980 Diabetes Care Volume 42, May 2019

Hertzel C. Gerstein,1,2 Vidhya Nair,1,3,4 Dysglycemia and the Density of the Ruchi Chaube,5 Heidi Stoute,5 and Coronary Vasa Vasorum Geoff Werstuck1,5 Diabetes Care 2019;42:980–982 | https://doi.org/10.2337/dc18-2483

OBJECTIVE This study was conducted to determine the relationship between dysglycemia and the coronary artery vasa vasorum density.

RESEARCH DESIGN AND METHODS The left anterior descending coronary artery was removed from 57 deceased individuals during autopsy, and the capillaries in the vessel wall were identified

using fluorescent immunohistochemical staining. HbA1c was determined in post- mortem whole blood for each individual. The density of the vasa vasorum in the intima-media and the adventitia was manually quantified and recorded by readers unaware of the individual’s other characteristics.

RESULTS Theindividuals with diabetes had a lowerdensity of thecoronary vasa vasorum than

those without diabetes. The higher the HbA1c, the lower the density of these vessels in the adventitia and entire vessel wall.

CONCLUSIONS Dysglycemia-induced damage to the vasa vasorum may promote ischemic heart disease in people with diabetes. 1Department of Medicine, McMaster University and Hamilton Health Sciences, Hamilton, Canada 2 Diabetespromotesretinal,renal,neurologic,vascular,cardiac,andotherorgandamage. PopulationHealthResearchInstitute, McMaster University and Hamilton Health Sciences, Ham- Although many reasons for this have been suggested, dysglycemia-mediated vascu- ilton, Canada lopenia due to capillary damage in various tissues would provide an overarching 3Department of Pathology and Molecular Med- explanation. In the walls of medium to large arteries, these damaged capillary beds icine, McMaster University and Hamilton Health would bethe vasa vasorum (1), a possibility supported byanimal models (2)and in vivo Sciences, Hamilton, Canada 4 measurements in humans (3,4). University of Ottawa and The Ottawa Hospital, Ottawa, Canada If the above hypothesis is true, the density of the vasa vasorum should be reduced in 5Thrombosis and Atherosclerosis Research Insti- the coronary arteries of people with diabetes. This cross-sectional autopsy study was tute, McMaster University and Hamilton Health therefore conducted to compare the coronary artery vasa vasorum in individuals with Sciences, Hamilton, Canada normal versus high HbA1c at the time of death and to assess the relationship between Corresponding author: Hertzel C. Gerstein, NOVEL COMMUNICATIONS IN DIABETES HbA1c and vasa vasorum density. [email protected] Received 4 December 2018 and accepted 30 January 2019 RESEARCH DESIGN AND METHODS This article contains Supplementary Data online Deceased individuals undergoing forensic autopsy whose families consented to a full at http://care.diabetesjournals.org/lookup/suppl/ or limited postmortem examination were included if they were aged 50 or older, their doi:10.2337/dc18-2483/-/DC1. coronary arteries could be sectioned and stained, a postmortem blood sample was © 2019 by the American Diabetes Association. available, and the autopsy occurred during the pathologist’s (V.N.) working hours. Readers may use this article as long as the work is properly cited, the use is educational and not Age, sex, history of diabetes, and cause of death were collected, and a postmortem for profit, and the work is not altered. More infor- whole-blood sample was sent for HbA1c. The study was approved by the Research mation is available at http://www.diabetesjournals Ethics Board at Hamilton Health Sciences and McMaster University. .org/content/license. care.diabetesjournals.org Gerstein and Associates 981

prediabetes prediabetes prediabetes

P = 0.017 P = P =

Figure 1—Dysglycemia and the density of the vasa vasorum. The measured density of the left anterior descending artery’s vasa vasorum in the adventitia (A), intima-media (B),andentirevesselwall(C) is illustrated for people with normoglycemia, prediabetes, or diabetes. D–F: The relationship between HbA1c and these measurements in the three anatomic spaces is illustrated.

Processing of Anatomic Samples (goat anti-rabbit IgG) that was conjugated no formal sample size calculations were Left anterior descending coronary ar- to Alexa Fluor 594 (Life Technologies), done. The study was designed to con- teries were formalin fixed, decalcified, diluted 1:250 in PBS (pH 7.4), and protected tinue until 20 individuals with diabetes and sectioned. The sections were em- from light for 2 h at room temperature. The were included, as defined above. It stop- bedded in paraffin, and the proximal sections were then washed four times (for ped after 19 individuals with diabetes portions were sectioned for future im- 5 min each) in PBS (pH 7.4) and counter- were included due to relocation of the munohistochemical staining. The slides stained for 2 min with the nuclear stain investigator pathologist. were then heat-fixed for 4 min, and DAPI (Invitrogen), diluted 1:5,000 in PBS paraffin was removed using xylene so- (pH 7.4). After being washed four times (for Statistical Analyses lution for 5 min. Slides were passed 5 min each) in PBS (pH 7.4), they were HbA1c was analyzed on the Bio-Rad Var- through serial concentrations of ethanol mounted with the Fluorescent Aqueous iant II Instrument in the Hamilton Health (100% for 15 min, 70% for 6 min, and 50% Mounting Medium (Sigma-Aldrich). To Sciences National Glycohemoglobin Stan- for 6min) and then rehydrated in distilled control for nonspecific fluorescence, pre- dardization Program–certified Clinical H2O for 20 min. immune IgG control was used instead of Research Laboratory. Diabetes was diag- the primary antibody. Slides were stored nosed based on a known diagnosis or $ Immunohistochemical Staining in the dark at 4°C until imaged. a postmortem HbA1c level 6.5% (48 Antigen unmasking solution (H-3300; mmol/mol). Groups were compared using Vector Laboratories) was boiled for 17 Vasa Vasorum Quantification ordinary one-way ANOVA, and multiple min, and the sections were then placed All specimens were interpreted without linear regression estimated the age- and in the boiling solution for 3 min. After knowledge of diabetes status or the sex-adjusted effect of HbA1c on capillary cooling for 20 min, they were 1)blocked HbA1c. The vasa vasorum were imaged density within the various layers. Statis- and permeabilized in 10% goat serum using a fluorescent microscope (Nikon tical analyses were done using GraphPad (Vector Laboratories) + 0.05% Triton-X Eclipse T), and three sections were Prism and SAS 9.4 software. 100 in PBS (pH 7.4) at room temperature stained per individual. Microvessel den- for 45 min, 2) rinsed in PBS (pH 7.4), and 3) sity (vessels/mm2) was quantified for each RESULTS incubated overnight at 4°C with rabbit anti- section using Nikon Instruments Software The coronary arteries of 57 individuals human von Willebrand factor polyclonal (NIS)-Elements, and the mean density was were analyzed (Supplementary Table 1). antibody (DAKO) that was diluted 1:200 calculated for each individual. Staining ex- Their mean age was 62.5 years, 36 in 10% goat serum + 0.05% Triton-X 100 ceeding a set fluorescence intensity was (63%) were men, their mean HbA1c was in PBS (pH 7.4). The sections were then counted in each of the intimal-medial and 6.25% (45 mmol/mol), and cancer or cor- transferred to 37°C for 1 h, washed four adventitial layers. onaryarterydiseasewaslistedasthe times (for 5 min each) with PBS (pH 7.4), Because the distribution of the density cause of death in 24 (42.1%). The mean and incubated with the secondary antibody of coronary vasa vasorum was unknown, (SD) capillary density in the adventitia, 982 Dysglycemia and the Vasa Vasorum Diabetes Care Volume 42, May 2019

intima-media, and the entire vessel wall and/or inflammation, angiogenesis, and tegrity of the data and the accuracy of the data was 33.2 (13.7), 18.5 (8.3), and 25.6 (9.8) subsequent plaque formation and rup- analysis. vessels/mm2, respectively. ture (11). If the link between capillary At the time of death, 22 individuals integrity and diabetes-related disease in References (38.6%) were normoglycemic, 17 (29.8%) other organs is confirmed by in vivo im- 1. Gerstein HC, Werstuck GH. Dysglycaemia, vasculopenia, and the chronic consequences had an HbA1c level consistent with pre- aging of organ-specific capillary beds and – – of diabetes. Lancet Diabetes Endocrinol 2013; diabetes (5.7 6.4% or 39 46 mmol/mol), measurement of tissue perfusion (12), 1:71–78 and 18 (31.6%) had a prior diagnosis of drugs that block the effect of dysglycemia 2. Veerman KJ, Venegas-Pino DE, Shi Y, Khan MI, diabetes or an HbA1c level $6.5% on capillaries may also reduce cardiovas- Gerstein HC, Werstuck GH. Hyperglycaemia is (48 mmol/mol). Within both the ad- cular and other diabetes consequences. associated with impaired vasa vasorum neovas- cularization and accelerated atherosclerosis in ventitia and the entire vessel wall Unique strengths of this study are that fi fi apolipoprotein-E de cient mice. Atherosclerosis (Supplementary Fig. 1), the capillary den- these ndings arose from a study explic- 2013;227:250–258 sity was significantly lower in people with itly designed to test a previously artic- 3. Wang J, Chen H, Sun J, et al. Dynamic contrast- diabetes versus each of the other groups ulated hypothesis (1) and are supported enhanced MR imaging of carotid vasa vasorum in (P , 0.005). In a multiple linear regres- by evidence from animal experiments (2) relation to coronary and cerebrovascular events. – sion model adjusted for age and sex, a 1% and the analysis of tissue (rather than Atherosclerosis 2017;263:420 426 4. Park KH, Sun T, Diez-Delhoyo F, et al. Asso- higher HbA1c was associated with a 3.2 ultrasound or other images) from un- ciation between coronary microvascular function 2 vessels/mm (95% CI 0.6, 5.8) lower selected individuals. However, the cross- and the vasa vasorum in patients with early adventitial capillary density (P = 0.017) sectional design and limited data mean coronary artery disease. Atherosclerosis 2016; and a 2.3 vessels/mm2 (95% CI 0.6, 4.2) that the possibility that findings are a 253:144–149 lower vessel wall capillary density (Fig. 1). consequence of coronary disease cannot 5. Ross S, Gerstein HC, Eikelboom J, Anand SS, Yusuf S, Pare´ G. Mendelian randomization anal- be excluded. Nevertheless, these find- ysis supports the causal role of dysglycaemia and CONCLUSIONS ings extend the list of capillary beds diabetes in the risk of coronary artery disease. This study was designed to test the damaged by diabetes from the eyes Eur Heart J 2015;36:1454–1462 previously published hypothesis that di- and kidneys to the vasa vasorum and 6. Ahmad OS, Morris JA, Mujammami M, et al. A Mendelian randomization study of the effect of abetes-related coronary artery disease is support the possibility that dysglycemia type-2 diabetes on coronary heart disease. Nat due to dysglycemia-mediated capillary may promote a similar vasculopenic pro- Commun 2015;6:7060 damage in the vasa vasorum of arteries cess in capillary beds in the heart muscle, 7. Benn M, Tybjaerg-Hansen A, McCarthy MI, (1). This hypothesis is supported by our skeletal muscle, brain, liver, and other Jensen GB, Grande P, Nordestgaard BG. Non- findings that people with diabetes had a organs. fasting glucose, ischemic heart disease, and myocardial infarction: a Mendelian randomi- lower density of the coronary vasa vaso- zation study. J Am Coll Cardiol 2012;59:2356– rum than those without diabetes and 2365 that the higher the HbA1c, the lower the Funding. This project was supported by an 8. Tancredi M, Rosengren A, Svensson AM, et al. density of these vessels in the adventitia operating grant from the Heart and Stroke Excess mortality among persons with type 2 Foundation of Canada (G-17-0017029). diabetes. N Engl J Med 2015;373:1720–1732 and entire vessel wall. The HbA1c range fi Duality of Interest. H.C.G. holds the McMaster- 9. Gerstein HC, Miller ME, Ismail-Beigi F, et al.; de ning prediabetes is much smaller Sanofi Population Health Institute Chair in Di- ACCORD Study Group. Effects of intensive gly- than that characterizing diabetes, which abetes Research and Care and has received caemic control on ischaemic heart disease: anal- likely accounts for the absence of a dif- research grant support from AstraZeneca, Eli ysis of data from the randomised, controlled ference between those with normogly- Lilly, Merck, and Sanofi; honoraria for speaking ACCORD trial. Lancet 2014;384:1936–1941 cemia and prediabetes. from AstraZeneca, Boehringer Ingelheim, Eli Lilly, 10. Zoungas S, Arima H, Gerstein HC, et al.; Novo Nordisk, and Sanofi; and consulting fees Collaborators on Trials of Lowering Glucose Several Mendelian randomization from Abbott, AstraZeneca, Boehringer Ingel- (CONTROL) Group. Effects of intensive glucose studies (5–7), epidemiologic evidence heim, Eli Lilly, Merck, Novo Nordisk, Janssen, control on microvascular outcomes in patients (8), and some trials have demonstrated and Sanofi. No other potential conflicts of in- with type 2 diabetes: a meta-analysis of individ- that dysglycemia is causally linked to terest relevant to this article were reported. ual participant data from randomised controlled fi trials. Lancet Diabetes Endocrinol 2017;5:431– ischemic heart disease (9) and other Author Contributions. H.C.G. wrote the rst draft of the manuscript. V.N., R.C., H.S., and 437 consequences of diabetes (10). These G.W. reviewed and edited the manuscript and 11. Parma L, Baganha F, Quax PHA, de Vries MR. findings suggest that one mechanism acquired and analyzed the samples. H.C.G., V.N., Plaque angiogenesis and intraplaque hemor- for this relationship could be glucose- and G.W. developed the protocol and analyzed rhage in atherosclerosis. Eur J Pharmacol 2017; – mediated vasculopenia in the adventitial the data. H.C.G. was responsible for the study 816:107 115 design and the decision to submit and publish 12. Buscombe JR. Exploring the nature of ath- capillary network of the coronary ar- the manuscript. H.C.G. is the guarantor of this eroma and cardiovascular inflammation in vivo teries. This may promote hypoxia- work and, as such, had full access to all the data using positron emission tomography (PET). Br J mediated damage to the vessel wall in the study and takes responsibility for the in- Radiol 2015;88:20140648