Short-term exposure to lipopolysaccharide is associated with microvascular contractile dysfunction in vivo

Life Sciences, Vol. 56, No. 15 pp. 1243-1249, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/95 $9.50 + .oO

Pergamon

0024-3205(95)00069-O

SHORT-TERM EXPOSURE TO LIPOPOLYSACCHARIDE IS ASSOCIATED MICROVASCULAR CONTRACTILE DYSFUNCTION IN VIVO

WITH

X.-p. Gao, H. Suzuki, CO. Olopade, and I. Rubinstein* Department

of Medicine, University of Illinois at Chicago, and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612-7323 (Received

in final form January

18, 199.5)

The purpose of this study was to determine whether short-term exposure of resistance arterioles to lipopolysaccharide in situ is associated with changes in vasomotor tone. Using intravital microscopy, we found that suffusion of Eschen’chia coli lipopolysaccharide (3 pg/ml) over hamster cheek pouch arterioles for 1 h was associated with a significant immediate biphasic response: vasoconstriction followed by vasodilation (~~0.05). The former was attenuated by indomethacin, and the latter by SK&F 108566, a selective, non-peptide an iotensin II receptor antagonist (~~0.05). The nitric oxide synthase inhibitor, N B -L-mtro arginine, had no significant effects on lipopolysaccharide-induced responses. Allopurinol, a scavenger of reactive oxygen species, significantly attenuated lipopolysaccharide-induced vasodilation. Acetylcholine- and nitroglycerin-induced vasodilation were significantly potentiated after lipopolysaccharide. These responses were recorded in the absence of any significant changes in systemic arterial blood pressure. Collectively, these data suggest that short-term exposure of the peripheral microcirculation to lipopolysaccharide in situ is associated with an ischemia-reperfusion-like injury. These changes may contribute to end organ failure observed several hours after exposure to lipopolysaccharide. Key Words:

microcirculation,

arterioles,

endothelium,

angiotensin

II, lipopolysaccharide,

prostaglandins

Despite the use of modern antibiotics and supportive care, gram-negative sepsis syndrome still remains a major cause of mortality in humans (1, 2). Administration of lipopolysaccharide, a component of the gram-negative bacterial cell wall, to experimental animals and humans has been shown to result in a sepsis-like syndrome (l-4). One characteristic function in the peripheral (2-4). The emergence of adversely affect prognosis

feature of this syndrome is profound depression of vascular contractile microcirculation manifested by vasodilation and refractory hypotension circulatory collapse in sepsis syndrome is an ominous sign thought to by compromising organ blood flow and metabolic status (1, 2,5).

Current concepts suggest that intrinsic changes in resistance arterioles in the peripheral circulation mediate vascular contractile dysfunction in sepsis syndrome (1, 5). However, these changes evolve typically over several hours after exposure to lipopolysaccharide. Whether vascular contractile dysfunction is already present early (i.e. within 1 h) in the course of endotoxemia is uncertain. This issue is clinically relevant because early therapeutic intervention in sepsis syndrome may improve survival (1). *Correspondence to: Dr. I. Rubinstein, Dept. of Medicine (M/C 787), University of Illinois Chicago, 840 S. Wood St., Chicago, IL 60612-7323. Tel. (312) 996-8039, Fax. (312) 996-4665.

at

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The purpose of this study was, therefore., to determine whether l-h exposure to lipopolysaccharide is associated with immediate changes in microvascular contractile function in the peripheral circulation in situ. Materials Preoaration of animals. Adult male golden Syrian hamsters (n=25) weighing 134&l g were anesthetized with sodium pentobarbital (6 mg/lOO g body weight; i.p.>. A tracheostomy was performed to facilitate spontaneous breathing. A catheter was placed in a femoral vein for injection of supplemental anesthesia (2-4 mg/lOO g body weight/h). A femoral artery was cannulated to measure blood pressure. Mean arterial blood pressure was 109+6 mmHg at the beginning, and 103&4 mm Hg at the end of the experiments (p>OS). Body temperature was monitored and maintained constant (37-38OC) throughout the experiments. To visualize the microcirculation of the cheek pouch, we used a method previously described in our laboratory (6, 7). Briefly, the cheek pouch was spread over a small plastic base plate and an incision was made in the skin to expose the cheek pouch membrane. The avascular connective tissue layer was cut away from the cheek pouch membrane and a plastic chamber was positioned over the base plate. This arrangement forms a triple-layered complex: the base plate, the upper chamber, and the cheek pouch membrane exposed between the two plates. The cheek pouch chamber was used to maintain the suffusion fluid. It was connected via a three-way valve to a reservoir that allowed continuous suffusion of the chamber with warm (37-380~) bicarbonate buffer that was bubbled continuously with 95% N2-5% CO:! (pH7.4). The chamber was also connected via a three-way valve to an infusion pump that allowed for the controlled administration of lipopolysaccharide and drugs into the suffusate. Measurement of arteriolar diameter. The cheek pouch was epi-illuminated with a fiberoptic light source and viewed through an Olympus microscope. The image was projected through the microscope and into a closed-circuit television system that consists of TV camera (Panasonic WV1.500), monitor (Panasonic TR-124 MA) and videotape recorder (Panasonic AG-1230). The luminal diameter of second order cheek pouch arterioles (51-62 pm) (6-8) was measured from the video display of the microscope image using a videomicrometer (VIA 100; Boeckeler Instruments, Tucson, AZ). In each animal, the same arteriolar segment was used to measure luminal diameter throughout the duration of the experiment. Effects of liponolysaccharide on arteriolar diameter. The cheek pouch was suffused with buffer for 30 min (equilibration period). Then, lipopolysaccharide (3 pg/ml) was suffused for 60 min. Arteriolar diameter was determined immediately before, every minute for 5 min and every 5 min for 55 min during suffusion of lipopolysaccharide, and for additional 45 min thereafter. In preliminary experiments, we determined that repeated applications of lipopolysaccharide (3 pg/ml) for 60 min before and after suffusion of saline (vehicle) for 45 min was associated with reproducible responses. Suffusion of saline for the entire duration of the experiment had no significant effects on arteriolar diameter. The concentration of lipopolysaccharide used in these experiments is based on previous studies in hamsters (8,9). Effects. To determine whether lipopolysaccharide-induced responses are mediated by prostaglandins, indomethacin (10 mg/kg), at a concentration known to inhibit cyclooxygenase in the cheek pouch (6, lo), was administered i.v. 30 min before suffusion of lipopolysaccharide (3 pglml). Changes in arteriolar diameter were determined as outlined above. In preliminary experiments, we found that administration of indomethacin alone had no significant effects on arteriolar diameter. Effects of SK&F 108566 on lipopolysaccharide-induced resDonses. To determine whether responses of cheek pouch arterioles to lipopolysaccharide are mediated, in part, by angiotensin II, SK&F 108566 ((E)-a-[[2-butyl-l-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methylene]-2thiophenepropanoic acid; 1 FM), a selective, non-peptide angiotensin II receptor antagonist (1 l), was suffused over cheek pouch arterioles for 30 min. Then, lipopolysaccharide (3 pg/ml) was suffused together with SK&F 108566 for 60 min. Changes in arteriolar diameter were determined as outlined above. In preliminary experiments, we found that suffusion of SK&F 108566 alone

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had no significant effects on arteriolar diameter. The concentratioti of SK&F 108566 used in these experiments was based on a previous report in the literature (11). Effects of @-L-nitro arginine on linonolvsaccharide-induced resnonses . To determine the role of the L-arginine/nitric oxide biosynthetic pathway in modulating lipopolysaccharide-induced responses, fl-L-nitro arginine (100 yM), a selective nitric oxide synthase inhibitor, was suffused over cheek pouch arterioles for 30 min. Then, lipopolysaccharide (3 pg/rnl) was suffused together with @-L-nitro arginine for 60 min. Changes in arteriolar diameter were determined as outlined above. In preliminary experiments, we found that suffusion of @-L-nitro arginine alone had no significant effects on arteriolar diameter. The concentration of @-L-nitro arginine used in these experiments was based on a previous report in the literature (5). Effects of alloourinol on lioonolvsaccharide-induced resoonses. To determine whether lipopolysaccharide-induced responses are mediated, in part, by reactive oxygen species, allopurinol (30 mg/kg), a scavenger of reactive oxygen species (12, 13), was administered iv. 30 min before suffusion of lipopolysaccharide (3 pglml) for 60 min. In preliminary experiments, we found that administration of allopurinol alone had no significant effects on arteriolar diameter. Changes in arteriolar diameter were determined as outlined above. The concentration of allopurinol used in these experiments was based on a previous study from our laboratory (12, 13). Effects of lipouolvsaccharide on acetvlcholine- and nitronlvcerin-induced vasodilation. To determine whether lipopolysaccharide-induced responses were related, in part, to non-specific effects on arteriolar wall, acetylcholine (10 PM), an endothelium-dependent vasodilator, and nitroglycerin (10 yM), an endothelium-independent vasodilator, were suffused for 5 min. Then, lipopolysaccharide (3 ug/ml) was suffused for 60 min followed, 45 min later, by application of acetylcholine and nitroglycerin. In another series of experiments, acet lcholine and nitroglycerin (each, 10 PM) were suffused for 5 min before and after suffusion of hJ -L-nitro arginine (100 PM) for 30 min. Changes in arteriolar diameter were determined after each intervention as outlined above. The concentrations of acetylcholine and nitroglycerin used in these experiments were based on previous studies in our laboratory (6,7). Drugs. Lipopolysaccharide (Escherichia coli serotype 011 l:B4), indomethacin, fi-L-nitro arginine, acetylcholine and allopurinol were purchased from Sigma Chemical Co. (St. Louis, MO). Nitroglycerin was purchased from Du Pont Pharmaceuticals (Wilmington, DE). SK&F 108566 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). All drugs were prepared daily before each experiment. Statistical analysis. Data are presented as meanfSEM. Analysis Newman-Keuls test for multiple comparisons were used to compare experimental groups. A p value of less than 0.05 was considered significant.

of variance and the within and between

Results Effects of lioonolvsaccharide on arteriolar diameter. Suffusion of lipopolysaccharide was associated with a biphasic response: significant vasoconstriction, which was maximal within 30 min, followed by significant vasodilation, which was maximal within 60 min (-17+ 4% and 25*7% change from baseline, respectively: Fig. 1; n=6; ~~0.05). These responses were reversible once suffusion of lipopolysaccharide was stopped. Effects of indomethacin on lioopolvsaccharide-induced resnonses. Indomethacin significantly attenuated lipopolysaccharide-induced vasodilation (25+7% before vs. -lf5% after indomethacin; Fig. 1; n=6; ~~0.05). However, indomethacin had no significant effects on lipopolysaccharide-induced vasoconstriction (-17*4% before vs. -14f4% after indomethacin; Fig. 1; n=6; ~~0.05). Effects of SK&F 108566 on lipopolvsaccharide-induced resuonses. SK&F 108566 significantly attenuated lipopolysaccharide-induced vasoconstriction (-17*4% before vs. -6+2% after SK&F 108566; Fig. 1; n=6; p
1246

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Lipopolysaccharide and Arteriolar Diameter

#

-60 !

60

30 TIME (min)

Fig. 1 Responses of cheek pouch arterioles to topical application of lipopolysaccharide (3 /.tg/ml) for 60 min alone (open bars), and in the presence of indomethacin (10 mg/kg; closed bars) and SK&F 108566 (0.1 PM; hatched bars). Values are meanfSEM; n=6; *p
Effects of fi-L-nitro arginine on linouolvsaccharide-induced resnonses. fl-L-nitro arginine had no significant effects on lipopolysaccharide-induced vasoconstriction (-34+4% before, vs. -3of3% after fl-L-nitro arginine; Fig. 2; n=4; p>O.5) or vasodilation (36+3% before, vs. 24+_10% after fl-L-nitro arginine; Fig. 2; n=4; p>O.5).

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SO-

3 !z

zo-

W

!z 3 &

*

60 -

*

40 -

O-20 -40 -60 ’

*

*

# 30

60 TIME (min)

Fig. 2 Responses of cheek pouch arterioles to topical application of lipopolysaccharide 3 yg/ml) for 60 min in the absence (open bars) and presence (closed bars) of N h -L-nitro arginine (100 PM). Values are meadSEM; n=4; *pO.5). However, it significantly attenuated lipopolysaccharideinduced vasodilation (35f4% before, vs. 12rtlO% after allopurinol; Fig. 3; n=4; ~~0.05).

Lipopolysaccharide

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E 5 2

SO-

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3

20O-

3

-20 -

T

-40 -

3

-60

s

Diameter

1241

*

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fz

and Arteriolar

-r

*

t

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30 TIME

(min)

Fig. 3 Responses of cheek pouch arterioles to topical application of lipopolysaccharide (3 pg/ml) for 60 min in the absence (open bars) and presence (closed bars) of allopurinol (30 mg/kg). Values are meat&EM; n=4; *p
Effects of linonolvsaccharide on acetvlcholine- and nitroelvcerin-induced vasodilation. Acetvlcholine induced siwificant vasodilation that was significantly potentiated after suffusion of lipocolysaccharide (29+5% before vs. 57fll% after lip~polysaccharide; Fig. 4; n=4; ~~0.05). Acetylcholine-induced vasodilation was significantly attenuated by @-L-nitro arginine (29+5% before vs. 8f2% after NG-L-nitro arginine; Fig. 4; n=4; p
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6 Acetylcholine

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Nitroglycerin

Fig. 4 Responses of cheek pouch arterioles to topical application of acetylcholine (10 j,tM) and nitroglycerin (10 PM) for 5 mm before (open bars) and after topical application of lipopolysaccharide (3 pg/ml) for 60 min (closed bars) or @-L-nitro arginine (100 PM; hatched bars). Values are meanSEM; n=4; *pcO.O5 in comparison to baseline; tpc0.05 in comparison to acetylcholine and nitroglycerin alone; #p
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Discussion The results of the present study show that 1 h exposure of resistance arterioles to lipopolysaccharide in situ is associated with an immediate biphasic response: vasoconstriction followed by vasodilation. The former was mediated, in part, by angiotensin II and the latter by dilator prostaglandins. Our data suggest that two mechanisms mediated the prostaglandin-induced vasodilation: a direct effect, because indomethacin abrogated lipopolysaccharide-induced vasodilation, and an indirect effect through release of reactive oxygen species, because allopurinol also attenuated this response. These responses were recorded in the absence of any significant changes in systemic arterial blood pressure. Collectively, these data suggest that short-term exposure of the peripheral microcirculation to lipopolysaccharide in situ is associated with an ischemia-reperfusion-like injury. These responses may contribute to tissue ischemia and end organ failure observed several hours after exposure to lipopolysaccharide. The hamster cheek pouch is a well-established animal model to study the effects of vasoactive mediators, including lipopolysaccharide, on microvascular contractile function in situ (6-14). In each hamster, we examined the contractile responses of the same segment of a secondorder arteriole to lipopolysaccharide and drugs. Because systemic arterial blood pressure was essentially unchanged during the course of these experiments, it is unlikely that the effects of the drugs used in this study to modulate lipopolysaccharide-induced responses were related to changes in microvascular pressure. In addition, the potential interactions between local regulatory mechanisms and the arteriolar segment under study, and the interanimal variability in arteriolar responses to lipopolysaccharide and drugs should not have had any systematic effects on the results, because each animal served as its own control. Previous studies showed that systemic administration of lipopolysaccharide over a relatively short period of time was associated with a significant increase in plasma concentrations of angiotensin II and eicosanoids (2, 3). However, the relationship between release of these proinflammatory mediators and vasomotor tone was not investigated in these studies. The results of the present study indicate that both angiotensin II and prostaglandins play a role in regulating vasomotor tone in the peripheral microcirculation early in the course of endotoxemia. Baker and Sutton (4) studied the effects of increasing concentrations of acetylcholine on resistance arterioles in the rat cremaster muscle in vivo before and 1 h after intra-arterial bolus administration of lipopolysaccharide. They found that maximum diameter in response to acetylcholine was greater post-lipopolysaccharide. The results of the present study support and extend these observations by showing that acetylcholine-induced vasodilation in the cheek pouch was potentiated after topical application of lipopolysaccharide. Like acetylcholine, nitroglycerin, a nitric oxide donor, induced a greater increase in arteriolar diameter post-lipopolysaccharide. Collectively, these data indicate that lipopolysaccharide-induced responses in the cheek pouch are not related to non-specific impairment of arteriolar reactivity. The mechanisms that mediate the release of angiotensin II, dilator prostaglandins and reactive oxygen species in the cheek pouch during suffusion of lipopolysaccharide were not investigated in the present study. It is conceivable that several cells in and around cheek pouch arterioles could be involved in this process. For example, lipopolysaccharide could activate perivascular mast cells and intravascular polymorphonuclear leukocytes to release proteinases, such as chymase and cathepsin G, respectively, that are known to convert angiotensin I to angiotensin II very effectively (16). Angiotensin I-converting enzyme is less likely to play a significant role in this putative cascade because lipopolysaccharide inhibits enzyme activity in endothelial cells (17). Finally, endothelial cells, polymorphonuclear leukocytes and other cells in the peripheral microcirculation have been shown to release prostaglandins and reactive oxygen species upon exposure to lipopolysaccharide (2, 6, 12, 13). Clearly, additional studies are indicated to determine the mechanisms whereby these compounds are generated and released in the cheek pouch during suffusion of lipopolysaccharide. In summary, we found that l-h exposure to lipopolysaccharide is associated with immediate and profound effects on vasomotor tone in the peripheral microcirculation in situ. We

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that these responses contribute to tissue ischemia and end organ failure in sepsis syndrome observed several hours after the inciting stimulus. suggest

Acknowledements This study was supported, in part, by grants from the National Institutes of Health (DE10347 and DE00386), The Laerdal Foundation for Acute Medicine and American Heart Association-Nebraska Affiliate (NE-92-GS-6). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17.

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