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Enhanced neutrophil superoxide anion production and its modification by beraprost sodium in spontaneously hypertensive rats
AJH
2001; 14:722–728
Enhanced Neutrophil Superoxide Anion Production and Its Modification by Beraprost Sodium in Spontaneously Hypertensive Rats Masami Ohmori, Yasuhiko Kitoh, Atsuhiro Kawaguchi, Kazuhiro Harada, Koh-ichi Sugimoto, and Akio Fujimura To clarify the function of polymorphonuclear leukocytes (PMN) in spontaneously hypertensive rats (SHR) and the effect of beraprost sodium (BS) on these functions, we examined superoxide anion (O2⫺) production and adherent activity by PMN, as well as modification of these functions by BS ex vivo and in vitro. In study 1, we measured PMN functions in 4-week-old SHR and WistarKyoto (WKY) rats. In study 2 (ex vivo), 14-week-old SHR received vehicle (n ⫽ 6) and BS (30 g/kg/day [n ⫽ 6] and 100 g/kg/day [n ⫽ 7]) once daily for 4 weeks. In study 3 (in vitro), PMN from 18-week-old SHR were incubated with BS (0.1 and 1 mol/L) and theophylline (200 mol/L), which is reported to inhibit the PMN O2⫺ production. Systolic blood pressure, platelet counts, and PMN O2⫺ production stimulated by phorbol ester myris-
E
tate acetate were significantly elevated in 4-week-old SHR compared with WKY (P ⬍ .05). Beraprost sodium decreased the ex vivo PMN O2⫺ production, serum superoxide dismutase activity, and platelet counts (P ⬍ .05); however, BS did not reduce the in vitro PMN O2⫺ production. These data support our hypothesis that the enhanced PMN function contributes to the cardiovascular damages during the early phase of SHR, and that BS has merit for preventing the O2⫺ related organ damages in this model. Am J Hypertens 2001;14:722–728 © 2001 American Journal of Hypertension, Ltd. Key Words: Polymorphonuclear leukocytes (PMN), spontaneously hypertensive rat (SHR), beraprost sodium (BS), superoxide anion (O2⫺), adhesion.
pidemiological study has shown that high counts of leukocytes, especially of polymorphonuclear leukocytes (PMN), is an important and independent risk factor for ischemic heart disease, together with hypertension, hypercholesterolemia, and smoking.1,2 Activated PMN not only adhere to endothelial cells and generate cytotoxic superoxide anion (O2⫺) and release proteolytic enzymes and cytokines (interleukin [IL]–1, IL-8, tumor necrosis factor), but also aggregate in capillary arteries, thereby reducing blood flow to the organs.3–5 Reactive free radicals produced by PMN oxidize low-density lipoprotein (LDL) cholesterol, which in turn damages the lipid bilayer of the membrane and cell matrix and contributes to the pathogenesis of atherosclerosis.6 It has also been shown that PMN are involved in the initiation and development of cardiovascular damages, especially in ischemia-reperfusion injury.7–10 The spontaneously hypertensive rat (SHR) is an animal model of essential hypertension with prominent cardiovascular damage.11 In primary hypertension (both in SHR and
in patients with essential hypertension), peripheral PMN counts and its function are higher than those in the agematched Wistar-Kyoto rats (WKY) and normotensive subjects.12–14 We recently reported that O2⫺ production by PMN increases in mature SHR, but not in other animal models such as NG-nitro-L-arginine methyl ester (LNAME)– and deoxycorticosterone acetate (DOCA)/salt– induced hypertension.15 However, it is still unknown whether the O2⫺ production by PMN increases from an early stage of SHR. Beraprost sodium (BS) is a stable, orally active prostacyclin (PGI2) analogue that is used mainly for the treatment of atherosclerotic diseases.16 Long-term dosing for BS improves survival rate and incidence of stroke in stroke-prone SHR.17 The mechanism is not fully understood, but the improvement of cerebral circulation by its potent anti-platelet and vasodilating properties is speculated. However, it is unclear whether BS suppresses the enhanced PMN functions in SHR. The aim of this study was to evaluate the ex vivo PMN
Received August 22, 2000. Accepted December 5, 2000. From the Department of Clinical Pharmacology, Jichi Medical School, Tochigi, Japan.
Address correspondence and reprint requests to Akio Fujimura, MD, PhD, Department of Clinical Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan; e-mail: [email protected]
0895-7061/01/$20.00 PII S0895-7061(01)01309-7
© 2001 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
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functions concerning the O2⫺ production and adhesion in young SHR and WKY, and to clarify the potential mechanism of protection by BS for cardiovascular events in mature SHR.
Materials and Methods Animals Young (4-week-old) male spontaneously hypertensive Izumo rats (SHR; n ⫽ 12), age-matched male WistarKyoto Izumo rats (WKY; n ⫽ 12), and mature (14-weekold [n ⫽ 19] and 18-week-old [n ⫽ 18]) male SHR were obtained from Disease Model Cooperative Research Association (Kyoto, Japan). These animals were maintained under specific pathogen-free conditions in the animal center of Jichi Medical School and were given free access to standard rat chow and tap water. Systolic blood pressure (SBP) was measured by a standard tail-cuff sphygmomanometer (KN-201, Natsume, Tokyo, Japan) in the awake state. The experiment was conducted in accordance with the Jichi Medical School Guide for Laboratory Animals. Study 1 Heparinized whole blood was obtained from aorta of 4week-old SHR and WKY under ether anesthesia. As blood volume obtained from each rat was small, three samples were mixed in this study; therefore, making a total of four in the SHR and WKY groups. Study 2 In the ex vivo study, 14-week-old SHR were divided into three groups and were treated with vehicle (n ⫽ 6), lowdose BS (30 g/kg/day; n ⫽ 6), or high-dose BS (100 g/kg/day; n ⫽ 7). Beraprost sodium (Toray, Kanagawa, Japan) and its vehicle were given orally once daily for 4 weeks. Study 3 In the in vitro study, PMN obtained from 18-week-old SHR were incubated with BS (0.1 and 1 mol/L) and theophylline (200 mol/L; Sigma Chemical, St Louis, MO) on a 96-well plastic plate for 20 min. PMN Isolation Peripheral PMN were separated by density gradient (250 g for 40 min at room temperature) with Lympholyte-M (Cederlane Laboratories, Hornby, Canada) after sedimentation with 3% dextran sulfate (75% v/v) for 20 min.15
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placed on fetal bovine serum (FBS)-coated, 96-well plates, at a total volume of 200 g/well: 1) PMN suspension contained 3 ⫻ 105 cells; 2) PMA or its vehicle; 3) SOD (200 U/mL, Sigma Chemical) or its vehicle; and 4) cytochrome-C (12 mg/mL, Sigma Chemical). After incubation for 60 min at 37°C, O2⫺ production was determined by measuring the absorbances at 540 and 550 nm in a microplate spectrophotometer system (SPECTRAmax 340, Molecular Devices, Sunnyvale, CA). The assay was performed in duplicate. Assay for Adherent Activity Adherence of PMN to the FBS-coated surface was determined as protein content assayed by Lowry method with minor modification.15 This parameter was determined by measuring the absorbance at 700 nm in a microplate spectrophotometer system (SPECTRAmax 340, Molecular Devices). The assay was performed in duplicate. Assay for Superoxide Dismutase An activity of serum SOD was determined by the nitrate method with minor modification.18 The following materials were mixed: 4 mL of solution containing 0.4 mmol/L hypoxanthine and 3 mmol/L hydroxylamine, 0.25 mL of standard SOD (256 U/mL), and 0.05 mL of serum sample. After incubation for 10 min at 37°C, 10 mU/mL of xanthine oxidase was added to the solution and incubated for 30 min at 37°C. Thereafter, we added 2 mL of solution containing 0.45 g/L sulfanic acid, as well as 10 mg/L N-1-naphthyl ethylene diamine dihydrochloride in 20% acetic acid. After incubation for 30 min at room temperature, serum SOD activity was determined by measuring the absorbance at 545 nm in a spectrophotometer. Cell Blood Counts and Leukocyte Differentiation Cell blood counts were obtained automatically (Sysmex CC-780, Toa Medical, Tokyo, Japan). Leukocyte differentiation was determined by skilled technicians using Wright-Giemsa staining. Statistical Analysis Data are expressed as means ⫾ SE. They were analyzed by one-way analysis of variance and paired t test as appropriate, using Stat View-J 4.02 (Abacus Concepts, Berkeley, CA). A value of P ⬍ .05 was considered statistically significant. Data for mature (16-week-old) SHR and WKY are also given for reference.15
Assay for Superoxide Anion Production
Results
A O2⫺ production by isolated PMN was determined by the method for superoxide dismutase (SOD)–inhibitable reduction of cytochrome C, with and without phorbol ester myristate acetate (PMA, 100 ng/mL, Sigma Chemical) as a stimulant, or its vehicle.15 The following materials were
Body weight in the young SHR was significantly lower than that in the young WKY (Table 1). The SBP was already elevated at 4 weeks in the SHR group. Although hematocrit in the young SHR was significantly lower than
Study 1
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Table 1.
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Body weight, blood pressure, and hematological profiles Young Rats (4 Weeks) WKY (n ⴝ 12)
Body weight (g) Systolic blood pressure (mm Hg) Total leukocyte counts (/L) PMN counts (/L) Lymphocyte counts (/L) Hematocrit (%) Platelet counts (⫻104/L)
SHR (n ⴝ 12)
117.4 ⫾ 1.1 109.9 P ⬍ .05 117.9 ⫾ 3.0 152.2 P ⬍ .05 4475 ⫾ 141 4892 791 ⫾ 120 1001 3520 ⫾ 183 3631 40.2 ⫾ 0.4 38.0 P ⬍ .05 57.6 ⫾ 0.7 66.2 P ⬍ .05
Mature Rats (16 Weeks) WKY (n ⴝ 6)
SHR (n ⴝ 6)
⫾ 1.0
392.1 ⫾ 5.4
⫾ 6.6
⫾ 4.0
127.9 ⫾
⫾ 3.0
⫾ ⫾ ⫾ ⫾
219 179 244 0.5
⫾ 1.4
9283 1471 7552 42.0
⫾ ⫾ ⫾ ⫾
361.3 P ⬍ 0.05 2.6 196.9 P ⬍ 0.05 933 6443 272 1864 1099 5653 1.6 40.7
38.8 ⫾ 7.3
⫾ ⫾ ⫾ ⫾
1644 276 1170 1.0
43.0 ⫾ 7.0
WKY ⫽ Wistar-Kyoto rats; SHR ⫽ spontaneous hypertensive rats; PMN ⫽ polymorphonuclear leukocytes. Data are given as means ⫾ SE.
that in the young WKY, the PMN count tended to be greater and platelet count was significantly greater in the SHR group. Such findings were also obtained in the mature SHR and WKY, but the differences did not reach statistical significance (Table 1). Although an unstimulated O2⫺ production of the young SHR and WKY group did not differ significantly, PMA-stimulated O2⫺ production in the young SHR was significantly greater than that in the young WKY (Fig. 1). Adhesive properties of PMN did not differ between the young SHR and WKY (Fig 2.). Similar animal model– dependent profiles of PMN were also observed in the mature SHR and WKY (Figs. 1 and 2). The PMA-stimulated O2⫺ productions in the mature groups were ⬎10 times greater than those in the younger groups. Study 2 There were no significant differences among the three groups in body weight or SBP at 14 or 18 weeks of age (Table 2). Platelet counts and serum SOD activity in the
BS-treated groups were significantly lower than those in the vehicle-treated group. Other parameters (leukocyte counts, differentiation, and hematocrit) did not differ among these groups. The O2⫺ production by unstimulated PMN was similar among the three groups (Fig. 3A). However, PMA-stimulated O2⫺ production was dose-dependently suppressed by BS. Study 3 The following results were obtained in the 18-week-old SHR group: SBP, 206.0 ⫾ 5.7 mm Hg; body weight, 315.8 ⫾ 2.5 g; leukocytes, 8917 ⫾ 413/L; PMN, 1973 ⫾ 159/L; lymphocytes, 6817⫾ 385/L; hematocrit, 47.4 ⫾ 1.2%; and platelets, 63.4 ⫾ 1.9⫻104/L. The BS did not significantly influence the nonstimulated and PMA-stimulated O2⫺ production in vitro (Fig. 3B). On the other hand, theophylline tended to suppress the nonstimulated O2⫺production by PMN (P ⫽ .06) and to suppress significantly the PMA-stimulated (P⬍ .05) O2⫺ production by PMN.
FIG. 1. Superoxide anion production by polymorphonuclear leukocytes in 4- and 16-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). PMA ⫽ phorbol ester myristate acetate.
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FIG. 2. Adhesion by polymorphonuclear leukocytes in 4- and 16-week-old male SHR and WKY. Abbreviations as in Fig. 1.
Discussion When PMN functions are evaluated, there is always a concern about unexpected influences on this physiological parameter, such as infection. Therefore, all animals were kept and blood samplings were performed in specifically pathogen-free rooms in this study. Because PMA is a standard stimulant for examining PMN functions,19 we used this agent in our study; PMA directly activates protein kinase C (PKC) and stimulates both the respiratory burst of PMN and the release of O2⫺, proteolytic enzymes and cytokines. Stimulated by many endogenous and exogenous substances, PMN adhere to endothelial cells, and subsequently generate cytotoxic O2⫺ and release enzymes and cytokines.4 Although the activated PMN and O2⫺ generated by PMN play a crucial role in host defense, they are also believed to be involved in cardiovascular diseases.6 Recently we reported that actual O2⫺ production by PMN in the SHR is higher than those in its counterpart, WKY, which does not depend on blood pressure elevation.15 Nakazono et al used SOD as a scavenger of O2⫺ and
showed that reactive oxygen species play a critical role in the pathogenesis of hypertension in SHR.20 These observations led us to speculate that the activated PMN might be involved in hypertensive organ damages in SHR. Study 1 showed that the peripheral PMN count is greater and their PMA-stimulated O2⫺ production is enhanced in younger SHR, which are similar to observations in mature SHR.15 The SOD activity in cardiac tissues and the content of uric acid, another scavenger, in the cerebral cortex are reported to be reduced in young SHR.21,22 Therefore, we think that the younger SHR are in a state of hyperproduction and hypoconsumption of O2⫺, which may lead to organ damage at an early stage in this animal model. Although O2⫺ production by PMN increased in the younger SHR in this study, a mechanism responsible for this phenomenon remains to be determined. In study 2, BS at a clinically relevant concentration decreased the ex vivo PMA-stimulated O2⫺ production by circulatory PMN in SHR. However, in the in vitro study, the O2⫺ production was not suppressed by BS but, rather, by theophylline, which is reported to inhibit its produc-
FIG. 3. Effect of beraprost sodium on the superoxide anion production by polymorphonuclear leukocytes (PMN) in mature SHR. A) Ex vivo study; B) in vitro study. Abbreviations as in Figs. 1 and 2.
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PMN FUNCTIONS AND EFFECT OF PGI2 ANALOGUE IN SHR
Table 2.
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Characteristics of SHR treated with beraprost sodium Beraprost Sodium
Parameter Body weight (g) 14 weeks 18 weeks SBP (mm Hg) 14 weeks 18 weeks Leukocyte counts (/L) PMN counts (/L) Lymphocyte counts (/L) Hematocrit (%) Platelet counts (⫻104/L) Serum SOD activity (U/mL)
Vehicle (n ⴝ 6) 317.5 ⫾ 7.9 340.7 ⫾ 5.8 201.7 210.0 6700 1428 5043 44.9 51.9
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3.8 2.4 1674 276 1529 1.2 5.7
20.2 ⫾ 0.3
30 g/kg/day (n ⴝ 6)
100 g/kg/day (n ⴝ 7)
312.3 ⫾ 10.0 332.5 ⫾ 6.7
314.3 ⫾ 11.3 341.5 ⫾ 10.3
211.8 ⫾ 3.7 205.8 ⫾ 2.4 6983 ⫾ 1328 1696 ⫾ 456 5003 ⫾ 1038 46.6 ⫾ 0.3 37.6 ⫾ 7.1 P ⬍ .05 P ⬍ .05 18.5 ⫾ 0.5 P ⬍ .05
208.4 201.3 6057 1158 4719 45.1 37.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3.3 3.0 1030 271 891 0.9 7.0
18.6 ⫾ 0.5
P ⬍ .05
SHR ⫽ spontaneously hypertensive rats; SBP ⫽ systolic blood pressure; SOD ⫽ superoxide dismutase; other abbreviation as in Table 1. Data are given as means ⫾ SE.
tion.23 These results suggest that BS indirectly suppresses the activated PMN function in SHR. A previous report showed that PGI2 and its analogue have little suppressive effects on PMN functions at physiologically relevant concentrations in vitro,24 which is similar to the present findings. Lipopolysaccharide-stimulated macrophages release not only tumor necrosis factor–␣ (TNF-␣), which has the priming effect on PMN, but also prostanoids such as PGI2 and PGE2, which conversely inhibit the release of TNF-␣ from macrophage.25 Thus, we speculate that BS suppressed the priming condition of PMN through the reduction of cytokine release from macrophages in SHR. Beraprost sodium also decreased the serum SOD activity in this study, which might be a compensatory change accompanying the reduced O2⫺ production by PMN. The reason for the discrepancy between PMN O2⫺ production and adhesion in SHR in this study is unclear. However, it is possible that signal transduction pathway are different between the two types of functions of PMN. Snyderman indicated that transduction signals for chemotaxis and secretion in leukocytes are discrete and can be modified independently.26 A more limited expression of adhesion molecule (CD11b/CD18) on PMN has been reported under platelet activating factor– or leukotrien B4– stimulated conditions in SHR.27 Such a mechanism might also explain the discrepancy observed in this study. There are diverse data concerning platelet count in mature SHR. Reed et al observed an increase in this variable,28 whereas others did not.12,13 However, to our knowledge, few data concerning platelet counts in young SHR have been available. The present study showed that peripheral platelet count increases at an early stage of SHR. Platelets release many endogenous factors such as
thromboxane A2, platelet-derived growth factor, serotonin, prostaglandins, protease, adenosine, and cytokines, which affect the activation and aggregation of PMN.29 Platelet also releases neutrophil-activating peptide 2 (NAP 2) by thrombin stimulation, which, in turn, activates PMN.30 This pathway is enhanced in SHR.31 We think that an elevated number of platelet might contribute to the enhanced O2⫺ production by PMN through the release of the above endogenous factors in young SHR. In study 2, peripheral platelet counts decreased after the repeated dosing of BS in SHR. Toda pointed out that BS reduces blood cell counts including platelet in normotensive rats.16 To our knowledge, this is the first report of BS-induced thrombocytopenia in hypertensive rats. The reason for this phenomenon is unclear in this study, but BS blunts the release of cytokines (IL-1, IL-6, and TNF-␣) from macrophage,32 which might decrease the maturation of megakaryocytic cells in bone marrow. The thrombocytopenia induced by BS might also contribute to the reduced O2⫺ production by PMN through the decreased release of PMN-modulating factors from platelets in SHR. The precise mechanisms of hypertensive organ damages remain unclear. We observed that the O2⫺ production by PMA-stimulated PMN increases at the developmental as well as established phases in SHR. Numaguchi et al reported the decreased prostacyclin receptor expression in the thoracic aorta of SHR compared to WKY.33 Yu et al showed that BS inhibits mRNA expression for collagen synthesis, which is weaker in SHR than in WKY.34 Based on these observations, we speculate that multiple factors such as the enhanced neutrophil functions and the reduced PGI2-mediated stimuli contribute to hypertensive organ damages.
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This study showed that the elevated PMN O2⫺ production was reduced by the repeated dosing of BS in SHR. Celiprolol, a -blocker, also suppresses the O2⫺ production by circulatory PMN in patients with essential hypertension.14 Several antihypertensive drugs have radical scavenging effects35,36 and, at a subdeppressor dose, can prevent organ damages in stroke-prone SHR.37 These drugs may have the merit for preventing the O2⫺-related hypertensive organ damage. In summary, the present study showed that O2⫺ production by PMN and platelet counts were elevated at early stages in SHR. In addition, BS was found to suppress the enhanced O2⫺ production by PMA-stimulated PMN, as well as to decrease circulatory platelet counts, which might contribute to the reduced cardiovascular damages reported in SHR with BS. Further studies are needed to clarify the mechanisms involved in these phenomena.
13.
14.
15.
16. 17.
18.
19.
Acknowledgments We thank Miss M. Hojo for assistance with the animal experiments, and Miss T. Kawaguchi and C. Fukushima for preparation of the manuscript.
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2001; 14:722–728
Enhanced Neutrophil Superoxide Anion Production and Its Modification by Beraprost Sodium in Spontaneously Hypertensive Rats Masami Ohmori, Yasuhiko Kitoh, Atsuhiro Kawaguchi, Kazuhiro Harada, Koh-ichi Sugimoto, and Akio Fujimura To clarify the function of polymorphonuclear leukocytes (PMN) in spontaneously hypertensive rats (SHR) and the effect of beraprost sodium (BS) on these functions, we examined superoxide anion (O2⫺) production and adherent activity by PMN, as well as modification of these functions by BS ex vivo and in vitro. In study 1, we measured PMN functions in 4-week-old SHR and WistarKyoto (WKY) rats. In study 2 (ex vivo), 14-week-old SHR received vehicle (n ⫽ 6) and BS (30 g/kg/day [n ⫽ 6] and 100 g/kg/day [n ⫽ 7]) once daily for 4 weeks. In study 3 (in vitro), PMN from 18-week-old SHR were incubated with BS (0.1 and 1 mol/L) and theophylline (200 mol/L), which is reported to inhibit the PMN O2⫺ production. Systolic blood pressure, platelet counts, and PMN O2⫺ production stimulated by phorbol ester myris-
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tate acetate were significantly elevated in 4-week-old SHR compared with WKY (P ⬍ .05). Beraprost sodium decreased the ex vivo PMN O2⫺ production, serum superoxide dismutase activity, and platelet counts (P ⬍ .05); however, BS did not reduce the in vitro PMN O2⫺ production. These data support our hypothesis that the enhanced PMN function contributes to the cardiovascular damages during the early phase of SHR, and that BS has merit for preventing the O2⫺ related organ damages in this model. Am J Hypertens 2001;14:722–728 © 2001 American Journal of Hypertension, Ltd. Key Words: Polymorphonuclear leukocytes (PMN), spontaneously hypertensive rat (SHR), beraprost sodium (BS), superoxide anion (O2⫺), adhesion.
pidemiological study has shown that high counts of leukocytes, especially of polymorphonuclear leukocytes (PMN), is an important and independent risk factor for ischemic heart disease, together with hypertension, hypercholesterolemia, and smoking.1,2 Activated PMN not only adhere to endothelial cells and generate cytotoxic superoxide anion (O2⫺) and release proteolytic enzymes and cytokines (interleukin [IL]–1, IL-8, tumor necrosis factor), but also aggregate in capillary arteries, thereby reducing blood flow to the organs.3–5 Reactive free radicals produced by PMN oxidize low-density lipoprotein (LDL) cholesterol, which in turn damages the lipid bilayer of the membrane and cell matrix and contributes to the pathogenesis of atherosclerosis.6 It has also been shown that PMN are involved in the initiation and development of cardiovascular damages, especially in ischemia-reperfusion injury.7–10 The spontaneously hypertensive rat (SHR) is an animal model of essential hypertension with prominent cardiovascular damage.11 In primary hypertension (both in SHR and
in patients with essential hypertension), peripheral PMN counts and its function are higher than those in the agematched Wistar-Kyoto rats (WKY) and normotensive subjects.12–14 We recently reported that O2⫺ production by PMN increases in mature SHR, but not in other animal models such as NG-nitro-L-arginine methyl ester (LNAME)– and deoxycorticosterone acetate (DOCA)/salt– induced hypertension.15 However, it is still unknown whether the O2⫺ production by PMN increases from an early stage of SHR. Beraprost sodium (BS) is a stable, orally active prostacyclin (PGI2) analogue that is used mainly for the treatment of atherosclerotic diseases.16 Long-term dosing for BS improves survival rate and incidence of stroke in stroke-prone SHR.17 The mechanism is not fully understood, but the improvement of cerebral circulation by its potent anti-platelet and vasodilating properties is speculated. However, it is unclear whether BS suppresses the enhanced PMN functions in SHR. The aim of this study was to evaluate the ex vivo PMN
Received August 22, 2000. Accepted December 5, 2000. From the Department of Clinical Pharmacology, Jichi Medical School, Tochigi, Japan.
Address correspondence and reprint requests to Akio Fujimura, MD, PhD, Department of Clinical Pharmacology, Jichi Medical School, Tochigi 329-0498, Japan; e-mail: [email protected]
0895-7061/01/$20.00 PII S0895-7061(01)01309-7
© 2001 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
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functions concerning the O2⫺ production and adhesion in young SHR and WKY, and to clarify the potential mechanism of protection by BS for cardiovascular events in mature SHR.
Materials and Methods Animals Young (4-week-old) male spontaneously hypertensive Izumo rats (SHR; n ⫽ 12), age-matched male WistarKyoto Izumo rats (WKY; n ⫽ 12), and mature (14-weekold [n ⫽ 19] and 18-week-old [n ⫽ 18]) male SHR were obtained from Disease Model Cooperative Research Association (Kyoto, Japan). These animals were maintained under specific pathogen-free conditions in the animal center of Jichi Medical School and were given free access to standard rat chow and tap water. Systolic blood pressure (SBP) was measured by a standard tail-cuff sphygmomanometer (KN-201, Natsume, Tokyo, Japan) in the awake state. The experiment was conducted in accordance with the Jichi Medical School Guide for Laboratory Animals. Study 1 Heparinized whole blood was obtained from aorta of 4week-old SHR and WKY under ether anesthesia. As blood volume obtained from each rat was small, three samples were mixed in this study; therefore, making a total of four in the SHR and WKY groups. Study 2 In the ex vivo study, 14-week-old SHR were divided into three groups and were treated with vehicle (n ⫽ 6), lowdose BS (30 g/kg/day; n ⫽ 6), or high-dose BS (100 g/kg/day; n ⫽ 7). Beraprost sodium (Toray, Kanagawa, Japan) and its vehicle were given orally once daily for 4 weeks. Study 3 In the in vitro study, PMN obtained from 18-week-old SHR were incubated with BS (0.1 and 1 mol/L) and theophylline (200 mol/L; Sigma Chemical, St Louis, MO) on a 96-well plastic plate for 20 min. PMN Isolation Peripheral PMN were separated by density gradient (250 g for 40 min at room temperature) with Lympholyte-M (Cederlane Laboratories, Hornby, Canada) after sedimentation with 3% dextran sulfate (75% v/v) for 20 min.15
723
placed on fetal bovine serum (FBS)-coated, 96-well plates, at a total volume of 200 g/well: 1) PMN suspension contained 3 ⫻ 105 cells; 2) PMA or its vehicle; 3) SOD (200 U/mL, Sigma Chemical) or its vehicle; and 4) cytochrome-C (12 mg/mL, Sigma Chemical). After incubation for 60 min at 37°C, O2⫺ production was determined by measuring the absorbances at 540 and 550 nm in a microplate spectrophotometer system (SPECTRAmax 340, Molecular Devices, Sunnyvale, CA). The assay was performed in duplicate. Assay for Adherent Activity Adherence of PMN to the FBS-coated surface was determined as protein content assayed by Lowry method with minor modification.15 This parameter was determined by measuring the absorbance at 700 nm in a microplate spectrophotometer system (SPECTRAmax 340, Molecular Devices). The assay was performed in duplicate. Assay for Superoxide Dismutase An activity of serum SOD was determined by the nitrate method with minor modification.18 The following materials were mixed: 4 mL of solution containing 0.4 mmol/L hypoxanthine and 3 mmol/L hydroxylamine, 0.25 mL of standard SOD (256 U/mL), and 0.05 mL of serum sample. After incubation for 10 min at 37°C, 10 mU/mL of xanthine oxidase was added to the solution and incubated for 30 min at 37°C. Thereafter, we added 2 mL of solution containing 0.45 g/L sulfanic acid, as well as 10 mg/L N-1-naphthyl ethylene diamine dihydrochloride in 20% acetic acid. After incubation for 30 min at room temperature, serum SOD activity was determined by measuring the absorbance at 545 nm in a spectrophotometer. Cell Blood Counts and Leukocyte Differentiation Cell blood counts were obtained automatically (Sysmex CC-780, Toa Medical, Tokyo, Japan). Leukocyte differentiation was determined by skilled technicians using Wright-Giemsa staining. Statistical Analysis Data are expressed as means ⫾ SE. They were analyzed by one-way analysis of variance and paired t test as appropriate, using Stat View-J 4.02 (Abacus Concepts, Berkeley, CA). A value of P ⬍ .05 was considered statistically significant. Data for mature (16-week-old) SHR and WKY are also given for reference.15
Assay for Superoxide Anion Production
Results
A O2⫺ production by isolated PMN was determined by the method for superoxide dismutase (SOD)–inhibitable reduction of cytochrome C, with and without phorbol ester myristate acetate (PMA, 100 ng/mL, Sigma Chemical) as a stimulant, or its vehicle.15 The following materials were
Body weight in the young SHR was significantly lower than that in the young WKY (Table 1). The SBP was already elevated at 4 weeks in the SHR group. Although hematocrit in the young SHR was significantly lower than
Study 1
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Table 1.
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Body weight, blood pressure, and hematological profiles Young Rats (4 Weeks) WKY (n ⴝ 12)
Body weight (g) Systolic blood pressure (mm Hg) Total leukocyte counts (/L) PMN counts (/L) Lymphocyte counts (/L) Hematocrit (%) Platelet counts (⫻104/L)
SHR (n ⴝ 12)
117.4 ⫾ 1.1 109.9 P ⬍ .05 117.9 ⫾ 3.0 152.2 P ⬍ .05 4475 ⫾ 141 4892 791 ⫾ 120 1001 3520 ⫾ 183 3631 40.2 ⫾ 0.4 38.0 P ⬍ .05 57.6 ⫾ 0.7 66.2 P ⬍ .05
Mature Rats (16 Weeks) WKY (n ⴝ 6)
SHR (n ⴝ 6)
⫾ 1.0
392.1 ⫾ 5.4
⫾ 6.6
⫾ 4.0
127.9 ⫾
⫾ 3.0
⫾ ⫾ ⫾ ⫾
219 179 244 0.5
⫾ 1.4
9283 1471 7552 42.0
⫾ ⫾ ⫾ ⫾
361.3 P ⬍ 0.05 2.6 196.9 P ⬍ 0.05 933 6443 272 1864 1099 5653 1.6 40.7
38.8 ⫾ 7.3
⫾ ⫾ ⫾ ⫾
1644 276 1170 1.0
43.0 ⫾ 7.0
WKY ⫽ Wistar-Kyoto rats; SHR ⫽ spontaneous hypertensive rats; PMN ⫽ polymorphonuclear leukocytes. Data are given as means ⫾ SE.
that in the young WKY, the PMN count tended to be greater and platelet count was significantly greater in the SHR group. Such findings were also obtained in the mature SHR and WKY, but the differences did not reach statistical significance (Table 1). Although an unstimulated O2⫺ production of the young SHR and WKY group did not differ significantly, PMA-stimulated O2⫺ production in the young SHR was significantly greater than that in the young WKY (Fig. 1). Adhesive properties of PMN did not differ between the young SHR and WKY (Fig 2.). Similar animal model– dependent profiles of PMN were also observed in the mature SHR and WKY (Figs. 1 and 2). The PMA-stimulated O2⫺ productions in the mature groups were ⬎10 times greater than those in the younger groups. Study 2 There were no significant differences among the three groups in body weight or SBP at 14 or 18 weeks of age (Table 2). Platelet counts and serum SOD activity in the
BS-treated groups were significantly lower than those in the vehicle-treated group. Other parameters (leukocyte counts, differentiation, and hematocrit) did not differ among these groups. The O2⫺ production by unstimulated PMN was similar among the three groups (Fig. 3A). However, PMA-stimulated O2⫺ production was dose-dependently suppressed by BS. Study 3 The following results were obtained in the 18-week-old SHR group: SBP, 206.0 ⫾ 5.7 mm Hg; body weight, 315.8 ⫾ 2.5 g; leukocytes, 8917 ⫾ 413/L; PMN, 1973 ⫾ 159/L; lymphocytes, 6817⫾ 385/L; hematocrit, 47.4 ⫾ 1.2%; and platelets, 63.4 ⫾ 1.9⫻104/L. The BS did not significantly influence the nonstimulated and PMA-stimulated O2⫺ production in vitro (Fig. 3B). On the other hand, theophylline tended to suppress the nonstimulated O2⫺production by PMN (P ⫽ .06) and to suppress significantly the PMA-stimulated (P⬍ .05) O2⫺ production by PMN.
FIG. 1. Superoxide anion production by polymorphonuclear leukocytes in 4- and 16-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). PMA ⫽ phorbol ester myristate acetate.
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FIG. 2. Adhesion by polymorphonuclear leukocytes in 4- and 16-week-old male SHR and WKY. Abbreviations as in Fig. 1.
Discussion When PMN functions are evaluated, there is always a concern about unexpected influences on this physiological parameter, such as infection. Therefore, all animals were kept and blood samplings were performed in specifically pathogen-free rooms in this study. Because PMA is a standard stimulant for examining PMN functions,19 we used this agent in our study; PMA directly activates protein kinase C (PKC) and stimulates both the respiratory burst of PMN and the release of O2⫺, proteolytic enzymes and cytokines. Stimulated by many endogenous and exogenous substances, PMN adhere to endothelial cells, and subsequently generate cytotoxic O2⫺ and release enzymes and cytokines.4 Although the activated PMN and O2⫺ generated by PMN play a crucial role in host defense, they are also believed to be involved in cardiovascular diseases.6 Recently we reported that actual O2⫺ production by PMN in the SHR is higher than those in its counterpart, WKY, which does not depend on blood pressure elevation.15 Nakazono et al used SOD as a scavenger of O2⫺ and
showed that reactive oxygen species play a critical role in the pathogenesis of hypertension in SHR.20 These observations led us to speculate that the activated PMN might be involved in hypertensive organ damages in SHR. Study 1 showed that the peripheral PMN count is greater and their PMA-stimulated O2⫺ production is enhanced in younger SHR, which are similar to observations in mature SHR.15 The SOD activity in cardiac tissues and the content of uric acid, another scavenger, in the cerebral cortex are reported to be reduced in young SHR.21,22 Therefore, we think that the younger SHR are in a state of hyperproduction and hypoconsumption of O2⫺, which may lead to organ damage at an early stage in this animal model. Although O2⫺ production by PMN increased in the younger SHR in this study, a mechanism responsible for this phenomenon remains to be determined. In study 2, BS at a clinically relevant concentration decreased the ex vivo PMA-stimulated O2⫺ production by circulatory PMN in SHR. However, in the in vitro study, the O2⫺ production was not suppressed by BS but, rather, by theophylline, which is reported to inhibit its produc-
FIG. 3. Effect of beraprost sodium on the superoxide anion production by polymorphonuclear leukocytes (PMN) in mature SHR. A) Ex vivo study; B) in vitro study. Abbreviations as in Figs. 1 and 2.
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PMN FUNCTIONS AND EFFECT OF PGI2 ANALOGUE IN SHR
Table 2.
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Characteristics of SHR treated with beraprost sodium Beraprost Sodium
Parameter Body weight (g) 14 weeks 18 weeks SBP (mm Hg) 14 weeks 18 weeks Leukocyte counts (/L) PMN counts (/L) Lymphocyte counts (/L) Hematocrit (%) Platelet counts (⫻104/L) Serum SOD activity (U/mL)
Vehicle (n ⴝ 6) 317.5 ⫾ 7.9 340.7 ⫾ 5.8 201.7 210.0 6700 1428 5043 44.9 51.9
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3.8 2.4 1674 276 1529 1.2 5.7
20.2 ⫾ 0.3
30 g/kg/day (n ⴝ 6)
100 g/kg/day (n ⴝ 7)
312.3 ⫾ 10.0 332.5 ⫾ 6.7
314.3 ⫾ 11.3 341.5 ⫾ 10.3
211.8 ⫾ 3.7 205.8 ⫾ 2.4 6983 ⫾ 1328 1696 ⫾ 456 5003 ⫾ 1038 46.6 ⫾ 0.3 37.6 ⫾ 7.1 P ⬍ .05 P ⬍ .05 18.5 ⫾ 0.5 P ⬍ .05
208.4 201.3 6057 1158 4719 45.1 37.3
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
3.3 3.0 1030 271 891 0.9 7.0
18.6 ⫾ 0.5
P ⬍ .05
SHR ⫽ spontaneously hypertensive rats; SBP ⫽ systolic blood pressure; SOD ⫽ superoxide dismutase; other abbreviation as in Table 1. Data are given as means ⫾ SE.
tion.23 These results suggest that BS indirectly suppresses the activated PMN function in SHR. A previous report showed that PGI2 and its analogue have little suppressive effects on PMN functions at physiologically relevant concentrations in vitro,24 which is similar to the present findings. Lipopolysaccharide-stimulated macrophages release not only tumor necrosis factor–␣ (TNF-␣), which has the priming effect on PMN, but also prostanoids such as PGI2 and PGE2, which conversely inhibit the release of TNF-␣ from macrophage.25 Thus, we speculate that BS suppressed the priming condition of PMN through the reduction of cytokine release from macrophages in SHR. Beraprost sodium also decreased the serum SOD activity in this study, which might be a compensatory change accompanying the reduced O2⫺ production by PMN. The reason for the discrepancy between PMN O2⫺ production and adhesion in SHR in this study is unclear. However, it is possible that signal transduction pathway are different between the two types of functions of PMN. Snyderman indicated that transduction signals for chemotaxis and secretion in leukocytes are discrete and can be modified independently.26 A more limited expression of adhesion molecule (CD11b/CD18) on PMN has been reported under platelet activating factor– or leukotrien B4– stimulated conditions in SHR.27 Such a mechanism might also explain the discrepancy observed in this study. There are diverse data concerning platelet count in mature SHR. Reed et al observed an increase in this variable,28 whereas others did not.12,13 However, to our knowledge, few data concerning platelet counts in young SHR have been available. The present study showed that peripheral platelet count increases at an early stage of SHR. Platelets release many endogenous factors such as
thromboxane A2, platelet-derived growth factor, serotonin, prostaglandins, protease, adenosine, and cytokines, which affect the activation and aggregation of PMN.29 Platelet also releases neutrophil-activating peptide 2 (NAP 2) by thrombin stimulation, which, in turn, activates PMN.30 This pathway is enhanced in SHR.31 We think that an elevated number of platelet might contribute to the enhanced O2⫺ production by PMN through the release of the above endogenous factors in young SHR. In study 2, peripheral platelet counts decreased after the repeated dosing of BS in SHR. Toda pointed out that BS reduces blood cell counts including platelet in normotensive rats.16 To our knowledge, this is the first report of BS-induced thrombocytopenia in hypertensive rats. The reason for this phenomenon is unclear in this study, but BS blunts the release of cytokines (IL-1, IL-6, and TNF-␣) from macrophage,32 which might decrease the maturation of megakaryocytic cells in bone marrow. The thrombocytopenia induced by BS might also contribute to the reduced O2⫺ production by PMN through the decreased release of PMN-modulating factors from platelets in SHR. The precise mechanisms of hypertensive organ damages remain unclear. We observed that the O2⫺ production by PMA-stimulated PMN increases at the developmental as well as established phases in SHR. Numaguchi et al reported the decreased prostacyclin receptor expression in the thoracic aorta of SHR compared to WKY.33 Yu et al showed that BS inhibits mRNA expression for collagen synthesis, which is weaker in SHR than in WKY.34 Based on these observations, we speculate that multiple factors such as the enhanced neutrophil functions and the reduced PGI2-mediated stimuli contribute to hypertensive organ damages.
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This study showed that the elevated PMN O2⫺ production was reduced by the repeated dosing of BS in SHR. Celiprolol, a -blocker, also suppresses the O2⫺ production by circulatory PMN in patients with essential hypertension.14 Several antihypertensive drugs have radical scavenging effects35,36 and, at a subdeppressor dose, can prevent organ damages in stroke-prone SHR.37 These drugs may have the merit for preventing the O2⫺-related hypertensive organ damage. In summary, the present study showed that O2⫺ production by PMN and platelet counts were elevated at early stages in SHR. In addition, BS was found to suppress the enhanced O2⫺ production by PMA-stimulated PMN, as well as to decrease circulatory platelet counts, which might contribute to the reduced cardiovascular damages reported in SHR with BS. Further studies are needed to clarify the mechanisms involved in these phenomena.
13.
14.
15.
16. 17.
18.
19.
Acknowledgments We thank Miss M. Hojo for assistance with the animal experiments, and Miss T. Kawaguchi and C. Fukushima for preparation of the manuscript.
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