Inhibition Or Deletion of 11 -HSD1 Does Not Increase Angiogenesis in Ischemic Retinopathy

RESEARCH LETTER:

Inhibition or deletion of 11-HSD1 does not increase angiogenesis in ischemic retinopathy

Callam T Davidsona,Anna R Dovera, Carmel M McVicarb, Roly Megawc,Josephine V Glennb, Patrick WF Hadokea, Alan W Stittb, Brian R Walkera

aUniversity/BHF Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, Scotland, UK.

bCentre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, UK.

cMRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UK

Correspondence:

Prof Brian R Walker, University/BHF Centre for Cardiovascular Science, The Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK

Tel: +44 (0)131 242 6770

Fax: +44 (0)131 242 6779

Email:

Word Counts:

Main Text: 1211

1 Figure

Key words: Angiogenesis,11β-HSD1, retinopathy, ischemia, glucocorticoid, neovascularisation

Abbreviations:

11β-HSD1 - 11β-hydroxysteroid dehydrogenase type I

PDR - Proliferative diabetic retinopathy

OIR – Oxygen-induced retinopathy

Introduction

11-Hydroxysteroid dehydrogenase type I (11-HSD1) regenerates active glucocorticoids (cortisol in humans, corticosterone in rodents) from inert 11-keto metabolites [1]. Selective 11-HSD1 inhibitors have been shown to be safe and effective in the treatment of type 2 diabetes [2], lowering intracellular glucocorticoid concentrations in liver and adipose tissue (and thereby enhancing insulin sensitivity) [3]. They also improve a number of features of the metabolic syndrome, including liver fat content [4], are potentially atheroprotective [5] and improve cognition [6]. Although no longer under development for blood glucose lowering alone, 11-HSD1 inhibitors are being re-profiled for additional indications and may yet be prescribed in patients with type 2 diabetes.

11-HSD1 is also expressed in smooth muscle cells throughout the vasculature [7]. By acting directly through glucocorticoid receptors in the blood vessel wall, glucocorticoids tonically inhibit angiogenesis [8]. Loss of 11-HSD1, and the resulting reduction of glucocorticoid action in blood vessels, is associated with enhanced angiogenesis in multiple sites and has been shown to be beneficial in the myocardium after coronary artery occlusion andin skin following wound incision [9].

Angiogenesis can also contribute to pathology, as seen in solid tumour development and ischemic retinopathies such as retinal vein occlusions and proliferative diabetic retinopathy (PDR). If 11-HSD1 inhibitors are to be used for chronic treatment in patients with type II diabetes, then there is a concern that they may accelerate the progression of inappropriateblood vessel growth in the retina and exacerbate progression to sight-threatening PDR.

This study tests the hypothesis that pharmacological inhibition or deletion of 11-HSD1 promotespathological retinal neovascularisation. Retinal vascular remodelling was induced using the oxygen induced retinopathy (OIR) model [10] in which exposure of neonatal mice to hyperoxia from postnatal days 7 to 12 causes obliteration and cessation of development of central retinal capillary beds so that, on return to normoxia, a potent pre-retinal neovascular response is induced. Expression and localisation of 11β-HSD1 in the retina was also investigated by immunohistochemistry.

Methods

Animals

All animal work was carried out under UK Home Office licence in accordance with the Animals (Scientific Procedures) Act, 1986.Timed matings were obtained from breeding pairs of wild type C57Bl/6J mice (Harlan Laboratories, UK), from pairs of 11-HSD1-/- mice homozygous for a disrupted hsd11b1 allele and congenic on a C57Bl/6J genetic background, and from pairs of littermate control 11-HSD1+/+ mice. Mice in this study came from two litters and included males and females in approximately equal proportions.

Oxygen Induced Retinopathy Model

Seven day old 11-HSD1-/- homozygous and C57Bl/6J mouse pups and their nursing dams were exposed to 75% oxygen (humidified medical grade oxygen controlled by a PROOX oxygen controller model 110; Reming Bioinstruments Co. Redfield, NY) for 5 days. On postnatal day 12 the mice were returned to room air. Mice were terminally anaesthetised on postnatal day 17 using sodium pentobarbital, and one eye from each pup was enucleated and fixed in 4% paraformaldehyde.

Treatments

In wild type mice, compound 544 (3-(1-adamantyl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-α]azepine), a potent and selective inhibitor of 11β-HSD1 [4] (obtained from Enamine Ltd., Ukraine), was administered (30 mg/kg dissolved in 5% cyclodextrin; i.p. injection) to pups twice daily from postnatal day 5 until postnatal day 17. Control mice were administered vehicle injections.

Retinal analysis

Retinal flat mounts were stained with isolectin B4 (Sigma)and the corresponding streptavidin Alexa Fluor488 (Invitrogen). Retinalanalysis was conducted by an observer blind to the groups from which samples were obtained. Flat-mounted retinas were visualised and imaged using confocal microscopy (Eclipse TE2000-U, Nikon, Japan). Avascular areas and pre-retinal neovascularisation were quantified across the whole retina using Lucia Version 4.60 software as previously described [10].

Immunohistochemistry

To investigate the localisation of 11β-HSD1 in the neonatal mouse eye, threeP15 wildtype C57Bl/6J pups were terminally anaesthetised with sodium pentobarbital. Eyes were enucleated and fixed for one hour in 4% paraformaldehyde. Retinal flat mounts were incubated overnight in block buffer (0.5% Triton-X, 1% normal donkey serum, 0.1mM calcium chloride in phosphate buffered saline) followed by incubation overnight with Isolectin B4 (Thermo Scientific)and 11β-HSD1 (a kind gift from Dr Scott Webster) antibodies, and then incubated overnight with the corresponding fluorescently-labelled secondary antibodies (Invitrogen). A negative control retina was incubated in block buffer in lieu of primary antibody. Sections were mounted using Fluoromount G (Southern Biotech) and imaged on an LSM710 Confocal Microscope (Zeiss, Oberkochen, Germany).

Statistical analysis

Data are presented as means ± SEM (where n indicates the number of animals in each group) and were compared between groups by unpaired Student's t tests using GraphPad Prism software. Significance was indicated by P<0.05.

Results

Exposure of pups to hyperoxia followed by return to room air (normoxia) produced well-demarcated areas of retinal ischemia (avascular regions) that drove discrete pre-retinal neovascularization as previously described [10]. Compound 544, a selective 11-HSD1 inhibitor, did not alter the extent of the avascular areabut significantly reduced the area of neovascularisation, with a corresponding increase in the area of normal vasculature (Fig. 1a). Representative images are shown in Figure 1c and 1d. In the experiment using 11-HSD1-/- and control mice, the avascular area was smaller than in the drug administration study. Genetic deletion of 11-HSD1 had no effect on the avascular area or neovascularisation (Fig. 1b).

Immunofluorescent staining showed no 11β-HSD1 in the retinal vasculature. A strong perivascular signal, present only in remnant hyaloid vessels,served as an effective positive antibody control (Fig. 1e-g).

Discussion

This study tested the hypothesis that 11β-HSD1 inhibition promotes pathological neovascularization in the retina. Unexpectedly, pharmacological 11-HSD1 inhibition in the OIR model was associated with markedly reduced retinal neovascularisation, while genetic deletion of the enzymeproduced no discernible difference in the vascular profile of the retina during OIR. The lack of effect of 11-HSD1 manipulation on retinal angiogenesis is likely to be explained by retinal vessels being a rare “privileged site” where 11-HSD1 is not expressed in the vessel wall. Expression of the enzyme in hyaloid vessels is more typical of the systemic circulation, but these vessels do not persist into adulthood and so are unlikely to be relevant in PDR.

The reduced neovascularisation seen after enzyme inhibition may reflect the more severe ischemia in retinas from mice in the drug administration compared with genetic modification experiments, as a consequence of the additional manipulation of twice daily intraperitoneal injections which required intermittent removal of animals from hyperoxic conditions. Importantly, the finding that 11β-HSD1 is not highly expressed in the blood vessels of developing retinas vessels suggests that off-target effects elicited by compound 544 may be responsible for the reduced neovascularisation compared with vehicle treatment. Alternatively it is possible that 11β-HSD1 is expressed in neovascular regions of the ischemic retina, a question which was not addressed in this study.

Conclusions

These data suggest that 11β-HSD1 inhibitors are unlikely to pose a threat to retinal angiogenesis.However, OIR is an imperfect model for PDR in which environmental manipulation is used to disrupt healthy retinal development rather than sustained hyperglycaemia leading to degeneration of the adult vasculature. Expression of 11β-HSD1 should be investigated in the adult human retinal vasculature in order to provide pragmatic reassurance that 11-HSD1 inhibition is a safe strategy in patients with type 2 diabetes at risk of progressive retinopathy. Until such data are reported, monitoring for retinopathy remains a sensible precaution in any future studies of 11-HSD1 inhibitors in patients with diabetes.

Acknowledgements

Dr Scott Webster (CVS, QMRI, Edinburgh) provided 11β-HSD1 antibody. Claire L Kitson (CEM, QUB, Belfast) assisted in obtaining and analysing samples.

FUNDING

We acknowledge the support of the British Heart Foundation Centre of Research Excellence.

DUALITY OF INTEREST

BRW and PWFH are inventors on relevant patents owned by the University of Edinburgh. BRW has consulted extensively with pharmaceutical companies developing 11-HSD1 inhibitors and is Principal Investigator for a programme developing novel 11-HSD1 inhibitors, funded by the Wellcome Trust and recently licenced to Actinogen Medical.

CONTRIBUTION STATEMENT

Brian R Walker is guarantor of the article. ARD, CTD, PWFH, AWS and BRW designed the studies, analysed data and wrote or edited the manuscript. CMMcV, JVG, CTD and RM obtained and analysed samples and data and reviewed the manuscript.

REFERENCES

1. Seckl JR, Walker BR. 11b-Hydroxysteroid dehydrogenase type 1 - a tissue-specific amplifier of glucocorticoid action.Endocrinology 2001;142:1371-1376

2. Anderson A, Walker BR: 11β-HSD1 Inhibitors for the Treatment of Type 2 Diabetes and Cardiovascular Disease. Drugs 2013;13: 1385-1393

3. RosenstockJ, Banarer S, Fonseca VA et al. The 11-Beta-Hydroxysteroid Dehydrogenase Type 1 Inhibitor INCB13739 Improves Hyperglycemia in Patients with Type 2 Diabetes Inadequately Controlled By Metformin Monotherapy. Diabetes Care2010;33:1516-1522

4. Stefan N, Ramsauer M, Jordan P et al. Inhibition of 11β-HSD1 with RO5093151 for non-alcoholic fatty liver disease: a multicentre, randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol. 2014;2: 406-416

5. Hermanowski-Vosatka A, Balkovec JM, Cheng K et al. 11b-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice. Journal of Experimental Medicine 2005;202:517-527

6. Sooy K, Noble J, McBride A, Binnie M, Yau JLW, Seckl JR, Walker BR, Webster SP: Cognitive and disease-modifying effects of 11ß-hydroxysteroid dehydrogenase type 1 inhibition in male Tg2576 mice, a model of Alzheimer’s disease. Endocrinology2015;156 : 4592-4603.

7. Walker BR, Yau JL, Brett LP et al. 11b-Hydroxysteroid dehydrogenase in vascular smooth muscle and heart: implications for cardiovascular responses to glucocorticoids. Endocrinology 1991;129:3305-3312

8. Logie JJ, Ali S, Marshall KM, Heck MM, Walker BR, Hadoke PW. Glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration. PLoS One2010;5:e14476

9. Small GR, Hadoke PWF, Sharif I et al. Preventing regeneration of glucocorticoids by 11b-hydroxysteroid dehydrogenase type 1 enhances angiogenesis. Proc Natnl Acad Sci USA 2005;102:12165-12170

10. Gardiner TA, Gibson DS, de Gooyer TE, de la Cruz VF, McDonald DM, Stitt AW. Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy. Am J Pathol 2005;166:637-644

Figure legends

Figure 111β-HSD1 in mouse retinal vasculature

a) In C57Bl/6J mice, administration of the selective 11-HSD1 inhibitor compound 544 (black bars) did not affect the avascular area induced by hyperoxia, but induced a marked reduction in neovascular area in the retina and a corresponding increase in the area covered with normal vasculature compared to vehicle treatment (white bars). b) In offspring of 11-HSD1-/- mice (black bars), or offspring of their littermate 11-HSD1+/+ controls (white bars), the avascular area was smaller than in wild type C57Bl/6J mice receiving twice daily injections and there were no differences in the neovascular response. Data are mean ± SEM. n=10-12 per group in panel A and n=8 per group in panel B. *** p<0.001. c), d) Flat-mounted retinal images following in vivo fluorescein angiography from a wild type mouse treated with vehicle (c) or the 11β-HSD1 inhibitor, compound 544 (d). a = optic disc; b = avascular (ischemic) area; c = neovasculature; d = normal vessels. Bar = 100μm. e) P15 mouse retina stained with isolectin B4 (endothelial cell marker – red) and 11β-HSD1 (green). 11β-HSD1 immunoreactivitywas absent in retinal vessels. Thin arrow indicatesoptic disc. Thick arrow indicates the 11β-HSD1-positive hyaloid vessels. X10 magnification, scale bar=100μm. f) Isolectin B4-labelled retinal vessels lacked 11β-HSD1 immunoreactivity.X40 magnification. Scale bar = 20μm g) Hyaloid vessels showed 11β-HSD1 immunoreactivity in perivascular cells.X40 magnification.Scale bar = 20μm. Note that immunoreactivitywasonly present in the superficial plane, not in the retina itself.