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. 2016 Aug 2;11(8):e0160442.
doi: 10.1371/journal.pone.0160442. eCollection 2016.

Biomarkers for Diabetic Retinopathy - Could Endothelin 2 Be Part of the Answer?

Nicolette Binz  1 , Elizabeth P Rakoczy  1   2 , Ireni S Ali Rahman  2 , Nermina N Vagaja  2 , Chooi-May Lai  1   2
Affiliations

Biomarkers for Diabetic Retinopathy - Could Endothelin 2 Be Part of the Answer?

Nicolette Binz et al. PLoS One. .

Abstract

Purpose: The endothelins are a family of three highly conserved and homologous vasoactive peptides that are expressed across all organ systems. Endothelin (Edn) dysregulation has been implicated in a number of pathophysiologies, including diabetes and diabetes-related complications. Here we examined Edn2 and endothelin receptor B (Endrb) expression in retinae of diabetic mouse models and measured serum Edn2 to assess its biomarker potential.

Materials and methods: Edn2 and Ednrb mRNA and Edn2 protein expression were assessed in young (8wk) and mature (24wk) C57Bl/6 (wild type; wt), Kimba (model of retinal neovascularisation, RNV), Akita (Type 1 diabetes; T1D) and Akimba mice (T1D plus RNV) by qRT-PCR and immunohistochemistry. Edn2 protein concentration in serum was measured using ELISA.

Results: Fold-changes in Edn2 and Ednrb mRNA were seen only in young Kimba (Edn2: 5.3; Ednrb: 6.0) and young Akimba (Edn2: 7.9, Ednrb: 8.8) and in mature Kimba (Edn2:9.2, Ednrb:11.2) and mature Akimba (Edn2:14.0, Ednrb:17.5) mice. Co-localisation of Edn2 with Müller-cell-specific glutamine synthetase demonstrated Müller cells and photoreceptors as the major cell types for Edn2 expression in all animal models. Edn2 serum concentrations in young Kimba, Akita and Akimba mice were not elevated compared to wt. However, in mature mice, Edn2 serum concentration was increased in Akimba (6.9pg/mg total serum protein) compared to wt, Kimba and Akita mice (3.9, 4.6, and 3.8pg/mg total serum protein, respectively; p<0.05).

Conclusions: These results demonstrated that long-term hyperglycaemia in conjunction with VEGF-driven RNV increased Edn2 serum concentration suggesting Edn2 might be a candidate biomarker for vascular changes in diabetic retinopathy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exist.

Figures

Fig 1
Fig 1
Edn2 (A) and Ednrb (B) fold changes in mRNA expression in young and mature retinae of wt, Akita, Kimba and Akimba mice. Data represented are fold changes in expression normalised against Ppia as the housekeeping gene. Differential expression was determined using delta delta Ct according to Livak and Schmittgen [23]. Fold changes in expression in Akita, Kimba and Akimba retinae represent increases or decreases in mRNA expression levels compared to wt retinae. N = 4 per group; *p<0.05 compared to wt and Akita
Fig 2
Fig 2. Cellular localisation of Edn2 in the young retina.
Edn2 expression was localised to Müller cells as demonstrated by co-localisation with glutamine synthetase (GS), a Müller cell-specific enzyme, and photoreceptor inner and outer segments (PIS, POS) in wt, Akita, Akimba and Kimba retinae (C, G, K, O; arrows and arrowhead). In Akita, Edn2 expression was mostly in the GCL, where Müller cell endfeet reside, the outer plexiform layer (OPL) and the outer nuclear layer (ONL; E, G). In Akimba and Kimba, Edn2 expression was mostly in the inner retina, the GCL and the inner plexiform layer as well as the photoreceptor inner and outer segments (IPL, PIS, POS; J, K, N, O). Co-localisation of Edn2 and GS was most pronounced in the GCL and Müller cell processes in the IPL of Kimba mice (O). Scale bar: 100μm
Fig 3
Fig 3. Cellular localisation of Edn2 in the mature retina.
Following 20 weeks of chronic hyperglycaemia in the mature Akita retina there was increased Edn2 staining in the IPL compared to young Akita retinae (F). Particularly in the mature Akimba retina GS staining was much reduced compared to the young retina, suggesting not only loss of photoreceptors (L) but also loss of Müller cells (I, arrowheads). Müller cell loss was also evident in mature Kimba retinae (M, arrowheads; O, arrows) compared to young Kimba retinae, but the loss was less pronounced. Scale bar: 100μm
Fig 4
Fig 4. Evidence of Müller cell gliosis in mature Akimba mice with a severe phenotype.
In the mature wt retina GFAP expression was localised and restricted to the astrocytes that reside on the GCL (arrowheads, C-D). In Akimba mice, GFAP expression was observed as thick processes in the GCL and long radial processes in the INL/OPL, indicating Müller cell gliosis (G-H). Edn2 only co-localised with GFAP in Müller cell processes in the INL/OPL (arrows, H) but not in the astrocytes of the GCL. Sections were counterstained with DAPI (B, F). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PIS/POS: photoreceptor inner and outer segments. Scale bar: 200μm
Fig 5
Fig 5. Serum Edn2 concentration in young and mature wt, Akita, Kimba and Akimba mice.
Serum concentrations did not differ between young wt, Akita, Kimba and Akimba mice (A). In wt mice, Edn2 serum concentration decreased with age. In mature mice (B), there was no significant difference in serum Edn2 concentration between wt, Akita and Kimba mice. However, circulating levels of Edn2 were significantly higher in mature Akimba mice (6.9pg/mg total retina protein) compared to wt (3.9pg/mg; p<0.01), Akita (3.8pg/mg; p<0.01) and Kimba (4.6pg/mg total retina protein; p<0.05) mice.
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MeSH terms

Grants and funding

This work was funded by the Juvenile Diabetes Research Foundation (USA), National Health and Medical Research Council (Australia) and WestPac grant-in-aid (Australia). N. Binz received financial support from the Lions Save-Sight Foundation of Western Australia through the foundation’s Brian King Postdoctoral Research Fellowship. I. Ali Rahman was the recipient of the Ministry of Education’s, Brunei Darussalam, Postgraduate Research Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.