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Ann Thorac Surg 2006;81:634-641
© 2006 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
b Division of Cardiology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Accepted for publication July 26, 2005.
* Address correspondence to Dr Sellke, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, 110 Francis St, Suite 2A, Boston, MA 02215 (Email: fsellke{at}caregroup.harvard.edu).
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Abstract |
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METHODS: Twelve Yucatan miniswine, fed either a normal (NORM, n = 6) or high cholesterol (HCHO, n = 6) diet for 13 weeks, underwent ameroid constrictor placement around the circumflex artery. Three weeks later, myocardial perfusion was quantified using isotope-labeled microspheres. Seven weeks after ameroid placement, coronary microvascular responses and myocardial perfusion were assessed. Vascular density was evaluated by PECAM-1 (CD-31) staining, and vascular endothelial growth factor, endothelial nitric oxide synthase, endostatin, and angiostatin protein levels were determined. Myocardial protein oxidation was quantified.
RESULTS: Coronary microvessels from HCHO pigs showed significant endothelial dysfunction. Baseline-adjusted myocardial flow at 7 weeks was significantly reduced in the HCHO animals (–0.002 ± 0.06 versus +0.23 ± 0.09 mL/min/g, HCHO versus NORM, p = 0.04). Endostatin expression was significantly increased in the HCHO pigs (2.2-fold, p = 0.001 versus NORM). There was a mild reduction in myocardial vascular endothelial growth factor expression (–29% ± 14%, p = 0.09) in HCHO animals, but no difference in expression of endothelial nitric oxide synthase and angiostatin. The HCHO animals demonstrated increased myocardial protein oxidation compared with the NORM group (+155% ± 21%, p = 0.03 versus NORM).
CONCLUSIONS: Ischemia-induced angiogenesis is inhibited in hypercholesterolemic pigs with a concomitant increase in endostatin expression and oxidative stress. These findings suggest that under conditions of hypercholesterolemia, coronary collateral development may be regulated by endogenous angiogenesis inhibitors such as endostatin as well as reactive oxygen species.
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Introduction |
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It has been shown that endothelium-derived nitric oxide (NO) production through the activation of tyrosine kinase receptors plays an important role in angiogenesis [3]. Reduction in bioavailable NO, either through decreased production or increased degradation, can therefore impair the angiogenic process. Although reactive oxygen species (ROS) are important in angiogenic signaling [4], excess ROS can rapidly combine with NO thereby decreasing its bioavailability [5]. This reduction in bioavailable NO has been observed in animal models as well as in patients with coronary artery disease with hypercholesterolemia, diabetes, hypertension, and other risk factors for endothelial dysfunction [6].
Studies in vivo and in vitro reveal a balance between angiogenic activators, such as VEGF or FGF, and inhibitors, such as endostatin and angiostatin, controlling endogenous angiogenic response [7]. Endostatin, first isolated from a murine hemangioendothelioma, has potent antiangiogenic activity by inhibiting proliferation and migration of endothelial cells in addition to inducing apoptosis [8]. Angiostatin, originally purified from the urine and serum of a mouse model of Lewis lung cell carcinoma [9], is also a potent antiangiogenic factor that induces apoptosis in endothelial cells and inhibits endothelial cell migration [10]. The relationship between these negative regulators of angiogenesis and hypercholesterolemia remains unclear.
To evaluate potential mechanisms of hypercholesterolemia induced impairment in angiogenesis, we studied the effects of diet-induced hypercholesterolemia and resulting endothelial dysfunction on the endogenous angiogenic response to chronic myocardial ischemia in Yucatan miniswine. The angiogenic response was assessed using isotope-labeled microspheres representing collateral-dependent perfusion of the ischemic territory, as well as by quantification of endothelial cell density in normocholesterolemic and hypercholesterolemic animals. In-vitro microvessel relaxation responses were used to evaluate endothelial function, and western blotting and immunohistochemistry were employed to evaluate the expression of proangiogenic and antiangiogenic mediators involved in the angiogenic process.
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Material and Methods |
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For all surgical procedures, anesthesia was induced with ketamine (10 mg/kg intramuscularly), thiopental (5 to 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 per minute. During the first procedure, the pericardium was opened through a left minithoracotomy, and microspheres (Biophysics Assay Laboratory, Worcester, Massachusetts) were injected into the left atrium during temporary occlusion of the LCx to determine the exact myocardial territory at risk. Next, a ferromagnetic ameroid constrictor (1.75 mm in internal diameter) was placed around the proximal left circumflex artery.
Three weeks after ameroid placement, the animals were anesthetized, and coronary angiography was performed through an 8F sheath (Cordis, Miami, Florida) introduced into the femoral artery using a catheter with the appropriate distal angulation. High atomic weight contrast was injected after selective cannulation of the right and left coronary ostia to confirm occlusion of the circumflex artery by the ameroid constrictor. Microspheres were injected in the left atrium to determine myocardial perfusion at rest and with atrial pacing (150 beats per minute). Seven weeks after ameroid placement, the animals were again anesthetized and the heart exposed through a sternotomy. Microspheres were then injected at rest and with pacing, and euthanasia was performed with injections of saturated potassium chloride solution. The heart was harvested, and two 1-cm-thick transversal slices were cut at the midventricular level, and then sectioned into eight segments identified clockwise starting from the anterior junction of the right and left ventricles. Samples from the anterior and left lateral walls were divided and rapidly frozen in liquid nitrogen (molecular studies), and kept in 4°C Krebs solution (microvessel reactivity studies) or fixed in 10% formalin (histologic studies). Separate samples were weighed and dried in a 60°C oven (microsphere perfusion analyses). Plasma cholesterol levels were from serum samples obtained at three time points (first surgery, second surgery, and harvesting) using an enzymatic method (AccelLAB, Montreal, Quebec, Canada).
In Vitro Assessment of Coronary Microvessel Reactivity
After cardiac harvest, epicardial coronary arterioles (80 to 180 µm in diameter and 1 to 2 mm in length) originating from branches of the left anterior descending (LAD) were dissected from the surrounding tissue with a x40 microscope and examined in isolated microvessel chambers as described previously [11]. The microvascular responses to sodium nitroprusside (SNP [1 nmol/L to 100 µmol/L], endothelium-independent cGMP-mediated vasodilator) and adenosine 5' diphosphate (ADP [1 nmol/L to 10 µmol/L], endothelium-dependent vasodilator) and VEGF (1 fM to 1 nM) were evaluated. Briefly, microvessels were cannulated with dual glass micropipettes and pressurized to 40 mm Hg using two burettes containing Krebs solution. Vessels were bathed in Krebs solution and preconstricted by 20% to 50% of the baseline diameter with the thromboxane A2 analog, U46619 (0.1 to 1 µM). All drugs were applied extraluminally. Relaxation responses were defined as the percent relaxation of the preconstricted diameter. Six vessels were examined in each group with uniform levels of preconstriction.
Myocardial Perfusion Analysis
Myocardial perfusion was determined during each procedure with isotope-labeled microspheres (ILMs [BioPAL, Worcester, Massachusetts]) by previously reported methods [12]. Briefly, gold-labeled microspheres were injected during temporary LCx occlusion at the time of ameroid placement to identify myocardial samples that originated from the LCx distribution (those with the lowest count of gold-labeled microspheres). Samarium and europium-labeled ILMs were used during the second procedure to determine baseline blood flow in the LCx territory 3 weeks after ameroid placement at rest and with pacing. Lutetium and lanthanum-labeled ILMs were injected at the final procedure, 7 weeks after ameroid placement. After euthanasia, 10 transmural left ventricular sections were collected for ILM assays in each animal, weighed, and dried. Each sample was exposed to neutron beams and microsphere densities 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 which showed the lowest count of gold-labeled microspheres by using the following equations:(1) crude blood flow (tissue sample) = [withdrawal rate (mL/min) / weight (tissue sample)] x [isotope counts (tissue sample) / isotope counts (reference blood sample)]; and (2) adjusted blood flow = crude blood flow (third surgery) – crude blood flow at baseline (second surgery).
Immunohistochemistry
Myocardial sections from the LCx territory of high cholesterol and normal diet were stained with anti–PECAM-1 (CD-31) antibody diluted to 1:600 (BD Biosciences Pharmingen, San Diego, California) as previously described [13]. 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 LCx territories of high cholesterol and normal diet animals. Cardiac endostatin was detected in paraffin sections stained with antiendostatin antibody diluted to 1:1000 (Upstate, Chicago, Illinois), followed by counterstaining with methyl green.
Western Blotting
Western blotting was performed as previously described [12]. Briefly, whole-cell lysates were isolated from the homogenized myocardial samples with a RIPA buffer (Boston Bioproducts, Worcester, Massachusetts) 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, Hercules, California). Forty micrograms of total protein were fractionated by 4% to 20% gradient, sodium dodecyl sulphate polyacrylamide gel electrophoresis (Invitrogen, San Diego, California) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, Massachusetts). Each membrane was incubated with specific antibodies as follows: anti-VEGF antibody diluted to 1:250 (Calbiochem, San Diego, California), antiendothelial nitric oxide synthase (eNOS) antibody diluted to 1:2500 (BD Biosciences, San Jose, California), antiendostatin antibody diluted to 1:1000 (Upstate, Chicago, Illinois), and antiangiostatin antibody diluted to 1:1000 (Calbiochem). Then the membranes were incubated for 1 hour in diluted appropriate secondary antibody (Jackson Immunolab, West Grove, Pennsylvania). Immune complexes were visualized with the enhanced chemiluminescence detection system (Amersham, Piscataway, New Jersey). Bands were quantified by densitometry of radioautograph films.
Detection of Protein Oxidation
Oxidized proteins were detected using a commercial kit (Oxyblot, Chemicon International, Temecula, California) employing methods suggested by the manufacturer. Tissue homogenates were first incubated with 2,4-dinitrophenylhydrazine and the carbonyl groups in the protein side chains were derivatized to 2,4-dinitrophenylhydrazone. The dinitrophenylhydrazone-derivatized protein samples were separated by 10% polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were incubated with primary antibody specific to the dinitrophenylhydrazone moiety of the proteins, followed by incubation with a horseradish peroxidase-linked secondary antibody. Immune complexes were visualized as described above.
Data Analysis
All results are expressed as mean ± SEM. Microvessel responses are expressed as percentage relaxation of the preconstricted diameter and were analyzed using two-way, repeated measures analysis of variance. Western blots were analyzed after digitalization of x-ray films using a flat-bed scanner (ScanJet 4c; Hewlett Packard, Palo Alto, California) and NIH ImageJ 1.33 software (National Institutes of Health, Bethesda, Maryland). Comparisons between samples were analyzed by unpaired, two-tailed t tests using GraphPad Prism 4 (GraphPad Software, San Diego, California). All p values less than 0.05 were considered significant.
Animal Care
Animals were cared for in compliance with the Harvard Medical Area Institutional Animal Care and Use Committee and in accordance with "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and "Guide for the Care and Use of Laboratory Animals" (NIH publication 5377-3, 1996).
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Results |
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Coronary Microvessel Reactivity
The diameter of coronary microvessels from the LAD territory was 145 ± 10 µm in the NORM group, and 135 ± 14 µm in the HCHO group. The percentage of contraction after the application of U46619 (1 x 10–6 – 3 x 10–6 M) was 39% ± 4% in the NORM group and 36% ± 3% in the HCHO group. Figure 1
shows the relaxation curves for increasing concentrations of vasodilators. Compared with the NORM group, animals in the HCHO group demonstrated impaired microvessel relaxation to endothelium-dependent vasorelaxants, ADP (p = 0.007), and endothelium-independent vasorelaxant, SNP (p = 0.02). The VEGF-mediated relaxation response was mildly impaired in HCHO animals but it was not statistically significant (p = 0.14).
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Comment |
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Best and colleagues [14] have demonstrated that high-cholesterol-diet–induced hypercholesterolemia impairs endothelium-dependent relaxation in pig coronary arteries. However, Shishido and associates [15] have reported that not only endothelial dysfunction but also smooth muscle cell dysfunction exists in chronically hypercholesterolemic Watanabe rabbit aortas. Similarly, in our study, we also found impaired vasorelaxation to both endothelial-dependent (ADP) and endothelium-independent (SNP) agents.
Reactive oxygen species can be generated from numerous sources within the cell including mitochondria, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase, xanthine oxidase, and eNOS uncoupling [6]. In the context of angiogenesis, they are released in response to angiogenic stimuli, for example, ischemia, and play an important role in angiogenic signaling [4]. However, ROS also rapidly combine with NO, forming peroxynitrite and can, therefore, reduce the amount of bioavailable NO [5]. Chronic, excessive production of ROS can also cause irreversible oxidation of cellular proteins leading sometimes to altered function [16]. The increased burden of oxidative stress in the HCHO group, as demonstrated by higher levels of oxidized proteins, combined with maintained levels of eNOS expression in both groups suggests that the reduced NO bioavailability and resulting endothelial dysfunction in these animals may be due to increased NO degradation, in the presence of ROS, rather than impaired NO synthesis.
The relationship between antiangiogenic factors, such as endostatin or angiostatin, and endothelial-dependent vasodilation was studied by Koshida and coworkers [17], who demonstrated that exogenous angiostatin treatment diminished endothelium-dependent vasodilation in rat heart arterioles in vitro. Although there are no data indicating a direct relationship between endostatin and endothelium-dependent vasodilation, several reports indicate that endostatin may inhibit VEGF signaling at different levels, including NOS activation [18], VEGF receptors [19], and extracellular signal regulated kinase activation [20].
That we did not observe any change in eNOS and angiostatin and observed only mild differences in VEGF should be interpreted with caution, as the protein expression was only evaluated 7 weeks after ameroid placement. It is possible that the levels of these angiogenic mediators were altered during the early hours or days after the induction of myocardial ischemia.
Endostatin was originally identified by O'Reilly and coworkers [8] from conditioned medium of a hemangio-endothelioma cell line as a highly active and endothelial specific angiogenic inhibitor. It is an endogenous 20kD protein that is a C-terminal fragment of collagen XVIII produced by proteolytic cleavage [8]. Endostatin can inhibit angiogenesis by blocking migration and proliferation of endothelial cells and by increasing apoptosis [21]. On the other hand, one of the proposed physiological effects of endostatin is antiatherosclerosis. In 1999, Moulton and colleagues [22] investigated endostatin as well as TNP-470, another substance known to inhibit the growth of capillaries, in apolipoprotein E-deficient mice fed a high cholesterol diet for 16 weeks. They found that endostatin significantly reduced intimal neovascularization and plaque growth. More recently, Moulton and colleagues [23] indicated that loss of collagen XVIII, the source of endostatin, enhanced neovascularization of aorta in the collagen XVIII knockout mouse. Hence, it is possible that endostatin is an important endogenous protective factor against atherosclerosis in hypercholesterolemia.
It has been shown that hypercholesterolemia inhibits angiogenesis induced by endogenous or exogenous growth factors in the heart as well as models of hindlimb ischemia [12, 24, 25]. It is generally accepted that endothelium-derived NO production, through the activation of tyrosine kinase receptors, plays an important role in angiogenesis [3], and that decreased NO bioavailability is critically linked to the diminished angiogenic response in hypercholesterolemia. This study demonstrates that reduced NO bioavailability, in the setting of hypercholesterolemia, is likely due to increased NO degradation, due to the presence of excessive ROS, rather than decreased NO production. It also suggests that the antiangiogenic protein, endostatin, may play an important role in regulating the angiogenic response in hypercholesterolemia. Whether a relationship between oxidative stress and endostatin expression exists is unclear and should be further evaluated. Future studies addressing the regulation of antiangiogenic factors in chronic diseases leading to coronary artery disease, for example, diabetes and hypertension, are also warranted.
The decreased endogenous myocardial angiogenic response observed in the presence of hypercholesterolemia may be due to increased oxidative stress as well as increased expression of endostatin. These data support the existing hypothesis that the balance between multiple proangiogenic and antiangiogenic factors ultimately affects the myocardial angiogenic process, particularly in the setting of hypercholesterolemic endothelial dysfunction.
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