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J Thorac Cardiovasc Surg 2007;134:1453-1460
© 2007 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

Insulin treatment enhances the myocardial angiogenic response in diabetes

^a Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Ontario, Canada
^b Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.

Read at the Eighty-seventh Annual Meeting of The American Association for Thoracic Surgery, Washington, DC, May 5-9, 2007.

Received for publication May 8, 2007; revisions received July 24, 2007; accepted for publication August 2, 2007.

^_* Address for reprints: Frank W. Sellke, Beth Israel Deaconess Medical Center, 110 Francis St, LMOB 2A, Boston, MA 02215. (Email: fsellke{at}caregroup.harvard.edu).

	Abstract

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Objective: Growth factor and cell-based angiogenesis are attractive therapeutic options for diabetic patients with end-stage coronary disease. Reduced collateral vessel formation observed in diabetes is associated with increased expression of anti-angiogenic proteins, angiostatin and endostatin. The aim of this study was to determine the effects of insulin treatment on the diabetic angiogenic response to chronic myocardial ischemia.

Methods: Yucatan miniswine were treated with alloxan (pancreatic ß-cell specific toxin, 150 mg/kg) and divided into two groups. In the diabetic group (DM, n = 8), blood glucose levels were kept greater than 250 mg/dL, and in the insulin-treated group (IDM, n = 6), intramuscular insulin was administered daily to keep blood glucose less than 150 mg/dL. A third group of age-matched swine served as nondiabetic controls (ND; n = 8). Eight weeks later, all animals underwent circumflex artery ameroid constrictor placement to induce chronic ischemia. Myocardial perfusion was assessed at 3 and 7 weeks after ameroid placement using microspheres. Microvascular function, capillary density, and myocardial expression of anti-angiogenic mediators were evaluated.

Results: Diabetic animals exhibited significant impairments in endothelium-dependent microvessel relaxation to adenosine diphosphate and substance P, which were reversed in insulin-treated animals. Collateral-dependent perfusion in the ischemic circumflex territory, which was profoundly reduced in diabetic animals (–0.18 ± 0.02 vs +0.23 ± 0.07 mL · min^–1 · g^–1; P < .001), improved significantly with insulin treatment (0.12 ± 0.05 mL · min^–1 · g^–1; P < .01). Myocardial expression of anti-angiogenic proteins, angiostatin and endostatin, showing a 4.3- and 3.6-fold increase in diabetic animals respectively (both P < .01 vs ND), was markedly reduced in insulin-treated animals (2.3- and 1.8-fold vs ND; both P < .01).

Conclusions: Insulin treatment successfully reversed diabetic coronary endothelial dysfunction and significantly improved the endogenous angiogenic response. These pro-angiogenic effects may be mediated through downregulation of anti-angiogenic mediators. Insulin therapy appears to be a promising modality to enhance the angiogenic response in diabetic patients.

	Introduction

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Drs Sellke and Boodhwani (left to right)

For more than a decade, cardiovascular researchers and clinicians have explored therapeutic angiogenesis, using growth factors or cell-based therapies, as a treatment option for patients with coronary artery disease. Despite early success in animal models, clinical trials of angiogenic therapies have yielded disappointing results.^1,2 One of the proposed explanations for the discordance between animal studies and clinical trials is the presence of various anti-angiogenic influences that are found in patients with coronary artery disease but are absent in young healthy animals in which the majority of preclinical experiments are conducted. These include, among others, diabetes, hypercholesterolemia, advanced age, and endothelial dysfunction. We have previously demonstrated that diet-induced hypercholesterolemia is associated with endothelial dysfunction as well as an impairment in the endogenous³ and growth factor–induced angiogenic response.^4,5 Advanced age has been associated with impaired growth factor signaling in patients with coronary artery disease.⁶

Diabetes is a commonly found comorbid condition in patients with coronary artery disease and is increasing in prevalence worldwide.⁷ Its presence is associated with accelerated atherosclerosis as well as an impaired angiogenic response to chronic ischemia, which has been demonstrated in patients, both angiographically⁸ and in autopsy studies.⁹ Although therapeutic angiogenesis appears to be an attractive treatment option in patients with diabetes, the evaluation of therapeutic modalities in the presence of diabetes is limited, in large part, by the lack of a large animal model of diabetes and chronic ischemia that can be used to evaluate clinically relevant end points. We¹⁰ have recently reported the creation and thorough validation of such a model of alloxan-induced diabetes in Yucatan miniswine. In this model, diabetes is induced with alloxan, a pancreatic ß-cell specific toxin. We demonstrated that a 15-week exposure to chronic hypoinsulinemic hyperglycemia (type I diabetes) was associated with many of the functional, microvascular, and molecular abnormalities associated with human diabetes, including endothelial dysfunction, impaired collateral-dependent myocardial perfusion, and alterations in the angiogenic signaling in the myocardium. In this study, we sought to determine the effects of glycemic control using parenteral insulin treatment on microvascular function and the endogenous angiogenic response to chronic myocardial ischemia.

	Materials and Methods

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General Experimental Sequence and Diabetes Induction
Twenty-two Yucatan miniswine of male sex (Sinclair Research Inc, Colombia, Mo) were used for the studies. Diabetes was induced when animals were 8 months old by a single intravenous injection of alloxan (150 mg/kg). Only animals that achieved and maintained blood glucose levels greater than 200 mg/dL were included in the diabetes group. Animals were fed ad libitum and no dietary modification was used. These animals were divided into two groups. In the diabetic group (DM, n = 8), fasting blood sugar was maintained between 250 and 400 mg/dL. In the insulin-treated group (IDM, n = 6), glycemic control was achieved (fasting blood sugar < 150 mg/dL) through daily intramuscular insulin (70% amorphous, 30% crystalline) administration. Fasting blood glucose was monitored every 2 days early after diabetes induction and 1 to 2 times per week thereafter by obtaining a small blood sample (50 µL) from a capillary bed in the ear. Diabetes was maintained for 8 weeks before surgical instrumentation. Age-matched miniswine (ND, n = 8) served as controls.

All animals underwent an identical experimental protocol involving three separate procedures on each animal. Anesthesia was performed as reported previously,³ and animals received humane care in compliance with the Harvard Medical Area Institutional Animal Care and Use Committee and the National Research Council’s "Guide for the Care and Use of Laboratory Animals," prepared by the Institute of Laboratory Animals and published by the National Institutes of Health (NIH publication No.5377-3, 1996). In brief, for all surgical procedures, anesthesia was induced with ketamine (10 mg/kg intramuscularly), thiopental (5-10 mg/kg intravenously), and thiopental 2.5% and maintained with a gas mixture of oxygen at 1.5 to 2 L/min and isoflurane at 0.75% to 3.0%. The animals were intubated and mechanically ventilated at 12 to 20 breaths/min.

The first procedure, performed via a small left anterolateral thoracotomy 8 weeks after diabetes induction, consisted of the placement of a 1.75-mm ameroid constrictor around the proximal circumflex artery and the injection of 1.5 x 10⁷ gold-labeled microspheres into the left atrium during temporary occlusion of the circumflex coronary artery, to subsequently allow for identification, by shadow labeling, of the myocardial territory at risk.

The second procedure, also performed via left anterolateral thoracotomy, 3 weeks after ameroid placement, consisted of 1.5 x 10⁷ lutetium microspheres injected in the left atrium during rest conditions and 1.5 x 10⁷ europium microspheres injected during rapid atrial pacing (150 beats/min) to allow for determination of baseline perfusion after ameroid closure. To document ameroid closure, we performed left coronary angiography through an 8F sheath (Cordis Corporation, Miami, Fla) surgically inserted in the femoral artery, using a catheter with the appropriate distal angulation and high atomic weight contrast (Mallinckrodt Inc, St Louis, Mo).

The third procedure was carried out 4 weeks after the second procedure and 7 weeks after ameroid placement. Sternotomy was performed, 1.5 x 10⁷ samarium microspheres were injected into the left atrium during rest conditions, and 1.5 x 10⁷ lanthanum microspheres were injected during pacing (150 beats/min). The animals were then humanely killed with 10 mL/kg of a saturated KCl solution administered intravenously. Cardiac samples were harvested and snap frozen for molecular studies, sectioned, weighed, and dried for myocardial microsphere analyses, and put in 4°C Krebs solution for in vitro assessment of coronary microvascular reactivity. Ameroid constrictors were resected along with a segment of circumflex artery and examined under low-power magnification to confirm occlusion.

In Vitro Assessment of Coronary Microvessel Reactivity
After cardiac harvest, epicardial coronary arterioles (80-150 µm in diameter and 1-2 mm in length) originating from branches of the left anterior descending and circumflex arteries were dissected from the surrounding tissue with a x40 dissecting microscope and examined in isolated organ chambers, as described previously.¹¹ The responses to sodium nitroprusside (1 nmol-100 µmol), an endothelium-independent cyclic guanosine monophosphate–mediated vasodilator, as well as adenosine 5'-diphosphate (ADP) (1 nmol/L–10 µmol/L), substance P (1 fmol/L–1 nmol/L), and vascular endothelial growth factor (VEGF, 1 fmol/L–1 nmol/L), three endothelium-dependent receptor-mediated vasodilators that act via bioavailable nitric oxide, were studied after precontraction by 20% to 50% of the baseline diameter with the thromboxane A₂ analog U46619 (0.1-1 µmol/L). Relaxation responses were defined as the percent relaxation of the precontracted diameter, and 6 to 8 vessels were examined from 6 to 8 animals in each group from the left anterior descending and the circumflex territories.

Assessment of Myocardial Perfusion
Myocardial perfusion was assessed during each procedure with isotope-labeled microspheres (BioPAL, Worcester, Mass) using methods previously reported.³ Isotope-labeled microspheres, 15 µm in diameter, of different isotopic mass were used at each experimental stage. Gold-labeled microspheres were injected during temporary circumflex occlusion at the time of ameroid placement to identify myocardial samples that originated from the circumflex coronary distribution (those with the lowest count of gold-labeled microspheres). Lutetium- and europium-labeled microspheres were used during the second procedure to determine baseline blood flow at rest and with atrial pacing at 150 beats/min. Samarium- and lanthanum-labeled microspheres were injected at rest and during pacing during the third procedure. Reference blood samples were obtained from the femoral artery during the second and third procedures. After the animals were humanely killed, 10 circumferential, transmural left ventricular sections were collected for assay of isotope-labeled microspheres in each animal, weighed, and dried at 60°C for 24 hours. Each sample was exposed to neutron beams and microsphere densities were measured in a gamma counter. Adjusted myocardial blood flow (at rest and with pacing), reflecting changes in lateral myocardial perfusion, was determined from the two myocardial samples that showed the lowest count of gold microspheres by the following equations:

Assessment of Myocardial Function
Before tissue harvest, a catheter was surgically inserted through the femoral artery, passed into the left ventricle, and left ventricular pressure was recorded over 10-second intervals. Global systolic and diastolic function was determined with Cardiosoft software (Sonosoft Inc, London, Ontario, Canada) by taking the first derivative of left ventricular pressure (dP/dt). Next, maximum +dP/dt (systolic function) and minimum –dP/dt (diastolic function) measurements were obtained and averaged over 15 to 20 cardiac cycles in each animal.

Immunohistochemistry
Myocardial sections from the circumflex territory of control and diabetic animals were stained with anti-platelet–endothelial cell adhesion molecule-1 (CD-31) antibody diluted to 1:600 (BD Biosciences, San Diego, Calif) as previously described.³ The sections were counterstained with methyl green and examined for capillary endothelial cell density in a triplicate, blinded fashion from 600 x 440 µm (0.264 mm²) cross-sectional fields randomly selected from the center of circumflex territories.

Western Blotting
Whole-cell lysates were isolated from the homogenized myocardial samples with a radioimmunoprecipitation assay buffer (Boston Bioproducts, Worcester, Mass) and centrifuged at 12,000g for 10 minutes at 4°C to separate soluble from insoluble fractions. Protein concentration was measured spectrophotometrically at a 595-nm wavelength with a DC protein assay kit (Bio-Rad Laboratories, Hercules, Calif). Forty to eighty micrograms of total protein were fractionated by 4% to 20% gradient, sodium dodecylsulfate polyacrylamide gel electrophoresis (Invitrogen, San Diego, Calif) and transferred to polyvinyl difluoride membranes (Millipore, Bedford, Mass). Each membrane was incubated with specific antibodies as follows: anti-VEGF antibody (dilution 1:250) (Calbiochem, San Diego, Calif), anti–endothelial nitric oxide synthase antibody (1:2500) (BD Biosciences, San Jose, Calif), anti-Tie-2 antibody (1:200) (Santa Cruz, Santa Cruz, Calif), anti-endostatin antibody (1:1000) (Upstate, Chicago, Ill), and anti-angiostatin antibody. Then the membranes were incubated for 1 hour in diluted appropriate secondary antibody (Jackson Immunolab, West Grove, Pa). Immune complexes were visualized with the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ). Bands were quantified by densitometry of radioautograph films.

Data Analysis
Data are reported as means ± SEM. Microvessel responses are expressed as percent relaxation of the preconstricted diameter and were analyzed by 2-way repeated measures analysis of variance, examining the relationship between vessel relaxation, log concentration of the vasoactive agent of interest, and the experimental group (SAS version 9.1; SAS Institute, Inc, Cary, NC). Immunoblots are expressed as a ratio of protein to loading band density and were analyzed after digitization and quantification of x-ray films with ImageJ 1.33 (National Institutes of Health, Bethesda, Md). Blots and isotope-labeled microsphere data were analyzed by analyses of variance. Bonferroni corrections were applied to multiple tests.

	Results

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Experimental Model
Diabetes induction was attempted in 17 animals, of which 14 (82%) demonstrated consistent blood glucose levels greater than 200 mg/dL. One animal died of renal failure and 2 failed to become hyperglycemic. Four animals in the DM group occasionally required low doses of insulin (10-20 u/day) to keep blood sugar levels less than 400 mg/dL. In the IDM group, all animals required large doses of daily insulin (average dose of 78 U/day) to achieve target fasting blood glucose levels. Three of the 6 IDM animals required twice a day dosing to achieve adequate glycemic control. Hypoglycemic episodes requiring treatment occurred in 2 animals on 4 occasions. Fasting blood glucose levels in the different groups throughout the experimental protocol are depicted in Figure 1. All animals survived the entire experimental protocol.

View larger version (10K): [in this window] [in a new window]   Figure 1. Blood glucose levels in no diabetes (ND), diabetes (DM), and insulin-treated diabetes (IDM) groups over the 7-week experimental protocol.

View larger version (25K): [in this window] [in a new window]   Figure 2. Coronary microvascular reactivity. Percent relaxation to increasing concentrations of vasodilating agents after preconstriction with U46619. Responses to endothelium-dependent vasodilators ADP (A), Substance P (B), and VEGF (C), as well as endothelium-independent vasodilator SNP (D) in the ischemic territory of ND (n = 8), DM (n = 8), and IDM (n = 6) animals. ADP, Adenosine 5' diphosphate; SNP, sodium nitroprusside; VEGF, vascular endothelial growth factor. *P < .001 vs ND. **P < .01 vs DM. P < .05 vs controls. ND, DM, and IDM as in Figure 1.

View larger version (11K): [in this window] [in a new window]   Figure 3. Myocardial perfusion. Isotope-labeled microspheres were used to determine baseline-adjusted circumflex territory myocardial blood perfusion in control (n = 8) and diabetic swine (DM, n = 8; IDM, n = 6) after circumflex ameroid occlusion. Adjusted circumflex territory perfusion, which was profoundly reduced in DM animals at rest (A) and with pacing (B), improved with insulin treatment, but still remained lower than controls. *P < .001 vs ND. **P < .001 vs DM and P < .05 vs ND. ND, DM, and IDM as in Figure 1.

View larger version (28K): [in this window] [in a new window]   Figure 4. Left ventricular (LV) function. Diabetic animals demonstrated reduced LV contractility and impaired LV relaxation. Insulin treatment resulted in significant improvement in LV systolic function (n = 4-6/group; *P < .001 vs ND; **P < .01 vs DM; P < .05 vs ND). ND, DM, and IDM as in Figure 1.

View larger version (47K): [in this window] [in a new window]   Figure 5. Endothelial cell density. CD31+ endothelial cell density per high power field (0.264 mm2) was determined in the ischemic territory of control (n = 8) and diabetic (DM, n = 8; IDM, n = 6) animals. Diabetic animals had reduced endothelial cell density compared with controls, which was significantly improved in insulin-treated diabetic animals. *P < .001 vs ND. **P < .001 vs DM. ND, DM, and IDM as in Figure 1.

Anti-angiogenic mediators: Angiostatin and endostatin
Figure 6 displays the results of Western blots of anti-angiogenic mediators, angiostatin and endostatin. The expression of both angiostatin (4.1 ± 1.3-fold; P < .01) and endostatin (3.6 ± 0.4-fold; P < .01) was profoundly increased in the myocardium of diabetic animals. IDM animals demonstrated a significant reduction in myocardial angiostatin (1.8 ± 0.2-fold vs ND; P < .05 for comparison of IDM vs DM) and endostatin (2.3 ± 0.2-fold vs ND; P < .01 for comparison of IDM vs DM) expression. Similar patterns of expression of the anti-angiogenic proteins were observed in both the ischemic circumflex and the nonischemic territories of the left anterior descending coronary artery.

View larger version (19K): [in this window] [in a new window]   Figure 6. Myocardial expression of anti-angiogenic mediators. Expression of angiostatin (A) and endostatin (B) was significantly increased in the myocardium of diabetic animals and was significantly reduced in insulin-treated diabetic animals. (n = 4-6/group). *P < .01. **P < .05. ND, DM, and IDM as in Figure 1.

View larger version (52K): [in this window] [in a new window]   Figure 7. Myocardial expression of pro-angiogenic mediators. A, Expression of growth factors, VEGF and Ang-1, their respective receptors, VEGFR2 and Tie-2, as well as downstream mediator eNOS were assessed. B, Quantitative analysis showed that diabetic animals had reduced myocardial expression of VEGF and Ang-1, but VEGFR2 and Tie-2 expression was unchanged. Insulin treatment caused an increase in VEGF and Ang-1 expression as well as a significant increase in Tie-2 expression. Ang-1, Angiopoiten-1; VEGF, vascular endothelial growth factor; eNOS, endothelial nitric oxide synthase (n = 4-6/group). *P < .05 v. ND. **P < .05 vs DM; P < .001 vs ND. ND, DM, and IDM as in Figure 1.

	Discussion

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Glycemic control remains the mainstay of treatment in diabetes and has been shown to improve both macrovascular and microvascular complications of diabetes.^12,13 Insulin treatment, with or without oral hypoglycemic agents, is the most common method used clinically to achieve glycemic control. Insulin has multifaceted effects on the myocardium, which mainly involve the regulation of fuel consumption, glucose transport, glycogen synthesis, and glycolysis.¹⁴ More relevant to vascular function, however, is the demonstrated ability of insulin to increase endothelial nitric oxide availability in the vasculature.¹⁵ Furthermore, the insulin receptor, which is present in the myocardium, is a tyrosine kinase receptor that shares many of the downstream mediators common to angiogenic growth factors and their receptors, for example PI3 kinase and mitogen-activated protein kinases.¹⁵ In addition to its direct effects on the myocardium and coronary vasculature, insulin exerts indirect effects through the reduction in systemic blood glucose levels. By reducing blood glucose levels, insulin can avoid the adverse effects of chronic hyperglycemia, which include increased oxidative stress, chronic inflammation, and the nonenzymatic glycation of proteins, particularly in the extracellular matrix.^16,17 The pro-angiogenic effects of insulin demonstrated in this study may be due to a combination of the aforementioned processes.

We found that although endothelium-independent microvessel relaxation to sodium nitroprusside was preserved in all groups, diabetes was associated with impaired microvascular response to ADP and substance P, implying endothelial dysfunction and reduced nitric oxide bioavailability. Treatment with insulin led to a complete reversal in this endothelial dysfunction. However, the impairment in VEGF-induced microvessel relaxation, observed in diabetes, was only partially improved in insulin-treated animals, suggesting a persistent, residual impairment in VEGF signaling in the coronary microvasculature. Examination of pro-angiogenic proteins at the molecular level revealed that expression of VEGF, Ang-1, and its receptor, Tie-2, was markedly enhanced with insulin treatment.

Angiostatin and endostatin, cleavage products of plasminogen and collagen XVIII, respectively, have been shown to have potent anti-angiogenic effects in vitro and in rodent models.^18,19 Weihrauch and associates²⁰ have associated increased angiostatin with hyperglycemia and impaired angiogenesis in dogs. The association between angiostatin and diabetes and diminished coronary collateral formation has also been shown in patients with coronary disease.^21,22 We¹⁰ have previously shown that both angiostatin and endostatin are upregulated many fold in the setting of diabetes. In this study, we found that insulin treatment caused a significant reduction but not complete normalization in angiostatin and endostatin expression. The expression of these anti-angiogenic mediators mirrored the functional finding of significantly improved but not normalized perfusion of the ischemic circumflex territory. These observations provide further evidence for the critical role of these anti-angiogenic mediators, both as markers and as potential novel targets for the modulation of the angiogenic response in diabetes. Furthermore, the effects of insulin on matrix metalloproteinases, which are responsible for the production of these anti-angiogenic proteins, needs to be further evaluated in this setting.

	Model Strengths and Limitations

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Our model of alloxan-induced diabetes largely mimics type I diabetes, that is, hypoinsulinemic hyperglycemia. Although various pathophysiologic and molecular aspects of the disease are common between type I and type II diabetes,²³ including chronic hyperglycemia, endothelial dysfunction, increased oxidative stress and inflammation, accelerated atherosclerosis, and diminished angiogenesis, other features may be different and may limit the generalizability of this model to type II diabetes and insulin resistance. Last, whereas this model of chronic ischemia provides physiologically relevant measures of perfusion as well as morphologic assessment of angiogenesis, it is limited by the fact that molecular markers are only assessed at a single time point, 7 weeks after ameroid placement, and therefore, acute changes in pro-angiogenic and anti-angiogenic mediators may not be completely captured in this model.

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Model Strengths and Limitations Conclusions References

 
Insulin treatment, targeted to achieve glycemic control, reverses coronary endothelial dysfunction and significantly improves but does not normalize the endogenous myocardial angiogenic response to chronic ischemia in the setting of diabetes. These effects may be mediated by the direct effects of insulin on the myocardium and coronary vasculature or indirectly through systemic reductions in blood glucose. Anti-angiogenic proteins, angiostatin and endostatin, are downregulated with insulin treatment and appear to play an important role in the diabetic angiogenic response. Insulin therapy appears to be a promising strategy to improve the results of growth factor or cell-based angiogenic therapies in diabetic patients.

	Footnotes

 
¹ Dr Sellke was supported by grant R01 HL69024 from the National Institutes of Health and by grant support from Ikaria Pharmaceuticals.

² Dr Boodhwani was supported by a grant from the National Institutes of Health (HL04095-06) and the Irving Bard Memorial Fellowship.

³ Dr Ruel reports grant support from Bristol Myers Squibb.

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