Type 2 Diabetic Mice Have Increased Arteriolar Tone and Blood Pressure
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动脉硬化血栓血管生物学 2005年第8期
From the Department of Physiology (Z.B., W.L., T.H.N., A.K., G.K.), New York Medical College, Valhalla, NY; the Department of Pathophysiology (A.K.), Semmelweis University, Budapest, Hungary; and the Division of Clinical Physiology Z.B., N.E., A.T.), Institute of Cardiology, University of Debrecen, Debrecen, Hungary.
Correspondence to Zsolt Bagi, MD, PhD, Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, 4004 Debrecen, PO Box 1, Hungary. E-mail bagizs@jaguar.unideb.hu
Abstract
Objective— Type 2 diabetes mellitus (T2-DM) is frequently associated with vascular dysfunction and elevated blood pressure, yet the underlying mechanisms are not completely understood. We hypothesized that in T2-DM, the regulation of peripheral vascular resistance is altered because of changes in local vasomotor mechanisms.
Methods and Results— In mice with T2-DM (C57BL/KsJ-db–/db–), systolic and mean arterial pressures measured by the tail cuff method were significantly elevated compared with those of control (db+/db–) animals (db/db, 146±5 and 106±2 mm Hg versus control, 133±4 and 98±4 mm Hg, respectively; P<0.05). Total peripheral resistance, calculated from cardiac output values (measured by echocardiography) and mean arterial pressure were significantly elevated in db/db mice (db/db, 25±6 versus control, 15±1 mm Hg[middot]mL–1[middot]min–1). In isolated, pressurized gracilis muscle arterioles (diameter 80 μm) from db/db mice, stepwise increases in intraluminal pressure (from 20 to 120 mm Hg) elicited a greater reduction in diameter than in control vessels at each pressure step (at 80 mm Hg, db/db, 66±4% versus control, 79±3%). The passive diameters of arterioles (obtained in Ca2+-free solution) and the calculated myogenic index were not significantly different in the 2 groups. The presence of the prostaglandin H2/thromboxane A2 receptor antagonist SQ29548 did not affect arteriolar diameters of control mice but reduced the enhanced arteriolar tone of db/db mice back to control levels (at 80 mm Hg, 80±4%). The inhibitor of cyclooxygenase-1 (COX-1), SC-560, did not affect the basal tone of arterioles, whereas NS-398, an inhibitor of COX-2, caused a significant shift in the arteriolar pressure–diameter curve of vessels from db/db mice (at 80 mm Hg, 76±3%) but not in those of control mice. Also, in aortas of db/db mice, expression of COX-2 was enhanced compared with controls.
Conclusions— Collectively, these findings suggest that in mice with T2-DM, the basal tone of skeletal muscle arterioles is increased because of an enhanced COX-2–dependent production of constrictor prostaglandins. These alterations in microvascular prostaglandin synthesis may contribute to the increase in peripheral resistance and blood pressure in T2-DM.
Here we report that mice with type 2 diabetes mellitus have elevated systolic blood pressures and increased peripheral vascular resistance. In type 2 diabetic mice, these alterations are associated with enhanced skeletal muscle arteriolar tone, which is likely attributable to increased release of COX-2–derived constrictor prostaglandins within the arteriolar wall.
Key Words: type 2 diabetes mellitus ? microvessels ? basal arteriolar tone ? cyclooxygenase-2
Introduction
Type 2 diabetes mellitus (T2-DM), which has reached epidemic proportions in Western countries, is associated with a markedly increased incidence of cardiovascular diseases, accounting for 70% of deaths in the diabetic population.1 However, the exact relations among T2-DM, obesity, and cardiovascular disease are not completely understood and have been the subject of some dispute. T2-DM is often part of an array of complex abnormalities referred to as the metabolic syndrome, which is frequently accompanied by elevated blood pressure.2
Several studies have demonstrated that vasomotor dysfunction of microvessels is an early manifestation of the vascular complications in T2-DM.3,4 Alterations in local vasoregulatory mechanisms intrinsic to the vascular wall, such as enhanced pressure-induced arteriolar tone5–7 and reduced endothelium-dependent dilation,6,8–11 have been reported previously as characteristic of T2-DM. Changes in the local vasoregulatory mechanisms of peripheral microvessels may significantly influence vascular resistance in T2-DM; however, the possible underlying mechanisms are still open to question.
Recently, a key role for low-grade vascular inflammation has received great attention in the development of diabetic vascular complications.12,13 Among other factors, prostaglandins (PGs) are important mediators of several inflammatory mechanisms14; however, it is also known that many PG derivatives have specific vasoactive properties, thereby contributing to the local regulation of arteriolar diameter.15,16 Early reports have already proposed a key role for altered vascular PG metabolism in diabetes-related changes in local vasoregulatory mechanisms.17 It was found that in mesenteric arteries of T1-DM dogs, exogenous arachidonic acid elicited thromboxane A2 (TxA2)–mediated constriction, whereas in control animals, it caused prostacyclin (PGI2)-dependent dilation.18 A recent study found that in aortas of T2-DM mice, phenylephrine-induced contraction was reduced and acetylcholine-induced relaxation was enhanced by the nonselective cyclooxygenase (COX) inhibitor indomethacin, indicating agonist-induced release of constrictor PGs.19 Although these studies suggested that the vascular synthesis of constrictor PGs is enhanced, the role of different COX isoforms and the functional consequences of altered PG synthesis affecting vascular resistance in T2-DM remain unclear.
It is known that COX-2 expression and activity are readily upregulated by inflammatory and physical stimuli.20 Recent biochemical studies have proposed a possible role for enhanced COX-2 expression in high glucose–induced alterations in constrictor prostanoid production in cultured endothelial cells.21 Also, it has been demonstrated that upregulation of COX isoforms is associated with a significant elevation of vascular PG synthesis.22 However, there are only a limited number of studies investigating alterations in vascular COX-2–dependent mechanisms in T2-DM, and little is known about the functional consequences of altered microvascular prostanoid synthesis.23 The aforementioned prompted us to investigate the cellular sources and the specific role of altered vascular prostanoid synthesis in the regulation of microvascular resistance in normal mice and mice with T2-DM.
Methods
We used 12- to 14-week-old, male db/db (C57BL/KsJ-db–/db–) and control heterozygous (C57BL/KsJ-db+/db–) mice in our experiments.11,24 Animals were fed standard chow and drank tap water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at New York Medical College.
Determination of Blood Pressure and Calculation of Total Peripheral Resistance
In conscious mice, systolic and diastolic blood pressures were measured by the tail-cuff method and mean arterial pressure was calculated. Total peripheral vascular resistance was also calculated from mean arterial pressure and cardiac output data, which were obtained by echocardiography in awake animals.25
Isolation of Gracilis Muscle Arterioles
Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Using microsurgery instruments and an operating microscope, we isolated a gracilis muscle arteriole (0.5 mm long) running intramuscularly and transferred it into an organ chamber containing 2 glass micropipettes filled with physiologic salt solution (PSS) composed of the following (in mmol/L): 110 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose, and 24.0 NaHCO3 equilibrated with a gas mixture of 10% O2 and 5% CO2, balanced with nitrogen, at pH 7.4. Vessels were cannulated on both ends, and micropipettes were connected with silicone tubing to an adjustable PSS reservoir. Inflow and outflow pressures were set to 80 mm Hg and continuously measured by a pressure servocontrol system (Living Systems Instrumentation). Temperature was set at 37°C by a controller. The internal arteriolar diameter at the midpoint of the arteriolar segment was measured by videomicroscopy with a microangiometer (Texas Instruments). Changes in arteriolar diameter and intraluminal pressure were continuously recorded with the Biopac-MP100 system connected to a computer and analyzed with AcqKnowledge data acquisition software (Biopac Systems, Inc).11,24
Pressure-Induced Arteriolar Response
After a 1-hour incubation period, spontaneous basal arteriolar tone developed in response to 80 mm Hg intraluminal pressure, without the use of any constrictor agent. Then, the change in diameter of arterioles was measured in response to stepwise increases in intraluminal pressure from 20 to 120 mm Hg. To obtain the passive arteriolar characteristics, pressure-induced arteriolar responses were reassessed in the presence of Ca2+-free PSS. Active arteriolar tone (in Ca2+-containing PSS) was expressed as a percentage of passive diameters (in Ca2+-free PSS). Myogenic index was also calculated, as described previously.26
Arteriolar Response to Arachidonic Acid
In separate experiments, at an intraluminal pressure of 80 mm Hg, arachidonic acid (10–9 to 10–7 mol/L, Cayman Chemicals) was applied to the superfusion solution. Each dose was incubated with the vessel for 10 minutes, and steady-state diameters were recorded. Acetylcholine (10–9 to 10–7 mol/L) and the NO donor sodium nitroprusside (SNP, 10–8 and 10–7 mol/L) were used to test the function of the endothelium and smooth muscle of arterioles. In separate experiments, the arteriolar endothelium was removed by air, and arachidonic acid–induced responses were obtained again. Endothelium denudation was ascertained by the loss of dilation to acetylcholine and the maintained dilation to the NO donor SNP.
Selective Inhibition of the PGH2/TxA2 Receptor and COX-1 and COX-2 Enzymes
After inhibition of PGH2/TxA2 receptors with SQ29548 (10–6 mol/L for 15 minutes, Cayman Chemicals), pressure- and arachidonic acid–induced arteriolar responses were obtained once more. For specific inhibition of COX isoenzymes, arterioles were incubated with the selective COX-1 inhibitor SC-560 (10–6 mol/L for 30 minutes, Cayman Chemicals) or with the selective COX-2 inhibitor NS-398 (10–5 mol/L for 30 minutes, Cayman Chemicals), and pressure- and arachidonic acid–induced arteriolar responses were reassessed.
Immunoblots
Mouse aortas were dissected from control and db/db mice, cleared of connective tissue, and briefly rinsed in ice-cold PSS. After the addition of 100 μL of sample buffer (from Sigma Inc), tissues were homogenized. Aliquots were separated by electrophoresis on a 10% polyacrylamide gel at 125 V for 1 hour and transferred onto a polyvinyl difluoride membrane. Immunoblot analysis was performed as described before.24 Antibodies used for detection (anti–COX-1 IgG, anti–COX-2 IgG) were obtained from Cayman Chemicals. Anti–?-actin IgG from Abcam Ltd was used for loading control. Signals were revealed with chemiluminescence and visualized autoradiographically. Optical density of bands was measured and quantified by Image J software.
Statistics
Data are expressed as mean±SEM. To obtain the passive diameter, arterioles were exposed to a Ca2+-free solution containing EGTA (10–3 mol/L) and 10–4 mol/L SNP. Statistical analyses were performed by a 2-way ANOVA for repeated measures, followed by the Tukey post hoc test or Student’s t test, as appropriate. P<0.05 was considered statistically significant.
Results
Previously, we had found that at 12 weeks of age, body weight, serum glucose, and serum insulin values of db/db mice were significantly elevated compared with age-matched wild-type animals, resembling data obtained from patients with obesity and T2-DM.11,24
Blood Pressure and Calculated Peripheral Resistance
Systolic and mean arterial pressures were significantly elevated in conscious db/db mice compared with wild-type mice (Table). Calculated peripheral vascular resistance (obtained from mean arteriolar pressure and cardiac output data) was significantly elevated in db/db mice compared with wild-type animals (Table).
Hemodynamic Parameters, Basal Tone, and Agonist-Induced Changes in Diameter of Isolated Skeletal muscle Arterioles of Mice
Pressure-Induced Arteriolar Response
After a 1-hour incubation period, spontaneous myogenic tone developed in isolated skeletal muscle arterioles without the use of any vasoactive agent. At 80 mm Hg, the diameter of arterioles of db/db mice was significantly reduced compared with that of arterioles from wild-type mice (Table). There were no significant differences between passive arteriolar diameters in the 2 groups of animals obtained in Ca2+-free PSS at 80 mm Hg (Table). Stepwise increases in intraluminal pressure from 20 to 120 mm Hg elicited significantly greater reductions in the diameter of arterioles from db/db mice compared with control vessels at each pressure step (Figure 1A and 1B). The passive pressure-diameter curves of arterioles (obtained in Ca2+-free solution) were not different in the 2 groups of animals (Figure 1A). The calculated myogenic index was also not significantly different in arterioles isolated from control and db/db mice (Figure 1C).
Figure 1. Diameters of skeletal muscle arterioles isolated from control (n=15) and db/db (n=15) mice in response to stepwise increases (20 to 120 mm Hg) in intraluminal pressure in the presence or absence of extracellular Ca2+ (A). Normalized arteriolar diameters of control (n=15) and db/db (n=15) mice are expressed as percentages of the passive diameter (B). Calculated myogenic index values of arterioles from control and db/db mice developed in response to stepwise increases in intraluminal pressure (C). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Role of COX Isoforms in Pressure-Induced Arteriolar Tone
To elucidate the role of PGs in pressure-induced arteriolar tone development in control and db/db mice, selective inhibitors of PG receptors and COX enzymes were used. The presence of the PGH2/TxA2 receptor antagonist SQ29548 did not affect the pressure-induced responses of arterioles of control mice, but it reduced the tone of arterioles of db/db mice back to control levels (Figure 2A and 2B). The presence of the selective inhibitor of cyclooxygenase-1 (COX-1), SC-560, did not affect the basal tone of arterioles in either control or db/db mice (Figure 2C and 2D). On the other hand, the presence of NS-398, a selective inhibitor of COX-2, caused a significant upward shift in the arteriolar pressure-diameter curve of vessels from db/db mice, but did not significantly affect that of arterioles isolated from control animals (Figure 2C and 2D).
Figure 2. Normalized arteriolar diameters of control (n=9) and db/db (n=9) mice in the absence or presence of SQ 29,548, a PGH2/TXA2 receptor antagonist (A and B); NS-398, a selective inhibitor of COX-2; and SC-560, a selective inhibitor of COX 1 (C and D). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Role of COX Isoforms in Arachidonic Acid–Induced Arteriolar Response
In the next series of experiments, we obtained arteriolar responses to exogenously administered arachidonic acid, the precursor of PGs, in the absence and presence of the specific inhibitors. In arterioles of control mice, arachidonic acid in a concentration-dependent manner elicited dilation; however, it caused significant constriction in arterioles isolated from db/db mice (Figure 3A). The presence of the PGH2/TxA2 receptor antagonist SQ29548 did not affect the arachidonic acid–induced dilation of arterioles of control mice but reduced the arachidonic acid–induced constriction in arterioles of db/db mice (Figure 3A). Also, we found that removal of the endothelium reduced arachidonic acid–induced arteriolar responses in control vessels but did not affect responses significantly in arterioles of db/db mice (Figure 3B). In control mice, arachidonic acid–induced dilations were significantly reduced by the selective inhibitor of COX-1, SC-560, but not by NS-398, a selective inhibitor of COX-2 (Figure 4A). In contrast, in arterioles of db/db mice, the arachidonic acid–induced constriction was significantly reduced by the COX-2 inhibitor NS-398, whereas the COX-1 inhibitor SC-560 had no effect (Figure 4B).
Figure 3. Arachidonic acid-induced changes in arteriolar diameters from control (n=7) and db/db (n=7) mice in the absence or presence of SQ 29,548, a PGH2/TXA2 receptor antagonist (A), and after endothelium removal (B, n=5). Data are mean±SEM. *Significant difference from control; #Significant difference from db/db arterioles (P<0.05). Abbreviations are as defined in text.
Figure 4. Arachidonic acid–induced changes in arteriolar diameters from control (n=7) and db/db (n=7) mice in the absence or presence of NS-398, a selective inhibitor of COX-2, and SC-560, a selective inhibitor of COX 1 (A and B). Data are mean±SEM. *Significant difference from control (P<0.05). Abbreviations are as defined in text.
Immunoblots
Western blot analysis was performed on aortas from both control and db/db mice. There were no significant differences in total COX-1 protein levels in the 2 groups, whereas COX-2 expression was significantly greater in aortas from db/db mice (Figure 5).
Figure 5. Western blot analysis of the expression of COX-1 (A and B) and COX-2 (C and D) in aortas from control and db/db mice. Anti–?-actin was used to normalize for loading variations. Bar graphs represent the summary of normalized densitometric ratios (n=5 for each group). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Discussion
The main findings of the present study are that mice with T2-DM have elevated systolic blood pressures and increased peripheral vascular resistance. In T2-DM mice, these alterations are associated with enhanced skeletal muscle arteriolar tone, which is likely to be attributable to increased release of COX-2–derived constrictor PGs within the skeletal muscle arteriolar wall.
T2-DM is frequently associated with elevated systemic blood pressure; however, the nature of the mechanisms have not yet been fully elucidated. A key role for altered regulation of microvascular resistance has been suggested by several earlier investigations that found specific impairment of microvascular vasoregulatory mechanisms in subjects with T2-DM.4 Accordingly, a reduced endothelium-dependent arteriolar vasodilation6,9–11 and/or an enhanced smooth muscle–dependent vasoconstriction5–7 in microvessels has been demonstrated in T2-DM, alterations that could influence total peripheral resistance.
In the present study, hemodynamic parameters were obtained first in awake mice. Using a tail-cuff method, we found a significant rise in systolic and mean arterial blood pressures in db/db mice compared with control animals (Table). Furthermore, by using echocardiography, a decreased cardiac output was also observed in db/db mice (Table). On the basis of these parameters, the total peripheral resistance was calculated and found to be significantly elevated in db/db mice compared with that of control animals (Table). This study is the first to demonstrate that at 12 weeks of age, in association with obesity, hyperglycemia, and hyperinsulinemia, db/db mice exhibit an enhanced peripheral vascular resistance and elevated systemic blood pressure. These results are in accordance with previous clinical observations of the prevalence of high blood pressure in patients with obesity and T2-DM.2,27
In the next series of experiments, we aimed to elucidate the possible underlying mechanisms responsible for the enhanced peripheral vascular resistance. It is well known that arterioles respond to an increase in transmural pressure by constriction, a response termed myogenic constriction, which plays a key role in the local regulation of tissue blood flow.28,29 Earlier it had been found that an elevation of systemic blood pressure is associated with a rise in resistance of the skin microcirculation in T2-DM patients.30 It has been proposed that enhanced arteriolar tone could protect the distal part of the microcirculation from the increased intraluminal pressure.31 Indeed, previous studies have demonstrated an enhanced tone in skeletal muscle5 and mesenteric6 arterioles of T2-DM animals. An enhanced arteriolar tone, especially in the skeletal muscle circulation, may further increase vascular resistance, which, if uncompensated, could ultimately result in the elevation of systemic blood pressure.
In the present study, we demonstrated that in response to stepwise increases in intraluminal pressure (from 20 to 120 mm Hg), the diameter of isolated skeletal muscle arterioles was significantly reduced in db/db compared with control mice, whereas the passive diameter (obtained in Ca2+-free solution) of arterioles was not significantly different between the 2 groups (Figure 1). These findings indicate that active tone is greater in the arterioles of db/db mice, which, however, is unlikely to be the result of an altered arteriolar compliance. Next, myogenic indexes28,29 were calculated, allowing us to assess the pressure-sensitive behavior of skeletal muscle arterioles. We found that myogenic indexes were not significantly different in the 2 groups, suggesting that mechanisms other than pressure-sensitive myogenic regulation are responsible for the reduction in the basal diameter of arterioles from db/db mice.
Arteriolar diameter is continuously modulated by dilator and constrictor factors, many of them intrinsic to the vascular wall. Early investigations reported enhanced release of a constrictor prostanoid from diabetic vessels.18 On the basis of previous observations, we hypothesized that vascular production of constrictor PGs is increased in T2-DM, thereby contributing to the reduced diameter of skeletal muscle arterioles of db/db mice. Indeed, we found that a PGH2/TxA2 receptor antagonist increased the diameter of arterioles of T2-DM mice back to control levels, whereas it did not affect the diameter of vessels from control animals (Figure 2A and 2B). These findings indicate that endogenous release of constrictor PGs, PGH2/TxA2, may be responsible for the reduced diameter of arterioles from T2-DM mice.
Recently, a role for prostanoid-mediated vascular inflammation has been shown to be associated with the development of vascular complications in T2-DM.13 Prostanoids generated by COXs from arachidonic acid20 are important mediators of several inflammatory mechanisms. Two isoforms of the COX enzyme, encoded by distinct genes, have been isolated in mammalian cells.20 COX-1 is constitutively expressed in most tissues, such as vascular endothelial cells, and is involved in the maintenance of cellular homeostasis.23 In contrast, under normal conditions, COX-2 is expressed only at low or undetectable levels but is readily upregulated by inflammatory, mitogenic, and physical stimuli.32 Only a limited number of biochemical studies have investigated alterations in COX-2–dependent mechanisms related to DM. In this context, it has been found that high-glucose treatment caused increases in expression of COX-2 protein in mesangial cells.33 Also, in cultured human endothelial cells, high-glucose treatment elicited an enhanced production of TxA2 in association with an upregulation of COX-2.21 Because in the db/db mouse model of T2-DM the level of vascular COX-2 expression is not known, we aimed to measure COX-2 expression in intact vessels from control and db/db mice. COX-2 protein levels in the aortas of T2-DM mice were markedly increased compared with those of control animals (Figure 5). Although COX-2 expression has been found to be associated with enhanced production of constrictor prostanoids under normal34 and certain pathologic22,35 conditions, the specific role of COX-1– and COX-2–dependent prostanoid synthesis in the mediation of arteriolar diameter changes in T2-DM has not yet been elucidated.
In control arterioles, selective inhibition of COX-1 did not affect the diameter of arterioles (Figure 2C), whereas it reduced arachidonic acid–induced dilation (Figure 4A). Because removal of the endothelium also reduced arachidonic acid–induced arteriolar responses in control vessels (Figure 3B), we concluded that dilator PGs, most likely endothelium-derived prostacyclin, were produced by the metabolism of arachidonic acid. In contrast, in arterioles of T2-DM mice, both basal tone (Figure 2D) and arachidonic acid–induced constrictions were reduced by the selective inhibitor of COX-2, but not that of COX-1, or endothelium removal (Figures 3B and 4B). We interpret these findings to mean that in arterioles of db/db mice, COX-2–dependent release of constrictor PGs, most likely PGH2/TxA2, derived primarily from vascular smooth muscle cells, mediate both the enhanced pressure- and arachidonic acid–induced reductions of arteriolar diameter.
Recently, an important role for reactive oxygen species in the regulation of arteriolar tone has received a great deal of attention.36 In T2-DM rats, reactive oxygen species have been proposed to play a role in myogenic activation of skeletal muscle arterioles.5 In this context, we previously found that in T2-DM (db/db) mice, owing to the reduced activity of vascular superoxide dismutase and catalase together with enhanced activation of vascular NAD(P)H oxidase, vascular production of superoxide was increased.24 Thus, one can speculate that vascular oxidative stress in T2-DM or other disease conditions37 may also be associated with alteration in COX-2–dependent synthesis of PGs. Indeed, recently it has been found that in high glucose–treated mesangial cells, mitochondrial superoxide production was associated with enhanced COX-2 expression.33 Also, in cultured human endothelial cells, high glucose elicited enhanced production of reactive oxygen species, resulting in increased production of TxA2, which was also associated with an upregulation of COX-2.21 However, in T2-DM, the interrelation between vascular oxidative stress and altered prostanoid metabolism needs to be addressed in future investigations.
Taken together, we propose an important role for COX-2–derived constrictor PGs in the altered regulation of skeletal muscle arteriolar resistance in T2-DM, obese mice. It still remains a question, however, whether and to what extent changes in arteriolar PG synthesis could contribute to the alterations of total peripheral resistance and blood pressure in T2-DM. Nevertheless, based on the present studies, alterations in COX-2–dependent and PG-mediated modulation of vasomotor function should be taken into consideration in future investigations of T2-DM.
Acknowledgments
This study was supported by a grant from the American Heart Association, Northeast Affiliate (0555897T), and National Institutes of Health grants HL-43023, HL-46813, OTKA T-034779, 048376, F-048837, and 711-K-84289.
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Belton OA, Duffy A, Toomey S, Fitzgerald DJ. Cyclooxygenase isoforms and platelet vessel wall interactions in the apolipoprotein E knockout mouse model of atherosclerosis. Circulation. 2003; 108: 3017–3023.
Cseko C, Bagi Z, Koller A. Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways. J Appl Physiol. 2004; 97: 1130–1137.
Wang D, Chabrashvili T, Wilcox CS. Enhanced contractility of renal afferent arterioles from angiotensin-infused rabbits: roles of oxidative stress, thromboxane prostanoid receptors, and endothelium. Circ Res. 2004; 94: 1436–1442.(Zsolt Bagi; Nora Erdei; A)
Correspondence to Zsolt Bagi, MD, PhD, Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, 4004 Debrecen, PO Box 1, Hungary. E-mail bagizs@jaguar.unideb.hu
Abstract
Objective— Type 2 diabetes mellitus (T2-DM) is frequently associated with vascular dysfunction and elevated blood pressure, yet the underlying mechanisms are not completely understood. We hypothesized that in T2-DM, the regulation of peripheral vascular resistance is altered because of changes in local vasomotor mechanisms.
Methods and Results— In mice with T2-DM (C57BL/KsJ-db–/db–), systolic and mean arterial pressures measured by the tail cuff method were significantly elevated compared with those of control (db+/db–) animals (db/db, 146±5 and 106±2 mm Hg versus control, 133±4 and 98±4 mm Hg, respectively; P<0.05). Total peripheral resistance, calculated from cardiac output values (measured by echocardiography) and mean arterial pressure were significantly elevated in db/db mice (db/db, 25±6 versus control, 15±1 mm Hg[middot]mL–1[middot]min–1). In isolated, pressurized gracilis muscle arterioles (diameter 80 μm) from db/db mice, stepwise increases in intraluminal pressure (from 20 to 120 mm Hg) elicited a greater reduction in diameter than in control vessels at each pressure step (at 80 mm Hg, db/db, 66±4% versus control, 79±3%). The passive diameters of arterioles (obtained in Ca2+-free solution) and the calculated myogenic index were not significantly different in the 2 groups. The presence of the prostaglandin H2/thromboxane A2 receptor antagonist SQ29548 did not affect arteriolar diameters of control mice but reduced the enhanced arteriolar tone of db/db mice back to control levels (at 80 mm Hg, 80±4%). The inhibitor of cyclooxygenase-1 (COX-1), SC-560, did not affect the basal tone of arterioles, whereas NS-398, an inhibitor of COX-2, caused a significant shift in the arteriolar pressure–diameter curve of vessels from db/db mice (at 80 mm Hg, 76±3%) but not in those of control mice. Also, in aortas of db/db mice, expression of COX-2 was enhanced compared with controls.
Conclusions— Collectively, these findings suggest that in mice with T2-DM, the basal tone of skeletal muscle arterioles is increased because of an enhanced COX-2–dependent production of constrictor prostaglandins. These alterations in microvascular prostaglandin synthesis may contribute to the increase in peripheral resistance and blood pressure in T2-DM.
Here we report that mice with type 2 diabetes mellitus have elevated systolic blood pressures and increased peripheral vascular resistance. In type 2 diabetic mice, these alterations are associated with enhanced skeletal muscle arteriolar tone, which is likely attributable to increased release of COX-2–derived constrictor prostaglandins within the arteriolar wall.
Key Words: type 2 diabetes mellitus ? microvessels ? basal arteriolar tone ? cyclooxygenase-2
Introduction
Type 2 diabetes mellitus (T2-DM), which has reached epidemic proportions in Western countries, is associated with a markedly increased incidence of cardiovascular diseases, accounting for 70% of deaths in the diabetic population.1 However, the exact relations among T2-DM, obesity, and cardiovascular disease are not completely understood and have been the subject of some dispute. T2-DM is often part of an array of complex abnormalities referred to as the metabolic syndrome, which is frequently accompanied by elevated blood pressure.2
Several studies have demonstrated that vasomotor dysfunction of microvessels is an early manifestation of the vascular complications in T2-DM.3,4 Alterations in local vasoregulatory mechanisms intrinsic to the vascular wall, such as enhanced pressure-induced arteriolar tone5–7 and reduced endothelium-dependent dilation,6,8–11 have been reported previously as characteristic of T2-DM. Changes in the local vasoregulatory mechanisms of peripheral microvessels may significantly influence vascular resistance in T2-DM; however, the possible underlying mechanisms are still open to question.
Recently, a key role for low-grade vascular inflammation has received great attention in the development of diabetic vascular complications.12,13 Among other factors, prostaglandins (PGs) are important mediators of several inflammatory mechanisms14; however, it is also known that many PG derivatives have specific vasoactive properties, thereby contributing to the local regulation of arteriolar diameter.15,16 Early reports have already proposed a key role for altered vascular PG metabolism in diabetes-related changes in local vasoregulatory mechanisms.17 It was found that in mesenteric arteries of T1-DM dogs, exogenous arachidonic acid elicited thromboxane A2 (TxA2)–mediated constriction, whereas in control animals, it caused prostacyclin (PGI2)-dependent dilation.18 A recent study found that in aortas of T2-DM mice, phenylephrine-induced contraction was reduced and acetylcholine-induced relaxation was enhanced by the nonselective cyclooxygenase (COX) inhibitor indomethacin, indicating agonist-induced release of constrictor PGs.19 Although these studies suggested that the vascular synthesis of constrictor PGs is enhanced, the role of different COX isoforms and the functional consequences of altered PG synthesis affecting vascular resistance in T2-DM remain unclear.
It is known that COX-2 expression and activity are readily upregulated by inflammatory and physical stimuli.20 Recent biochemical studies have proposed a possible role for enhanced COX-2 expression in high glucose–induced alterations in constrictor prostanoid production in cultured endothelial cells.21 Also, it has been demonstrated that upregulation of COX isoforms is associated with a significant elevation of vascular PG synthesis.22 However, there are only a limited number of studies investigating alterations in vascular COX-2–dependent mechanisms in T2-DM, and little is known about the functional consequences of altered microvascular prostanoid synthesis.23 The aforementioned prompted us to investigate the cellular sources and the specific role of altered vascular prostanoid synthesis in the regulation of microvascular resistance in normal mice and mice with T2-DM.
Methods
We used 12- to 14-week-old, male db/db (C57BL/KsJ-db–/db–) and control heterozygous (C57BL/KsJ-db+/db–) mice in our experiments.11,24 Animals were fed standard chow and drank tap water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at New York Medical College.
Determination of Blood Pressure and Calculation of Total Peripheral Resistance
In conscious mice, systolic and diastolic blood pressures were measured by the tail-cuff method and mean arterial pressure was calculated. Total peripheral vascular resistance was also calculated from mean arterial pressure and cardiac output data, which were obtained by echocardiography in awake animals.25
Isolation of Gracilis Muscle Arterioles
Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Using microsurgery instruments and an operating microscope, we isolated a gracilis muscle arteriole (0.5 mm long) running intramuscularly and transferred it into an organ chamber containing 2 glass micropipettes filled with physiologic salt solution (PSS) composed of the following (in mmol/L): 110 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 5.0 glucose, and 24.0 NaHCO3 equilibrated with a gas mixture of 10% O2 and 5% CO2, balanced with nitrogen, at pH 7.4. Vessels were cannulated on both ends, and micropipettes were connected with silicone tubing to an adjustable PSS reservoir. Inflow and outflow pressures were set to 80 mm Hg and continuously measured by a pressure servocontrol system (Living Systems Instrumentation). Temperature was set at 37°C by a controller. The internal arteriolar diameter at the midpoint of the arteriolar segment was measured by videomicroscopy with a microangiometer (Texas Instruments). Changes in arteriolar diameter and intraluminal pressure were continuously recorded with the Biopac-MP100 system connected to a computer and analyzed with AcqKnowledge data acquisition software (Biopac Systems, Inc).11,24
Pressure-Induced Arteriolar Response
After a 1-hour incubation period, spontaneous basal arteriolar tone developed in response to 80 mm Hg intraluminal pressure, without the use of any constrictor agent. Then, the change in diameter of arterioles was measured in response to stepwise increases in intraluminal pressure from 20 to 120 mm Hg. To obtain the passive arteriolar characteristics, pressure-induced arteriolar responses were reassessed in the presence of Ca2+-free PSS. Active arteriolar tone (in Ca2+-containing PSS) was expressed as a percentage of passive diameters (in Ca2+-free PSS). Myogenic index was also calculated, as described previously.26
Arteriolar Response to Arachidonic Acid
In separate experiments, at an intraluminal pressure of 80 mm Hg, arachidonic acid (10–9 to 10–7 mol/L, Cayman Chemicals) was applied to the superfusion solution. Each dose was incubated with the vessel for 10 minutes, and steady-state diameters were recorded. Acetylcholine (10–9 to 10–7 mol/L) and the NO donor sodium nitroprusside (SNP, 10–8 and 10–7 mol/L) were used to test the function of the endothelium and smooth muscle of arterioles. In separate experiments, the arteriolar endothelium was removed by air, and arachidonic acid–induced responses were obtained again. Endothelium denudation was ascertained by the loss of dilation to acetylcholine and the maintained dilation to the NO donor SNP.
Selective Inhibition of the PGH2/TxA2 Receptor and COX-1 and COX-2 Enzymes
After inhibition of PGH2/TxA2 receptors with SQ29548 (10–6 mol/L for 15 minutes, Cayman Chemicals), pressure- and arachidonic acid–induced arteriolar responses were obtained once more. For specific inhibition of COX isoenzymes, arterioles were incubated with the selective COX-1 inhibitor SC-560 (10–6 mol/L for 30 minutes, Cayman Chemicals) or with the selective COX-2 inhibitor NS-398 (10–5 mol/L for 30 minutes, Cayman Chemicals), and pressure- and arachidonic acid–induced arteriolar responses were reassessed.
Immunoblots
Mouse aortas were dissected from control and db/db mice, cleared of connective tissue, and briefly rinsed in ice-cold PSS. After the addition of 100 μL of sample buffer (from Sigma Inc), tissues were homogenized. Aliquots were separated by electrophoresis on a 10% polyacrylamide gel at 125 V for 1 hour and transferred onto a polyvinyl difluoride membrane. Immunoblot analysis was performed as described before.24 Antibodies used for detection (anti–COX-1 IgG, anti–COX-2 IgG) were obtained from Cayman Chemicals. Anti–?-actin IgG from Abcam Ltd was used for loading control. Signals were revealed with chemiluminescence and visualized autoradiographically. Optical density of bands was measured and quantified by Image J software.
Statistics
Data are expressed as mean±SEM. To obtain the passive diameter, arterioles were exposed to a Ca2+-free solution containing EGTA (10–3 mol/L) and 10–4 mol/L SNP. Statistical analyses were performed by a 2-way ANOVA for repeated measures, followed by the Tukey post hoc test or Student’s t test, as appropriate. P<0.05 was considered statistically significant.
Results
Previously, we had found that at 12 weeks of age, body weight, serum glucose, and serum insulin values of db/db mice were significantly elevated compared with age-matched wild-type animals, resembling data obtained from patients with obesity and T2-DM.11,24
Blood Pressure and Calculated Peripheral Resistance
Systolic and mean arterial pressures were significantly elevated in conscious db/db mice compared with wild-type mice (Table). Calculated peripheral vascular resistance (obtained from mean arteriolar pressure and cardiac output data) was significantly elevated in db/db mice compared with wild-type animals (Table).
Hemodynamic Parameters, Basal Tone, and Agonist-Induced Changes in Diameter of Isolated Skeletal muscle Arterioles of Mice
Pressure-Induced Arteriolar Response
After a 1-hour incubation period, spontaneous myogenic tone developed in isolated skeletal muscle arterioles without the use of any vasoactive agent. At 80 mm Hg, the diameter of arterioles of db/db mice was significantly reduced compared with that of arterioles from wild-type mice (Table). There were no significant differences between passive arteriolar diameters in the 2 groups of animals obtained in Ca2+-free PSS at 80 mm Hg (Table). Stepwise increases in intraluminal pressure from 20 to 120 mm Hg elicited significantly greater reductions in the diameter of arterioles from db/db mice compared with control vessels at each pressure step (Figure 1A and 1B). The passive pressure-diameter curves of arterioles (obtained in Ca2+-free solution) were not different in the 2 groups of animals (Figure 1A). The calculated myogenic index was also not significantly different in arterioles isolated from control and db/db mice (Figure 1C).
Figure 1. Diameters of skeletal muscle arterioles isolated from control (n=15) and db/db (n=15) mice in response to stepwise increases (20 to 120 mm Hg) in intraluminal pressure in the presence or absence of extracellular Ca2+ (A). Normalized arteriolar diameters of control (n=15) and db/db (n=15) mice are expressed as percentages of the passive diameter (B). Calculated myogenic index values of arterioles from control and db/db mice developed in response to stepwise increases in intraluminal pressure (C). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Role of COX Isoforms in Pressure-Induced Arteriolar Tone
To elucidate the role of PGs in pressure-induced arteriolar tone development in control and db/db mice, selective inhibitors of PG receptors and COX enzymes were used. The presence of the PGH2/TxA2 receptor antagonist SQ29548 did not affect the pressure-induced responses of arterioles of control mice, but it reduced the tone of arterioles of db/db mice back to control levels (Figure 2A and 2B). The presence of the selective inhibitor of cyclooxygenase-1 (COX-1), SC-560, did not affect the basal tone of arterioles in either control or db/db mice (Figure 2C and 2D). On the other hand, the presence of NS-398, a selective inhibitor of COX-2, caused a significant upward shift in the arteriolar pressure-diameter curve of vessels from db/db mice, but did not significantly affect that of arterioles isolated from control animals (Figure 2C and 2D).
Figure 2. Normalized arteriolar diameters of control (n=9) and db/db (n=9) mice in the absence or presence of SQ 29,548, a PGH2/TXA2 receptor antagonist (A and B); NS-398, a selective inhibitor of COX-2; and SC-560, a selective inhibitor of COX 1 (C and D). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Role of COX Isoforms in Arachidonic Acid–Induced Arteriolar Response
In the next series of experiments, we obtained arteriolar responses to exogenously administered arachidonic acid, the precursor of PGs, in the absence and presence of the specific inhibitors. In arterioles of control mice, arachidonic acid in a concentration-dependent manner elicited dilation; however, it caused significant constriction in arterioles isolated from db/db mice (Figure 3A). The presence of the PGH2/TxA2 receptor antagonist SQ29548 did not affect the arachidonic acid–induced dilation of arterioles of control mice but reduced the arachidonic acid–induced constriction in arterioles of db/db mice (Figure 3A). Also, we found that removal of the endothelium reduced arachidonic acid–induced arteriolar responses in control vessels but did not affect responses significantly in arterioles of db/db mice (Figure 3B). In control mice, arachidonic acid–induced dilations were significantly reduced by the selective inhibitor of COX-1, SC-560, but not by NS-398, a selective inhibitor of COX-2 (Figure 4A). In contrast, in arterioles of db/db mice, the arachidonic acid–induced constriction was significantly reduced by the COX-2 inhibitor NS-398, whereas the COX-1 inhibitor SC-560 had no effect (Figure 4B).
Figure 3. Arachidonic acid-induced changes in arteriolar diameters from control (n=7) and db/db (n=7) mice in the absence or presence of SQ 29,548, a PGH2/TXA2 receptor antagonist (A), and after endothelium removal (B, n=5). Data are mean±SEM. *Significant difference from control; #Significant difference from db/db arterioles (P<0.05). Abbreviations are as defined in text.
Figure 4. Arachidonic acid–induced changes in arteriolar diameters from control (n=7) and db/db (n=7) mice in the absence or presence of NS-398, a selective inhibitor of COX-2, and SC-560, a selective inhibitor of COX 1 (A and B). Data are mean±SEM. *Significant difference from control (P<0.05). Abbreviations are as defined in text.
Immunoblots
Western blot analysis was performed on aortas from both control and db/db mice. There were no significant differences in total COX-1 protein levels in the 2 groups, whereas COX-2 expression was significantly greater in aortas from db/db mice (Figure 5).
Figure 5. Western blot analysis of the expression of COX-1 (A and B) and COX-2 (C and D) in aortas from control and db/db mice. Anti–?-actin was used to normalize for loading variations. Bar graphs represent the summary of normalized densitometric ratios (n=5 for each group). Data are mean±SEM. *Significant difference (P<0.05). Abbreviations are as defined in text.
Discussion
The main findings of the present study are that mice with T2-DM have elevated systolic blood pressures and increased peripheral vascular resistance. In T2-DM mice, these alterations are associated with enhanced skeletal muscle arteriolar tone, which is likely to be attributable to increased release of COX-2–derived constrictor PGs within the skeletal muscle arteriolar wall.
T2-DM is frequently associated with elevated systemic blood pressure; however, the nature of the mechanisms have not yet been fully elucidated. A key role for altered regulation of microvascular resistance has been suggested by several earlier investigations that found specific impairment of microvascular vasoregulatory mechanisms in subjects with T2-DM.4 Accordingly, a reduced endothelium-dependent arteriolar vasodilation6,9–11 and/or an enhanced smooth muscle–dependent vasoconstriction5–7 in microvessels has been demonstrated in T2-DM, alterations that could influence total peripheral resistance.
In the present study, hemodynamic parameters were obtained first in awake mice. Using a tail-cuff method, we found a significant rise in systolic and mean arterial blood pressures in db/db mice compared with control animals (Table). Furthermore, by using echocardiography, a decreased cardiac output was also observed in db/db mice (Table). On the basis of these parameters, the total peripheral resistance was calculated and found to be significantly elevated in db/db mice compared with that of control animals (Table). This study is the first to demonstrate that at 12 weeks of age, in association with obesity, hyperglycemia, and hyperinsulinemia, db/db mice exhibit an enhanced peripheral vascular resistance and elevated systemic blood pressure. These results are in accordance with previous clinical observations of the prevalence of high blood pressure in patients with obesity and T2-DM.2,27
In the next series of experiments, we aimed to elucidate the possible underlying mechanisms responsible for the enhanced peripheral vascular resistance. It is well known that arterioles respond to an increase in transmural pressure by constriction, a response termed myogenic constriction, which plays a key role in the local regulation of tissue blood flow.28,29 Earlier it had been found that an elevation of systemic blood pressure is associated with a rise in resistance of the skin microcirculation in T2-DM patients.30 It has been proposed that enhanced arteriolar tone could protect the distal part of the microcirculation from the increased intraluminal pressure.31 Indeed, previous studies have demonstrated an enhanced tone in skeletal muscle5 and mesenteric6 arterioles of T2-DM animals. An enhanced arteriolar tone, especially in the skeletal muscle circulation, may further increase vascular resistance, which, if uncompensated, could ultimately result in the elevation of systemic blood pressure.
In the present study, we demonstrated that in response to stepwise increases in intraluminal pressure (from 20 to 120 mm Hg), the diameter of isolated skeletal muscle arterioles was significantly reduced in db/db compared with control mice, whereas the passive diameter (obtained in Ca2+-free solution) of arterioles was not significantly different between the 2 groups (Figure 1). These findings indicate that active tone is greater in the arterioles of db/db mice, which, however, is unlikely to be the result of an altered arteriolar compliance. Next, myogenic indexes28,29 were calculated, allowing us to assess the pressure-sensitive behavior of skeletal muscle arterioles. We found that myogenic indexes were not significantly different in the 2 groups, suggesting that mechanisms other than pressure-sensitive myogenic regulation are responsible for the reduction in the basal diameter of arterioles from db/db mice.
Arteriolar diameter is continuously modulated by dilator and constrictor factors, many of them intrinsic to the vascular wall. Early investigations reported enhanced release of a constrictor prostanoid from diabetic vessels.18 On the basis of previous observations, we hypothesized that vascular production of constrictor PGs is increased in T2-DM, thereby contributing to the reduced diameter of skeletal muscle arterioles of db/db mice. Indeed, we found that a PGH2/TxA2 receptor antagonist increased the diameter of arterioles of T2-DM mice back to control levels, whereas it did not affect the diameter of vessels from control animals (Figure 2A and 2B). These findings indicate that endogenous release of constrictor PGs, PGH2/TxA2, may be responsible for the reduced diameter of arterioles from T2-DM mice.
Recently, a role for prostanoid-mediated vascular inflammation has been shown to be associated with the development of vascular complications in T2-DM.13 Prostanoids generated by COXs from arachidonic acid20 are important mediators of several inflammatory mechanisms. Two isoforms of the COX enzyme, encoded by distinct genes, have been isolated in mammalian cells.20 COX-1 is constitutively expressed in most tissues, such as vascular endothelial cells, and is involved in the maintenance of cellular homeostasis.23 In contrast, under normal conditions, COX-2 is expressed only at low or undetectable levels but is readily upregulated by inflammatory, mitogenic, and physical stimuli.32 Only a limited number of biochemical studies have investigated alterations in COX-2–dependent mechanisms related to DM. In this context, it has been found that high-glucose treatment caused increases in expression of COX-2 protein in mesangial cells.33 Also, in cultured human endothelial cells, high-glucose treatment elicited an enhanced production of TxA2 in association with an upregulation of COX-2.21 Because in the db/db mouse model of T2-DM the level of vascular COX-2 expression is not known, we aimed to measure COX-2 expression in intact vessels from control and db/db mice. COX-2 protein levels in the aortas of T2-DM mice were markedly increased compared with those of control animals (Figure 5). Although COX-2 expression has been found to be associated with enhanced production of constrictor prostanoids under normal34 and certain pathologic22,35 conditions, the specific role of COX-1– and COX-2–dependent prostanoid synthesis in the mediation of arteriolar diameter changes in T2-DM has not yet been elucidated.
In control arterioles, selective inhibition of COX-1 did not affect the diameter of arterioles (Figure 2C), whereas it reduced arachidonic acid–induced dilation (Figure 4A). Because removal of the endothelium also reduced arachidonic acid–induced arteriolar responses in control vessels (Figure 3B), we concluded that dilator PGs, most likely endothelium-derived prostacyclin, were produced by the metabolism of arachidonic acid. In contrast, in arterioles of T2-DM mice, both basal tone (Figure 2D) and arachidonic acid–induced constrictions were reduced by the selective inhibitor of COX-2, but not that of COX-1, or endothelium removal (Figures 3B and 4B). We interpret these findings to mean that in arterioles of db/db mice, COX-2–dependent release of constrictor PGs, most likely PGH2/TxA2, derived primarily from vascular smooth muscle cells, mediate both the enhanced pressure- and arachidonic acid–induced reductions of arteriolar diameter.
Recently, an important role for reactive oxygen species in the regulation of arteriolar tone has received a great deal of attention.36 In T2-DM rats, reactive oxygen species have been proposed to play a role in myogenic activation of skeletal muscle arterioles.5 In this context, we previously found that in T2-DM (db/db) mice, owing to the reduced activity of vascular superoxide dismutase and catalase together with enhanced activation of vascular NAD(P)H oxidase, vascular production of superoxide was increased.24 Thus, one can speculate that vascular oxidative stress in T2-DM or other disease conditions37 may also be associated with alteration in COX-2–dependent synthesis of PGs. Indeed, recently it has been found that in high glucose–treated mesangial cells, mitochondrial superoxide production was associated with enhanced COX-2 expression.33 Also, in cultured human endothelial cells, high glucose elicited enhanced production of reactive oxygen species, resulting in increased production of TxA2, which was also associated with an upregulation of COX-2.21 However, in T2-DM, the interrelation between vascular oxidative stress and altered prostanoid metabolism needs to be addressed in future investigations.
Taken together, we propose an important role for COX-2–derived constrictor PGs in the altered regulation of skeletal muscle arteriolar resistance in T2-DM, obese mice. It still remains a question, however, whether and to what extent changes in arteriolar PG synthesis could contribute to the alterations of total peripheral resistance and blood pressure in T2-DM. Nevertheless, based on the present studies, alterations in COX-2–dependent and PG-mediated modulation of vasomotor function should be taken into consideration in future investigations of T2-DM.
Acknowledgments
This study was supported by a grant from the American Heart Association, Northeast Affiliate (0555897T), and National Institutes of Health grants HL-43023, HL-46813, OTKA T-034779, 048376, F-048837, and 711-K-84289.
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