealed several important mechanisms of hyperglycaemic memory. The transcription factor mSin3A is persistently activated upon short cellular exposure to high glucose, regulating angiopoietin-2, an angiogenic growth factor and regulator of diabetic pericyte dropout . Short exposure to high glucose also induces epigenetic modifications and subsequent downregulation of antioxidant defence proteins (e.g. mediated by the sustained recruitment of SET7 methyltransferase to the NFκB p65 promoter ).
Hyperglycaemic memory has mainly been studied in short-term (cell culture) or long-term animal models, such as the studies by Kowluru . Therefore, the aim of this study was to evaluate a novel model of intermediate-term hyperglycaemic memory using islet transplantation to enable the study of subsequent early structural alterations in the neurovascular unit of the retina in diabetic retinopathy.
Male C57BL/6J mice (Charles River, Frankfurt, Germany) were housed under a 12 h light–dark cycle with free access to food and water. Experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Regional Commission in Karlsruhe, Germany.
Eight-week-old streptozotocin (STZ)-induced diabetic mice (diabetic control [DC]), 8-week-old STZ-induced diabetic mice receiving isogenic pancreatic islet transplantation (Tx) after 6 weeks of diabetes (DC+Tx) and age-matched controls (non-diabetic control [NC]) were analysed after 6 weeks (6W) and 12 weeks (12W). Use of isogenic animals and sterile conditions ensured the absence of immune responses and surgical complications. All mice were euthanised under general anaesthesia.
Diabetes was induced by intraperitoneal injection of STZ (160 mg/kg body weight; Sigma-Aldrich, Munich, Germany) in 8-week-old mice. Stable hyperglycaemia was confirmed 7 days after injection by blood glucose >16.7 mmol/l. Body weight and blood glucose were measured throughout the experiment (BGStar; Sanofi-Aventis, Frankfurt am Main, Germany; limited to 33.3 mmol/l). HbA1c was measured using affinity chromatography (In2it; Biorad, Munich, Germany).
Transplantation of pancreatic islet cells was performed by the Clinical Research Unit, Giessen (TL). Islets were taken from 8-week-old male C57BL/6J mice (Janvier Labs, Saint-Berthevin, France). Pancreatic islet-cell isolation was performed using pancreatic collagenase digestion and handpicked selection . About 300 islets were injected below the kidney capsule of the recipient mice after 6 weeks of diabetes induction. To support a basal insulin release of the islet graft, insulin-releasing pellets (LinBit; LinShin Canada, Toronto, ON, Canada), each releasing insulin at 0.1 U/24 h, were placed subcutaneously below the mid dorsal skin. The number of pellets placed in each recipient mouse, estimated by the targeted reduction of blood glucose levels (< 13.9 mmol/l), was two to four.
Retinas were isolated after overnight fixation of frozen eyes (−80°C) in 4% buffered formalin and were digested using a trypsin-based digestion method. Four to six retinas from each group were analysed morphometrically. In each retina, ten fields were randomly selected using ×400 magnification and CellF analysing software (Olympus, Hamburg, Germany). The cell numbers were normalised to relative capillary density (cell number/mm2 capillary area).
Frozen eyes were dissected and the retinas were extracted and immediately suspended in Trizol reagent (Invitrogen, Carlsbad, CA, USA). Zirconium oxide beads (1 mm, RNase-free; Next Advance, Averill Park, NY, USA) and a Bullet Blender (Next Advance) were used for retinal homogenisation. RNA was extracted using Trizol. RNA quality was verified using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantity measured by spectroscopy using an Infinite200 PRO NanoQuant System (Tecan, Männedorf, Switzerland).
Gene expression profiling
Gene expression profiling (n = 5 or 6 per group) was performed using Affymetrix GeneChip Mouse Gene 2.0 ST Array. Biotinylated antisense cRNA was then prepared in accordance with a standard labelling protocol. All protocols and equipment were from Affymetrix (High Wycombe, UK).
A custom CDF Version 19 with Entrez-based gene definitions was used to annotate the arrays. The raw fluorescence intensity values were normalised applying quantile normalisation and robust multi-array average (RMA) background correction. ANOVA was performed to identify differentially expressed genes (SAS JMP10 Genomics v6; SAS Institute, Cary, NC, USA). A false-positive rate of a = 0.05 with correction for false discovery rate was taken as the level of significance (GEO: GSE87433). Metabolic memory genes were identified, if they were regulated positively or negatively, in both NC12W → DC12W and NC12W → DC+Tx@12W. Analysis of the resulting gene list was performed using DAVID bioinformatics database (version 6.7, https://david-d.ncifcrf.gov/) using the categories UP_TISSUE and GOTERM_CC_FAT.
Metabolic data and pericyte numbers are expressed as mean ± SEM and were analysed using one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Animals were assigned to experimental groups by simple randomisation. Experimenters were blind to group assignment and outcome assessment. No data, samples or animals were excluded from the reported results.
To assess microvascular changes, the number of pericytes per retinal capillary area (PC/mm2) was determined. Compared with healthy controls (1981 ± 404 PC/mm2), the pericyte coverage of the retinal vasculature was significantly reduced in diabetic mice (1571 ± 383 PC/mm2, p < 0.001) and transplanted mice (1606 ± 268 PC/mm2, p < 0.001) (Fig. 1b). Endothelial cells were not affected by hyperglycaemic conditions or transplantation (data not shown).
Expression changes of cytoskeletal and nuclear proteins
Fold change vs NC12W
DEAD (Asp-Glu-Ala-Asp) box polypeptide 51
FYVE, RhoGEF and PH domain containing 4
PDZ and LIM domain 7
UTP23, small subunit (SSU) processome component
Centrosomal protein 57
Cysteine and glycine-rich protein 3
Echinoderm microtubule-associated protein like 5
Four and a half LIM domains 3
Microtubule-associated protein 1 A
Mitogen-activated protein kinase 1 interacting protein 1
Interferon activated gene 211
nei like 2 (E. coli)
Poly (ADP-ribose) polymerase family, member 2
Myosin, light chain 12B, regulatory
Dynein light chain LC8-type 1
Stromal antigen 3
Syntrophin, gamma 2
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5
UTP23, small subunit (SSU) processome component
Interferon activated gene 211
Lysine (K)-specific methyltransferase 2E
Nuclear receptor subfamily 6, group A, member 1
Poly (ADP-ribose) polymerase family, member 2
Cyclin-dependent kinase 8
Serine/arginine-rich splicing factor 1
Zinc finger protein 326
In our study, we demonstrated that changes in gene expression and microvascular damage in the neurovascular unit of the retina in a model of diabetes persisted after euglycaemic re-entry, indicating hyperglycaemic memory.
Euglycaemic re-entry of islet-transplanted mice was confirmed by blood glucose and HbA1c. The reduction in HbA1c levels mimics the effect seen in diabetic patients on intensive insulin treatment and appears to be greater than the reduction following sole insulin treatment in animals, which in rodents bears the disadvantage of glucose variability. However, restoration of euglycaemia did not correct microvascular damage, as reflected by sustained reduction in pericyte coverage of the retinal vascular network. This persistence of microvascular damage is considered to be caused by hyperglycaemic memory. Pericytes are an important component of the neurovascular unit, placed at the intersection of the neuroglial and vascular compartments in the retina and are also known to be the first cell type to be damaged under hyperglycaemic conditions .
Genes meeting the criteria for hyperglycaemic memory were attributed to the cytoskeletal and nuclear compartments. The cytoskeleton has been shown to be involved in the pathogenesis of diabetic microvascular complications [8, 9]. More importantly, sustained changes in nuclear factors have been reported to have a large-scale impact on gene expression, resulting in changes in antioxidative defence mechanisms to hyperglycaemic stress .
Our mouse model of hyperglycaemic memory revealed persistent changes in both gene expression patterns and early structural alterations in the neurovascular unit of the diabetic retina.
The datasets generated during and/or analysed during the current study are available in the GEO repository, GSE87433, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=idmbysgctluxviv&acc=GSE87433.
This study was supported by the Deutsche Forschungsgemeinschaft (International Research Training group 1874-1 DIAMICOM and the Deutsche Diabetes Gesellschaft).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
PF made substantial contributions to acquisition of data and drafting the article. AS, CS, MK, PW, AD, TL and GM made substantial contributions to analysis and interpretation of data, and revising the article critically for important intellectual content. H-PH made substantial contributions to conception and design, and revising the article critically for important intellectual content. All authors gave final approval of the version to be published. H-PH is responsible for the integrity of the work as a whole.
- 1.The Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) Research Group (2015) Effect of intensive diabetes therapy on the progression of diabetic retinopathy in patients with type 1 diabetes: 18 years of follow-up in the DCCT/EDIC. Diabetes 64:631–642CrossRefGoogle Scholar