© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Free radicals mediate endothelial dysfunction of coronary arterioles in diabetes
aVA Medical Center, Highway 6 West, Iowa City, IA 52246, USA
bDepartment of Internal Medicine and the Cardiovascular Center, University of Iowa College of Medicine, Iowa City, IA 52242, USA
* Corresponding author. Tel.: +1-319-339-7102; fax: +1-319-339-7135 kevin.dellsperger{at}med.va.gov
Received 8 December 1999; accepted 5 April 2000
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Abstract |
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Previous studies have demonstrated that vascular responses to acetylcholine (ACh) are impaired in diabetes mellitus (DM). Objective: Since reactive oxygen species (ROS) generation is increased in various disease states including DM, and a direct reaction between nitric oxide (NO) and superoxide anion has been demonstrated, we tested the hypothesis that inhibition of ROS will restore coronary microvascular responses to ACh in a dog model of DM (alloxan 60 mg/kg, i.v., 1 week prior to study). Methods: Changes in coronary microvascular diameters in diabetic (blood glucose >200 mg%) and normal animals to ACh (1–100 µM, topically) in the presence and absence of superoxide dismutase and catalase were measured using intravital microscopy coupled to stroboscopic epi-illumination and jet ventilation. Results: In diabetic animals in the absence of ROS scavengers, ACh induced coronary microvascular dilation was impaired when compared to normal animals (ACh 100 µM: DM=25±5%; normal=64±13%, P<0.05). Topical application of SOD (250 U/ml) and catalase (250 U/ml) restored to normal ACh induced coronary microvascular responses in DM while having no affect in normal animals. Responses to adenosine and nitroprusside were not different between normal and diabetic groups. Conclusions: These data provide direct evidence that oxygen-derived free radicals contribute to impaired endothelium-dependent coronary arteriolar dilation in diabetic dogs in vivo.
KEYWORDS Diabetes; Coronary circulation; Free radicals; Microcirculation; Acetylcholine; Endothelial function
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Diabetes mellitus (DM) is a potent risk factor for the development of coronary atherosclerosis. While much is known regarding the association of diabetes with epicardial conduit coronary function [1–4], relatively little is known regarding vasodilatory responses in coronary arterioles from subjects with diabetes. Diabetes has been shown to impair endothelium-dependent relaxation in rabbit aorta in vitro [5,6], and the cerebral circulation in vivo [7,8]. Tesfamariam and colleagues have shown that hyperglycemia impairs endothelium-dependent relaxation in large vessels in vitro [5,9]. Coronary microvessels <100 µm are critically responsible for the regulation of coronary blood flow [10–12]. Since prior investigations have shown that vasomotor regulation differs greatly between large (100–400 µm) and small (<100 µm) coronary microvessels, the relevance of comparing studies in large vessels to studies in the coronary microcirculation of diabetic animals is uncertain. Our group has shown that diabetes or chronic hyperglycemia is associated with impaired coronary microvascular responses to reduced perfusion pressure [13].
Other laboratories using in vitro methods have shown that administration of free radical scavengers restores endothelial function in rabbit aorta exposed to elevated glucose concentrations [9]. Elevated ambient glucose concentrations may result in glycosylation of native superoxide dismutase, leading to impaired function of the enzyme [14]. Inhibition of native superoxide dismutase with diethyldithiocarbamate results in localization of superoxide anion production in the endothelium and in the adventitia of rabbit aorta [15].
The goal of the present study is to determine whether topical administration of free radical scavengers can restore endothelium-dependent vasodilation in coronary microvessels in diabetic dogs in vivo. We tested whether superoxide dismutase and catalase administered topically would restore coronary arteriolar dilator responses to the endothelium-dependent agent acetylcholine in diabetic dogs. We also assessed the coronary arteriolar responses to topical acetylcholine in the presence and absence of superoxide dismutase and catalase in non-diabetic animals.
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2 Methods |
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The investigation conforms with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). These studies were approved by the University of Iowa and Department of Veterans Affairs animal care and use committees.
2.1 Model of diabetes
Adult mongrel dogs (n=22, body weight=6.9±0.5 kg) were injected with alloxan monohydrate (60 mg/kg, i.v.). Alloxan was prepared in 6% solution with citrate buffer (pH 4.5), as described by Engerman et al. [16] and as previously used in our laboratory [13]. Animals were allowed only water during the 24 h prior to alloxan injection and were fed immediately afterward. However, in three dogs, the animals were not fasted and they served as an alloxan injected non-diabetic control group. Hydration status, electrolytes, renal function, and glucose were closely monitored between injection and experiment days. All experiments involving diabetic animals were performed 1 week following alloxan injection. Animals were included in the diabetic group if their blood glucose was greater than 200 mg/dl at the mid-week measurement. Animals with volume depletion, electrolyte disturbances, or renal dysfunction were excluded from study (n=6 dogs).
2.2 General surgical preparation
On the day of the experiment, after fasting from the evening prior, the animals were sedated with ketamine (15 mg/kg, s.q.) and acepromazine (0.2 mg/kg, s.q.), and anesthetized with 
-chloralose (60 mg/kg, i.v.) and urethane (150 mg/kg, i.v.). Additional doses of -chloralose/urethane were given as needed to maintain surgical depth of anesthesia. Femoral arteries (PE-205) and one femoral vein (PE-150) were cannulated for hemodynamic monitoring, measurement of arterial blood gases, and for drug and fluid administration. Dogs were ventilated with a high frequency jet ventilator synchronized to the cardiac cycle, as previously described [17–19]. Respiratory pressure, inspiratory–expiratory ratio, end expiratory pressure, and FiO2 were adjusted to maintain physiological blood gases and pH at all times.
A left thoracotomy was performed, and the heart was suspended in a pericardial cradle. The left atrial appendage was cannulated (PE-150) for administration of fluorescein-labeled dextran. A 5 French catheter (Millar Instruments Houston, TX, USA) was placed in the left ventricle via the left atrial appendage for recording of left ventricular pressure and dP/dt. Snares were placed around the descending thoracic aorta and the inferior vena cava to control the aortic pressure. The epicardial surface was kept moist by suffusion of warmed Krebs solution [(mM); NaCl 118.3, KCl 4.7, CaCl2 2.5, MgPO4 1.2, NaHCO3 25, KH2PO4 1.2], bubbled with 20% O2, 5% CO2 and 75% N, at 2 ml/min. Body temperature was maintained (37±1°C) with a servocontrolled thermal blanket.
2.3 Microvascular preparation
Measurements of the coronary microvessels were obtained in the beating left ventricle using intravital microscopy (Zeiss, Germany) with epi-illumination of the cardiac surface by a computer controlled strobe (Chadwick-Helmuth, Almonte, CA, USA). Using the LV dP/dt signal, the strobe was triggered to flash once per cardiac cycle in late diastole. Fluorescein isothiocyanate dextran (molecular weight 487 000, Sigma, St. Louis, MO, USA) was injected into the left atrium to illuminate internal microvascular diameter and to differentiate arterioles from venules by sequence of illumination. A Zeiss Neofluora objective (6.3x, n.a.=0.02) was used; when coupled with a 6.3x relay lens, microvascular diameters can be measured with a resolution of 2.5 µm. Digital images were captured with a video camera and were stored on computer disk. The images were later recalled on a high resolution monitor; using a digitizing tablet and a computer, the microvascular diameters were measured in microns. Details of the system have been described previously [17–19]. All vessel measurements represent the mean of measurements from at least three image frames at each experimental condition.
2.4 Drugs
Acetylcholine (Sigma) was prepared in normal saline as a 1 mM stock solution. Acetylcholine was superfused on to the surface of the heart via a side port in the suffusion solution to achieve final concentrations of 1, 10 and 100 µM. Adenosine (Sigma) was prepared in normal saline as a stock solution 10 mM. Adenosine was superfused on to the surface of the heart via a side port in the suffusion solution to achieve final concentration of 10 and 100 µM, and 1 mM. Nitroprusside (Sigma) was prepared in normal saline as a 1 mM stock solution. Nitroprusside was superfused on to the surface of the heart via a side port in the suffusion solution to achieve final concentration of 1, 10 and 100 µM. Superoxide dismutase (Sigma) and catalase (ICN) were prepared in normal saline to achieve final concentrations on the cardiac surface of 250 units/ml for each.
2.5 Protocols
After the surgical preparation, at least 30 min was allowed for stabilization of monitored variables. The microvascular field of study was identified and coronary arterioles were verified by injection of fluorescein labeled dextran. Hemodynamics, microvascular diameters, and blood gases were measured at baseline. Sodium nitroprusside (100 µM) was administered topically to establish that the preparation had vascular tone. A 30-min washout period was allowed following this dose of sodium nitroprusside, and measurements were repeated to ensure that the preparation had returned to the baseline state. Diabetic and normal animals were studied according to the following protocols.
2.5.1 Protocol 1
In this protocol, the objective was to evaluate the effect of alloxan on vascular responses independent of diabetes. In three dogs injected with alloxan, but not rendered diabetic, following the general surgical preparation and a 30-min stabilization period, control measurements of blood gases, blood glucose, hemodynamics, and microvascular diameters (six vessels) were made. Following the dose of nitroprusside and subsequent washout period, control measurements were repeated. The dose–response relationship to acetylcholine (1, 10 and 100 µM) was studied.
2.5.2 Protocol 2
To evaluate the mechanism of endothelial dysfunction, microvascular responses to acetylcholine in normal (n=5 dogs, 11 vessels) and diabetic (n=6 dogs, 14 vessels) dogs were studied with and without superoxide dismutase and catalase. Following the general surgical preparation and a 30-min stabilization period, control measurements of blood gases, blood glucose, hemodynamics, and microvascular diameters were made. Following the dose of nitroprusside and subsequent washout period, control measurements were repeated. The animals received either superoxide dismutase (250 units/ml, topically) and catalase (250 units/ml, topically) or their vehicle (normal saline, topically), and measurements were repeated. Then, the dose–response study to acetylcholine (1, 10 and 100 µM) was carried out followed by a 30-min washout period. Baseline measurements were then repeated. Either vehicle or superoxide dismutase and catalase, whichever was not applied initially, was then administered. Measurements were repeated and a second acetylcholine concentration–response curve was constructed.
2.5.3 Protocol 3
Smooth muscle microvascular responses to adenosine and nitroprusside were studied in diabetic and normal dogs. Following the general surgical preparation and a 30-min stabilization period, control measurements of blood gases, blood glucose, hemodynamics, and microvascular diameters were made. The dose–response relationship to adenosine was studied in normal (n=3 dogs, six vessels) and diabetic (n=3 dogs, four vessels) dogs. In separate animals, a dose–response relationship to nitroprusside (1, 10 and 100 µM) was studied in normal (n=5 dogs, nine vessels) and diabetic (n=4 dogs, eight vessels) dogs.
Upon completion of the protocol, the animal was sacrificed with a bolus of saturated potassium chloride solution after a large dose of -chloralose.
2.6 Statistical analysis
All values are presented as mean±standard error of the mean. One-way analysis of variance with repeated measures was used to evaluate the changes in hemodynamic variables, blood gases and microvascular diameters.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Protocol 1: alloxan injected, non-diabetic dogs
In these animals blood glucose was not elevated (glucose at 1 week=99±14 mg/dl) when compared to baseline (glucose=85±10 mg/dl; P=NS). Mean baseline diameter of the coronary microvessels was 75±15 µm. Coronary microvascular responses to acetylcholine were not different from the normal responses without SOD and catalase in protocol 2 (acetylcholine, µM: 1: 18±5 µm; 10: 45±9 µm; 100: 51±8 µm; P=NS compared to normal dogs).
3.2 Protocol 2: baseline measurements
Blood glucose was elevated in all diabetic animals when compared to baseline [baseline glucose: 80±4 mg/dl; 3 days after injection (nonfasting): 333±37 mg/dl]. None of the following measurements were out of the normal range in the animals treated with alloxan: electrolytes, BUN, creatinine, pH, pCO2 and pO2.
Eleven vessels in five normal animals (mean body weight 6.2 kg) and fourteen vessels in six diabetic animals (mean body weight 5.9 kg) were studied.
3.3 Protocol 2: hemodynamics
There were no differences in baseline hemodynamics measured during the protocol. Mean arterial pressure was maintained constant throughout each experiment using aortic and inferior venal caval snares as necessary. Table 1 shows the hemodynamic data and arterial blood gas measurements from normal animals studied. There are no significant differences in the measured variables between the groups during the protocol. The normal animals had baseline blood glucose measurements of 88±12 mg/dl, which varied less than 5% throughout the protocol. Table 2 shows hemodynamic, blood glucose, and arterial blood gas measurements from diabetic animals studied. There are no significant differences between the groups in the protocol.
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3.4 Protocol 2: microvascular diameters
The coronary arteriolar dilation to 100 µM nitroprusside was similar between the normal and diabetic groups (normal: 64±13%, diabetic: 58±5%, P=NS). In the normal animals (baseline diameter=66±7 µm), topically applied acetylcholine resulted in a dose-dependent coronary arteriolar dilation, which was not altered by topical application of superoxide dismutase and catalase (Fig. 1). In the diabetic group of animals (baseline diameter=diabetic 68±7 µm), the acetylcholine responses were blunted during vehicle administration (Fig. 2). However, during topical application of superoxide dismutase and catalase, responses to acetylcholine were similar to those in normal animals (Fig. 2).
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3.5 Protocol 3: smooth muscle function
There were no differences in baseline hemodynamics or blood gases during the protocols (data not shown). In the diabetic group, glucose was elevated (347±30 mg/dl) at 1 week. Coronary microvascular responses to adenosine in both normal and diabetic animals are shown in Fig. 3A. Responses were not different between the two groups of animals (P=NS). Responses in coronary microvessels following nitroprusside application were not different between the normal and diabetic dogs (Fig. 3B, P=NS).
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4 Discussion |
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There are two principle findings of this study. First, we demonstrated in an in vivo beating heart that dilation of epicardial coronary arterioles to the endothelium-dependent vasodilator, acetylcholine, is impaired in diabetic animals while responses to adenosine and nitroprusside are intact. Second, topical application of superoxide dismutase and catalase restores acetylcholine vasodilatory responses in diabetic coronary arterioles to normal.
Several mechanisms of impaired endothelium-dependent dilation have been suggested in diabetes [20]. These include: (1) decreased production of NO; (2) impaired transit of NO from the site of production to its site of action on the vascular smooth muscle; (3) concomitant release of an endothelium-derived constricting factor; (4) impaired response in vascular smooth muscle to NO and (5) increased destruction of NO by oxygen-derived free radicals. Since dilation to endothelium-independent NO donor compounds, e.g. nitroprusside (Fig. 3B), have been shown to be normal [13], it is unlikely that vascular smooth muscle is unresponsive to NO. Because methods to directly measure NO concentration are not available in the microcirculation in vivo, it is difficult to determine whether NO production and/or transit are normal in diabetes. There is some evidence for release of an endothelium-derived constricting factor in diabetes. Increased production of vasoconstrictor prostanoids in diabetes have been described in rabbit aorta [5,21] and in isolated coronary artery rings [22]. Mayhan et al. have shown that dilation to endothelium-dependent agonists in cerebral microcirculation in diabetic rat can be restored with the endoperoxide receptor antagonist, SQ29548 [8]. Tesfamariam et al. described increased generation of endothelium-derived vasoconstrictor prostanoids in rabbit aortae exposed to elevated glucose [6]. They demonstrated that indomethacin or SQ29548 could restore impaired endothelium-dependent responses in vitro.
In this study, we provide evidence for reactive oxygen species destroying NO prior to its exerting its vasodilator effect. These studies were performed in vivo by direct visualization of coronary arterioles in the beating left ventricle. These data are consistent with studies performed in vitro by Tesfamariam et al. who showed that free radical scavengers restore endothelial function in diabetic rabbit aorta [9].
The source of free radical production is unclear. Several potential mechanisms have been suggested, including auto-oxidation of glucose [23], cyclooxygenase metabolism [24–28], mitochondrial oxidation [29], NAD(P)H oxidase [15], advanced glycosylation end products [30–32], and G protein activation [33]. Which reactive oxygen species is responsible for endothelial dysfunction is also unclear. Hydrogen peroxide, hydroxyl radical, and/or superoxide anion may be responsible for endothelial dysfunction in diabetes.
The cellular site of free radical production is yet to be determined. In this study superoxide dismutase and catalase applied topically restored endothelial function at the given doses in previous studies. With topical application of both superoxide dismutase and catalase, not only is superoxide anion scavenged, but a primary product of the dismutase reaction — hydrogen peroxide — is also destroyed. Since superoxide dismutase and catalase are highly charged molecules, it is unlikely that topical application of these agents would scavenge free radicals generated on the luminal side of the vascular basement membrane. Additionally, free radicals generated intracellularly would not likely be affected using these methods.
These studies may have important implications for the treatment of diabetic patients with coexisting coronary artery disease. Since previous studies in diabetic animals demonstrated impaired large vessel vasodilatory responses, and since our data show impaired microvascular responses to a putative endogenous vasodilator, acetylcholine, it is likely that there is less vasodilatory compensation for endogenous constrictor influences. Thus, the net effect would produce greater degrees of ischemia for the same degree of obstructive coronary disease.
In summary, epicardial coronary microvascular dilatation to acetylcholine is impaired in diabetic dogs. Topical application of superoxide dismutase and catalase can completely restore coronary arteriolar dilatory responses to acetylcholine in diabetic dogs in vivo. This provides direct evidence that oxygen-derived free radicals are at least in part responsible for impaired endothelium-dependent coronary arteriolar dilation in diabetic dogs in vivo.
Time for primary review 55 days.
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Acknowledgements |
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NIH NHLBI RO1 HL 51308, Heartland Affiliate Grant in Aid and the VA/Juvenile Diabetes Foundation Diabetes Research Center. Dr. Ammar was the recipient of an AHA, Iowa Affiliate, Post-doctoral Fellowship Award. Drs. Gutterman and Dellsperger are Established Investigators of the American Heart Association.
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