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Effect of Hyperglycemia on Myocardial Perfusion in Diabetic Porcine Models and Humans

Background Diabetes mellitus (DM) causes macro- and microvasculopathy, but data on cardiac microvascular changes in large animals are scarce. We sought to determine the effect of DM on macro- and microvascular changes in diabetic pigs and humans. Methods Eight domestic pigs (4 with type I diabetes and 4 controls) underwent coronary angiography with optical coherence tomography (OCT; at baseline and 1 and 2 months), coronary computed tomography angiography, cardiac magnet resonance (CMR) imaging, and histologic examination. Results The diabetic pigs had more irregular capillaries with acellular capillaries and a smaller capillary diameter (11.7 ± 0.33 μm vs. 13.5 ± 0.53 μm; P < 0.001) than those of the control pigs. The OCT showed no significant epicardial stenosis in either group; however diabetic pigs had a greater intima-media thickness. CMR results showed that diabetic pigs had a lower relative upslope at rest (31.3 ± 5.9 vs. 37.9 ± 8.1; P = 0.011) and during stress (18.0 ± 3.0 vs. 21.6 ± 2.8; P = 0.007) than the control pigs, implying decreased myocardial perfusion. Among the 79 patients with ST elevation myocardial infarction, 25 had diabetes and they had lower myocardial perfusion on CMR as well. Conclusion DM causes microvascular remodeling and a decrease in myocardial perfusion in large animals at a very early stage of the disease course. Early and effective interventions are necessary to interrupt the progression of vascular complications in diabetic patients.


INTRODUCTION
Diabetes mellitus (DM) is a well-known risk factor, not only for coronary artery disease (CAD), but also for the development of diabetic cardiomyopathy. The diabetic cardiomyopathy develops independently of CAD and hypertension and causes increased cardiovascular morbidity and mortality. 1,2 DM causes chronic vascular changes in both micro-and macrovascular systems and ultimately leads to multi-organ dysfunction. Regarding the macrovascular changes, DM is associated with accelerated atherosclerosis Previous studies have reported a "paradoxical" absence of significant stenosis in epicardial arteries in patients with significant ischemic symptoms and positive exercise test, 3 -5 implying the existence of coronary microvasculopathy. Diabetic cardiomyopathy is known to be associated with abnormal coronary microcirculation which is characterized by reduced levels of bioavailable nitric oxide. 6,7 However, the characteristics of abnormal coronary microcirculation is not well defined with the current diagnostic modalities.
Altered glucose homeostasis, metabolic, humoral, and hemodynamic disturbances contribute to the initiation and progression of vasculopathy in diabetic patients. 8 Suggested mechanisms include hyperglycemia-induced apoptosis or structural changes in vascular endothelial (VE) cells and cardiomyocytes through inflammation, oxidative stress, glycated products, or protein kinase C activation. 1,2 Recently, we found a novel mechanism where abnormal intercellular signaling in endothelial cells decreased the capillary diameter and induced microvascular rarefaction in diabetic conditions in a human in-vitro model of angiogenesis and in the retina, heart, skeletal muscle, and kidney of mice. 9, 10 The microvascular changes in DM has not been evaluated in a large animal model or human. The diagnosis of coronary microvasculopathy in patients with DM is challenging because macrovascular changes often coexist and the histologic examination of microvessels is not feasible. In the present study, we sought to determine the effect of DM on macro-and microvascular abnormalities in diabetic pigs using the state-of-the-art diagnostic modalities. In addition, we also validated our findings in diabetic patients using cardiac magnet resonance (CMR) imaging.

Study design and animals
A total of 8 domestic female pigs weighing between 20 and 25 kg were used for this study. Type I DM was induced in four pigs by intraperitoneal injection of streptozotocin at a dose of 150 mg/kg. 11, 12 Consecutive measurements of serum glucose were performed at predetermined time points. Sustaining blood glucose levels > 250 mg/dL were considered as having diabetes. If blood glucose exceeds 400 mg/dL, 4 to 8 units of long-acting insulin (Lantus ® ; Sanofi-Aventis, Paris, France) were injected to avoid acute complications such as diabetic ketoacidosis or hyperosmolar coma. The pigs underwent coronary angiography (CAG) with optical coherence tomography (OCT) at 1 and 2 months and CMR imaging and coronary computed tomography angiography (CCTA) at 2 months. After invasive and noninvasive imaging the pigs were sacrificed for histologic analyses (Fig. 1A).
A 6-French vascular sheath was introduced into the femoral artery using the Seldinger technique under sonographic guidance. Under fluoroscopic guidance, 6-French Judkin right 3.5 guiding catheters were positioned in the left coronary ostium. After the intracoronary administration of nitroglycerin (200 μg), CAG was performed. Intracoronary administration of 5,000 units of unfractionated heparin was given to prevent thrombus formation.

OCT
OCT images were acquired using the OCT system consisting of an optical fiber, a proximal low pressure occlusion balloon catheter (Helios™ Goodman; Advantec Vascular Corp™, Sunnyvale, CA, USA), and an OCT system mobile cart containing the optical imaging engine and computer for signal acquisition, analysis, and image reconstruction (M2CV OCT Imaging System; LightLab, Westford, MA, USA).
The left anterior descending artery (LAD) and left circumflex artery were wired with a 0.014inch Runthrough guide wire (Terumo, Tokyo, Japan) and then replaced by an OCT ImageWire™ and the occlusion balloon. The occlusion balloon was pulled-back and positioned in a proximal-segment of the arteries. Inflation was performed gently up to 0.4 to 0.7 atmosphere with a dedicated inflation device. The vessel segments were imaged using an automated pullback (1.0 mm/sec) from the distal to proximal segments. Images were acquired at 15 frames per second and displayed in a real-time 2D array at different transverse positions.

CCTA
All pigs underwent CCTA with a 64-slice multi-detector computed tomography (CT) scanner (Brilliance 64; Philips Medical Systems, Best, The Netherlands) in the prone-position. Prior to CCTA, all animals with a baseline heart rate > 70 beats/min received 10 to 30 mg of intravenous esmolol (Jeil Pharm, Seoul, Korea). CCTA was performed with a 64 × 0.625 mm section collimation, 420-ms rotation time, 120-kV tube voltage, and 800-mA tube current. During CCTA acquisition, a bolus of 80 mL iomeprol (Iomeron 400; Bracco, Milan, Italy) was injected intravenously (4 mL/sec). The animals' electrocardiogram (ECG) was simultaneously recorded to allow for retrospective segmental data reconstruction. Images were initially reconstructed at the mid-diastolic phase (75% of R-R interval) of the cardiac cycle. Compartment analysis was performed by using the cardiac CT viewer tool of the workstation (IntelliSpace Portal ISP, version 8.0; Philips Medical Systems). Center lines were rotated to the sagittal plane to acquire suitable short-axis pig heart image. Hounsfield units (HU) of region of interest were measured using the dedicated software in the 16 segments (American Heart Association [AHA] model, true apex not imaged) of the heart, ascending and descending aorta, and left ventricular (LV) cavity in 2 or 3 phases of the heart cycle. We calculated the ratio of HU of the myocardial segments to the averaged HU of the aorta and LV cavity.

CMR imaging and perfusion
All CMR studies were performed using a 1.5 T magnetic resonance (MR) imaging scanner (Siemens Avanto, Erlangen, Germany or Intera CV release 10; Philips Medical Systems) with a phased array cardiac coil. Cine images were acquired retrospectively with ECG-gated steadystate free precession (SSFP) sequences (image matrix 128 × 128, read field of view [FOV] 340 mm, phase FOV 75%-100% of the FOV read, echo time 1.12 ms, flip angle 70°) with a slice thickness of 8 mm and intersection gaps of 12-15 mm.
We performed pharmacological stress and rest perfusion studies. After the acquisition of cine scans at rest in the standard views, adenosine was intravenously infused at a dose of 140 μg/kg/min for up to 6 minutes via a 20-gauge cannula using a syringe pump (Graseby ® 3500; Smiths Medical, Minneapolis, MN, USA). Within the last minute of infusion, a bolus of gadodiamide (Omniscan; GE healthcare, Chicago, IL, USA) was injected (dose, 0.1 mmol/ kg; injection rate, 4 mL/sec) and stress perfusion MR images were obtained with a gradientecho sequence by using saturation-recovery SSFP. After 15 to 25 minutes, an identical MR perfusion scan was performed at rest.
Data were analyzed using a dedicated software (QMass; Medis, Leiden, the Netherlands). First-pass perfusion images at stress and rest states were analyzed semiquantitatively. The AHA 16-segment model was used (true apex not imaged), resulting in a total of 32 segments per pig (16 for the stress examination and 16 for the resting examination). Signal intensity (SI)-time curves were generated for all segments. The relative upslope at rest or at stress (RRU or SRU) was calculated by the maximum upslope of the LV myocardium divided by the maximum upslope of the LV cavity at stress and rest states. The myocardial perfusion reserve index (MPRI) was calculated as the ratio of the segmental upslope values during adenosine stress and rest (MPRI = SRU/RRU).

Immunofluorescence staining and confocal microscopic analysis
After euthanasia of the pigs, the coronary arteries were perfused with phosphate-buffered saline (PBS) and 2% paraformaldehyde (PFA) in PBS at room temperature. The isolated tissues were embedded in an optimal cutting temperature compound (Sakura Finetek,

DM and Cardiac Microvasculopathy in Large Animals
Torrance, CA, USA) and stored in a −80°C deep freezer. Frozen sagittal sections were cut into 25 to 30 μm slices using a cryostat (HM 550 MP; Microm, Walldorf, Germany). The sections were fixed with 4% PFA in PBS for 10 minutes and permeabilized with 10% fetal bovine serum and 0.5% Triton-X100 in PBS for 4 to 6 hours. We incubated the slices with tetramethyl rhodamine isothiocyanate-conjugated Bandeiraea simplicifolia 1-lectin (Sigma Aldrich, St. Louis, MO, USA) and fluorescein isothiocyanate-conjugated alpha smooth muscle actin (Sigma Aldrich) overnight at 4°C. We also stained the slices overnight at 4°C with specific primary antibody, anti-VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with fluorescent-tagged secondary antibodies (Invitrogen, Carlsbad, CA, USA) overnight at 4°C. The images were acquired by a confocal laser scanning microscope system (LSM 710; Carl Zeiss AG, Oberkochen, Germany) and processed with Zen 2008 (Carl Zeiss AG) and ImageJ (NIH, Bethesda, MD, USA) software. A water-or oilimmersion objective (40× or 63×, 1.4 numerical aperture) with the pinhole set for a section thickness of 0.8 to 1.2 μm (1 airy unit in each channel) was used. To visualize the threedimensional reconstructed image, a Z-stack of 20-μm thickness was obtained. Diode 405 nm, Multi-Argon 488 nm, HeNe 543 nm, and HeNe 633 nm laser lines were selected and images were sequentially acquired using separate laser excitations to avoid cross-talk between different fluorophores.

Immunofluorescence quantification of microvasculopathy in the myocardium
For comparative analysis we created images under same conditions of light, contrast, and magnification. Total capillary length and average diameter of the capillaries in images of tissues from mid-ventricular six segments of each pig were measured with ImageJ software (NIH). Therefore, 24 fields for each group were selected for image analysis. For evaluation of the capillary diameter at various points of interest in z-stack images, we measured the width of the lectin-positive area that was perpendicular to the capillary length. As consequence, the capillary diameter in this study corresponds to the abluminal diameter.

Patients
We retrospectively enrolled patients with ST-segment elevation myocardial infarction (STEMI) who underwent CMR 5 to 7 days and 6 months after the onset of myocardial infarction (MI) from January 2005 to November 2012. We then compared the myocardial perfusion in non-infarct segments between patients with and without DM.

Statistical analysis
Continuous variables are expressed as mean ± standard deviation or mean ± standard error and categorical variables are presented as absolute numbers and proportions (%). Overall comparisons between groups were performed with the Student's t-test or the Wilcox rank sum test for continuous variables and the χ 2 or Fisher exact test for categorical variables. In the MR perfusion experiments, we measured RRU, SRU, and MPRI values for 16 segments according to the AHA 16-segments model in all animals and tested whether the measured values differed between the groups. Because the measured values may also be influenced by the segments, the segment was treated as a factor as well. As consequence, we included both segments and groups as factors in the analyses and applied the two-way analysis of variance (ANOVA) to evaluate 'the effect of the groups' after adjusting 'the effect of the segments.' To check the normality of the data, we analyzed the residuals of each models using the following two methods. First, we confirmed that SRU and MPRI satisfy the normal distribution with the Shapiro tests. Second, we also visually confirmed the normality using histogram with normal density and QQ-plot. Both

Ethics statement
The present study protocol was reviewed and approved by the Institutional Review Board (IRB) of Seoul National University Bundang Hospital (IRB No. B-1703-388-102). Informed consent was waived because of the retrospective design and inability to obtain by the IRB.
The current study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (IACUC No. BA1111-093/071-01). All animals were treated according to the study protocol and the principles of care of laboratory animals.

Microvascular changes
There was no difference in pigs' weights between the groups on days 0 and 56 (Fig. 1B). Both groups showed similar weight gain during the experiment. Blood glucose level in the diabetic pigs was significantly higher than that in the control pigs during the experiment (Fig. 1C).
For the evaluation of microvasculature, we first evaluated the capillary anatomy. The two components of interest were the capillary lengths and diameter ( Fig. 1D and E). There was no difference in the capillary lengths (129.1 ± 0.44 μm vs. 129.4 ± 0.48 μm; P = 0.365) between diabetic and control pigs; however, diabetic pigs had a significantly shorter capillary diameter (11.7 ± 0.33 μm vs. 13.5 ± 0.53 μm; P < 0.001) as we previously reported ( Fig. 1F and G). We also found that VE-cadherin expression in the diabetic capillary was decreased and discontinued compared to that in the control ( Fig. 1H and I, Supplementary Videos 1 and 2). Overall, the capillaries in the diabetic pigs were more irregular and disconnected; the capillary diameter was highly variable with frequent acellular capillaries, so called string vessels.

MR perfusion
The myocardial perfusion by CMR at rest and during stress was measured in the sixteen myocardial segments in the diabetic and control pigs ( Fig. 2A and B). Diabetic pigs had lower mean values of RRU and SRU in most segments. However, MPRI showed no difference. We calculated estimated mean of the RRU, SRU, and MPRI of the 16 segments (Fig. 2C). Diabetic pigs had significantly lower RRU than control pigs (31.3 ± 5.9 vs. 37.9 ± 8.1; P = 0.011) (Fig. 2D). Diabetic pigs also had lower SRU for myocardial perfusion than control pigs (18.0 ± 3.0 vs. 21.6 ± 2.8; P = 0.007). However, there was no difference in MPRI between diabetic and control pigs (0.59 ± 0.05 vs. 0.62 ± 0.08; P = 0.380).

CT angiography
Myocardial SI in the arterial phase of the CT angiography represents myocardial texture and the amount of contrast in the myocardial microvessels. Therefore, we measured the HU of 16

DM and Cardiac Microvasculopathy in Large Animals
segments of the left ventricle in the arterial phases and compared them to that of the aorta (Fig. 3A). CT perfusion showed similar results in that the estimated mean ratio of myocardial SI to aortic SI was significantly lower in the diabetic pigs compared to the control pigs (0.43 ± 0.05 vs. 0.48 ± 0.02; P < 0.001) (Fig. 3B).

CAG and optical coherent tomography
According to the protocol, CAG and OCT were performed at three time points: baseline, 1 month, and 2 months (Fig. 4A). The coronary artery of the pigs increased with their growth. There was no difference in the vessel size of the proximal LAD between diabetic and control pigs (at baseline: 5.59 ± 0.60 mm vs. 5.68 ± 1.26 mm, P = 0.906; at 8 weeks: 7.31 ± 1.34 mm vs. 7.34 ± 1.47, P = 0.983) (Fig. 4B)

Myocardial flow analysis in patients with MI
We retrospectively enrolled 79 patients with STEMI who underwent CMR. Baseline characteristics, MI-culprit lesions, baseline LV function, and infarct size did not differ between the patients with and without diabetes ( Table 1). Patients with diabetes reported the duration of DM as median 3 and interquartile range 1-10 years. They had a higher serum glucose 250 ± 113 mg/dL vs. 146 ± 34 mg/dL and hemoglobin A1c (HbA1c) level (7.61% ± 0.28% vs. 5.95% ± 0.11%; P < 0.001) at the time of MI. Patients with diabetes had lower RRU and SRU than those without diabetes, whereas MPRI showed no difference (Fig. 5), as we had observed in the pig model.
During follow-up, despite a significant decrease in HbA1c, patients with diabetes had higher HbA1c than those without diabetes (6.78% ± 0.18% vs. 5.92% ± 0.09%; P < 0.001), indicating adequate diabetes control. The follow-up CMR revealed that LV end-systolic volume in patients with DM was significantly larger than that in non-DM patients (55.8 ± 14.9 mL vs. 61.5 ± 14 mL; P = 0.047). LV end-diastolic volume (142 ± 48 mL vs. 130 ± 29 mL, P = 0.267) was numerically larger and ejection fraction (55% ± 14% vs. 61% ± 14%, P = 0.180) was numerically lower in DM patients than in non-DM. Kaplan-Meier analysis showed that the composite of all-cause death, re-MI, and revascularization did not differ between the groups during a median follow-up of 3 years (Fig. 6).

DISCUSSION
In this study, we evaluated the effect of DM on microvascular and macrovascular changes in diabetic pigs using state-of-the-art diagnostic tools including CAG, OCT, CCTA, and CMR, among others, and found the following: 1) DM caused alterations in capillary structures, 2) without causing significant narrowing in the epicardial coronary artery during the in-vivo experiment period, 3) suggesting that the observed decreased myocardial microvascular perfusion is the result of microvascular changes. In addition, 4) we also showed that STEMI patients with diabetes also had decreased myocardial perfusion. To the best of our knowledge, our study is the first showing DM-related changes in microvascular structure and defects in myocardial microvascular perfusion independent of epicardial CAD in a largeanimal model.
We recently reported a novel mechanism in that abnormal intercellular signaling in endothelial cells induces microvascular rarefaction in diabetic mice. 9,10 Briefly, Notch ligand Jagged1 is a key molecule which is involved in the diabetic microvasculopathy. Elevated Jagged1 reduces the nuclear localization of Notch1 intracellular domain and leads to dysregulation of VE-cadherin and N-cadherin in endothelial cells. Diabetic capillary changes began within a few days in vitro and after 4 to 6 weeks in vivo. Interestingly, the changes were reversible by modulation of Jagged1.
In the current study, we could reproducibly show the capillary changes along with downregulation of VE-cadherin in the diabetic porcine model. VE-cadherin is a junctional adhesion molecule that stabilizes the established endothelium. 13 Likewise, the reduced expression and disruption of VE-cadherin correlated with the observed capillary changes such as a smaller capillary diameter, frequent presence of acellular capillaries, and disruptions and rarefication of the capillary.
In fluid hemodynamics, at a constant pressure, the flow is mainly determined by the resistance. Accordingly, in coronary circulation, the coronary flow will be determined by microvascular resistance. These structural changes of capillaries can lead to capillary dysfunction and increase in myocardial resistance in the coronary microcirculation. We repeatedly showed a decrease in myocardial perfusion by CCTA and CMR in the absence of significant epicardial stenosis, suggesting that the reduced myocardial perfusion in subjects with diabetes is mainly due to microvascular changes. In humans, histologic examination  was not feasible; however, we also observed similar decrease in myocardial perfusion in diabetic STEMI patients as in the diabetic pigs. Taken together, our study provides reasonable evidence that the diabetes-induced capillary changes occur in small and large animals, as well as in humans.
While the capillary changes in the animal models begins at an early phase of hyperglycemia, the time span in humans is yet to be determined: it may begin in the early stage of DM considering the rapid changes in in-vitro and animal models 9,10 ; however, clinically significant microvascular complications such as retinopathy or nephropathy develop over 7 years in patients with type 1 DM. 14 It is also of note that, until now, we do not know how to define a clinically significant myocardial microvasculopathy. Furthermore, we did not find a difference in clinical mid-term outcome between diabetic and non-diabetic STEMI patients. This was not unexpected considering the small number of patients of this study and the good control of DM reflected by reduction in HbA1c (7.6% at baseline and 6.8% at follow-up). Of note, we found that LV end-systolic volume was getting increased during follow-up in the DM group, which might be affected by the diminished myocardial perfusion. The natural course and long-term effect on the myocardial function and clinical outcomes should be further investigated.  In humans, functional and structural abnormalities of the coronary microcirculation are often observed in diabetic patients with normal coronary arteries. 15 -17 The coronary flow reserve (CFR) is the ratio between hyperemic and resting coronary flow like MPRI in our study. In the absence of significant stenosis of the epicardial coronary arteries, the reduction of CFR has been reported to represent coronary microvascular dysfunction. 18 A reduction in CFR and myocardial blood flow was associated with increased cardiac mortality in diabetic patients without coronary artery stenosis. 19 - 22 In the present study, we examined the segmental myocardial perfusion in the porcine heart two months after the induction of DM and observed impaired myocardial blood flow at rest and stress but not the decrease in MPRI in contrast to previous reports. 22 In line with our previous reports, 9 we think that diabetic microvasculopathy progresses at an early stage of hyperglycemia due to capillary remodeling which does not involve arteriole or epicardial CAD and thus does not impair the CFR or MPRI in the porcine model.
Currently, OCT is the clinically applicable imaging modality with the highest spatial resolution for the visualization of the coronary anatomy. In this study, OCT displayed no hemodynamically significant stenosis, but it did show a greater IMT in the coronary arteries of diabetic pigs. IMT represents early atherosclerotic changes of a vessel. Vascular remodeling is a homeostatic response to changes in flow and circumferential stretch to maintain or restore normal shear stress and wall. 23 Theoretically, decreased coronary flow due to impaired microcirculation can lead to a decrease in vessel diameter 24 and may also accelerate atherosclerosis. Whether the increase in IMT in diabetic pigs is a result of hemodynamic alteration or a result of hyperglycemia-induced changes needs further investigation. In addition, due to the limited duration of the experiment, it is unknown whether longer exposure to hyperglycemia would have led to significant macrovascular changes as well. Nonetheless, the decrease in perfusion in the present study is not affected by the epicardial arterial changes.
In our previous studies, we could reverse microvascular abnormalities by modulating the intercellular signaling, which could be a potential therapeutic target. 9,10 Therefore, a non- invasive monitoring tool for the development or improvement of microvasculopathy in patients with diabetes is of clinical interest and is challenging. We showed that CMR showed decreased myocardial perfusion in diabetic patients, thus CMR may be a good candidate for the non-invasive assessment of microvascular changes. However, STEMI patients group may not be considered as the same target group in the animal experiment because of co-existing CAD and the possible microvasculature alteration in the non-infarct area affected by the infarcted territories. Ideally, the human experiment should have been conducted in subjects without significant CAD. Because the number of diabetic patients is not large enough, we did not perform additional subgroup analyses according to the presence of significant CAD. Nonetheless, further studies with an adequate sample size are warranted before applying the CMR findings to clinical practice.
In conclusion, DM causes microvascular remodeling and a decrease in myocardial perfusion in large animals and humans, which occurs at a very early stage of the disease course. Early and effective interventions are necessary to interrupt the progression of vascular complications in diabetic patients.