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J Appl Physiol 94: 2313-2322, 2003. First published February 21, 2003; doi:10.1152/japplphysiol.00047.2002
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Vol. 94, Issue 6, 2313-2322, June 2003

Mesenteric microvascular inflammatory responses to systemic hypoxia are mediated by PAF and LTB4

Alfred J. Casillan, Norberto C. Gonzalez, Jennifer S. Johnson, Dawn R. S. Steiner, and John G. Wood

Department of Molecular and Integrative Physiology, The University of Kansas Medical Center, Kansas City, Kansas 66160


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Systemic hypoxia produces a rapid microvascular inflammatory response characterized by increased reactive oxygen species (ROS) levels, leukocyte-endothelial adherence and emigration, and increased vascular permeability. The lipid inflammatory mediator leukotriene B4 (LTB4) is involved in the early hypoxia-induced responses (ROS generation and leukocyte adherence). Whether other lipid inflammatory mediators participate in this phenomenon is not known. The objective of these experiments was to study the role of platelet-activating factor (PAF) in the microvascular inflammatory response to hypoxia and its potential interactions with LTB4 in this response. Intravital microscopy was used to examine mesenteric venules of anesthetized rats. We found that WEB-2086, a PAF receptor antagonist, completely prevented the increase in ROS levels and leukocyte adherence during a brief reduction in inspired PO2 to anesthetized rats; administration of either WEB-2086 or the LTB4 antagonist LTB4-DMA attenuated leukocyte emigration and the increase in vascular permeability to the same extent during prolonged systemic hypoxia in conscious rats. Furthermore, no additive effect was observed in either response when both antagonists were administered simultaneously. This study demonstrates a role for PAF in the rapid microvascular inflammatory response to hypoxia, as well as contributions of PAF and LTB4 to the slowly developing responses observed during sustained hypoxia. The incomplete blockade of the hypoxia-induced increases in vascular permeability and leukocyte emigration by combined administration of both antagonists indicates that factors in addition to LTB4 and PAF participate in these phenomena.

leukocyte-endothelial adhesive interactions; leukocyte emigration; microcirculation; intravital microscopy; inflammatory mediators


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PREVIOUS STUDIES FROM OUR laboratory have demonstrated that systemic hypoxia after a reduction in inspired PO2 causes a rapid microvascular inflammatory response characterized by an increase in the number of adherent leukocytes within mesenteric venules of anesthetized rats (37, 48). In addition, leukocyte emigration and increased vascular permeability occur in the mesenteric microcirculation of conscious rats exposed to the same inspired PO2 for 4 h (46). These responses are accompanied by an increase in reactive oxygen species (ROS)-dependent fluorescence (37, 38, 47) and are blocked by antioxidants and by exogenous nitric oxide (NO) administration (46, 47), suggesting that alterations in the ROS-NO balance may play a role in this process. A potential consequence of changes in this balance is local formation of lipid inflammatory mediators, which can occur rapidly and promote microvascular inflammation (22, 25, 40). Evidence to support this view is provided by our laboratory's recent demonstration that leukotriene B4 (LTB4) contributes to the rapid increases in leukocyte-endothelial adhesive interactions during 10 min of systemic hypoxia (37). However, the potential involvement of LTB4, or of other lipid inflammatory mediators, in microvascular inflammatory responses during a more prolonged period of hypoxia is not known. To our knowledge, there are no reports supporting a role for any lipid inflammatory mediator in hypoxia-induced changes in leukocyte emigration or increased vascular permeability.

The overall objective of this study was to continue our work on the role of lipid inflammatory mediators in the microvascular inflammatory response to hypoxia. Specifically, we investigated whether platelet-activating factor (PAF), a proinflammatory phospholipid, participates in leukocyte recruitment and changes in vascular permeability during systemic hypoxia. Hypoxia has been reported to stimulate PAF release from endothelial cells in vitro (12) and to promote PAF-dependent neutrophil adherence to cultured endothelial cells (2). In the present study, intravital microscopy was used to measure microvascular ROS levels, to directly visualize adhesive interactions of circulating leukocytes with mesenteric venules, and to measure vascular permeability to albumin. Experiments were designed to examine whether 1) administration of WEB-2086, a PAF receptor antagonist, attenuates the increases in ROS and in leukocyte adherence within mesenteric venules of anesthetized rats during a brief reduction in inspired PO2 and 2) administration of WEB-2086 and the LTB4 receptor antagonist LTB4-dimethyl amide (DMA), alone or in combination, attenuate leukocyte emigration and the increase in vascular permeability during more prolonged systemic hypoxia in conscious rats.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Sprague-Dawley rats weighing 225-370 g were purchased from Sasco (Omaha, NE). All surgical and experimental procedures were approved by the Animal Care and Use Committee of the University of Kansas Medical Center. The University of Kansas is accredited by the American Association for the Accreditation of Laboratory Animal Care. Guidelines set by the National Institutes of Health and the Public Health Service Policy on the humane use and care of laboratory animals were followed at all times.

Surgical Procedures

Experiments in anesthetized rats. After an overnight fast with free access to water, rats were anesthetized by an injection of urethane (1.5 g/kg im). Polyethylene cannulas (PE-50) were inserted into a jugular vein and a carotid artery. A tracheotomy was performed, and the trachea was intubated by using polyethylene tubing (PE-240). The abdomen was opened along the midline by using a radiocautery (Harvard Apparatus, Holliston, MA). Lactated Ringer solution was infused via the jugular vein (2 ml/h), while blood pressure was continuously measured by connecting the carotid artery cannula to a digital blood pressure monitor (Micro-Med, Louisville, KY). Experiments were begun after a postoperative stabilization period of ~20 min.

Experiments in conscious rats. On the day before the experiments, rats were given a short-acting anesthetic (xylazine-ketamine, 2 mg/kg im), and polyethylene catheters (PE-50) were placed into a jugular vein and a carotid artery. The catheters were tunneled subcutaneously to the back of the neck, exteriorized, cut at a length of 2 in., and then flame-sealed. Once these surgical procedures were completed, the animals were placed on a heating pad to maintain their body temperature during recovery from the anesthetic. The rats were then placed into individual cages and fasted overnight with free access to water.

Adhesive Interactions of Circulating Leukocytes in Mesenteric Venules

The animal was positioned on a Plexiglas sheet on the stage of a Zeiss Axiovert inverted microscope. A section of the small intestine was carefully exteriorized and positioned over a glass coverslip on the Plexiglas sheet to view a mesenteric venule. Mesenteric venules were selected on the basis of the following criteria: 1) straight, unbranched vessels at least 100 µm in length; 2) diameters of 20-40 µm; and 3) no adjacent vessels within 100 µm of the venule. The exposed intestinal loop was covered with Saran wrap to prevent drying and to minimize the effect of ambient O2 on the mesentery. Images of mesenteric venules (×40 objective, ×10 eyepiece) were recorded by using a S-VHS videocassette recorder (JVC, Elmwood Park, NJ) with a time-date generator and a video camera (Panasonic, Secaucus, NJ). Fluorescent images were obtained by use of an intensified charge-coupled device (ICCD) camera (C2400, Hamamatsu Photonics, Shizuoko, Japan) with an excitation wavelength of 420-490 nm and an emission wavelength of 520 nm. To minimize photobleaching, the duration of recordings was <15 s in a given area of the microcirculation.

Venular diameter was measured by using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX). An optical Doppler velocimeter (Microcirculation Research Institute) was used to measure the centerline red blood cell (RBC) velocity in venules. Average RBC velocity was calculated as the centerline velocity divided by 1.6 (14). Wall shear rate, which represents the physical force generated at the vessel wall due to movement of blood, was calculated as 8 × (average RBC velocity/venular diameter) (19). During playback of video recordings, the number of adherent leukocytes was determined in each minute by counting the number of leukocytes that remained stationary for longer than 30 s.

Measurement of Leukocyte Emigration Across Mesenteric Venules

Leukocyte emigration was assessed by counting the number of leukocytes in an area defined by a length 100 µm parallel to the venule and a width 40 µm perpendicular to the venule. Emigration was then expressed as the number of extravascular leukocytes per 4 × 103 µm2. Three to five venules in each rat were analyzed to determine numbers of emigrated leukocytes, and these values were then averaged to obtain a single estimate for each animal.

Measurement of Vascular Permeability Index

Vascular permeability to albumin was measured as described by our laboratory (39, 46) and others (17, 22, 23). Fluorescence intensity from the FITC-labeled albumin was measured during playback of the recorded images. A digital image-analysis program (NIH Image 1.61) was used to measure the fluorescence intensity in three contiguous areas within the venule and in three areas in the adjacent perivascular regions. Each of these analyzed areas was a circle with a diameter equal to that of the venule. The measurement of fluorescence intensity was carried out in the following way: the first circle to be measured by image analysis was placed in the center of the vessel; after this, two additional circles, one on each side of the first circle, were placed inside the vessel; the three circles were immediately adjacent to one another. Circles of equal diameter were placed on the perivascular space on each side of the vessel adjacent to each of the intravascular circles. This approach provides an average value of fluorescence intensity over a fairly large area of the intra- and extravascular fields. In this manner, the influence of the heterogeneous microvascular leakage of albumin on the estimation of the fluorescence intensity was minimized. The values obtained were then averaged, and a vascular permeability index was calculated as the ratio of extravascular to intravascular fluorescence intensity. Values for the vascular permeability index from three to five venules in each rat were averaged to obtain a single estimate of vascular permeability in that animal (46).

Measurement of Microvascular ROS Levels

As previously reported (37, 38, 47), the oxidant-sensitive probe dihydrorhodamine (DHR) was used to quantitate ROS levels within mesenteric venules. Background fluorescence of mesenteric venules before injection of DHR did not differ between experimental groups. DHR fluoresces by forming rhodamine 123 when oxidized, principally by hydrogen peroxide-dependent reactions. The fluorescence produced by rhodamine 123 was detected by using an ICCD camera (Hamamatsu Photonics). Recordings of the DHR fluorescence were made during brief intervals (<15 s) to avoid light-induced activation of the probe. The fluorescence intensity was later measured during playback of videotapes by use of image-analysis software (NIH Image, 1.61). The intensity of the fluorescent signal was measured in five contiguous circles of 5-µm diameter along each side of the vessel and was averaged to obtain a single estimate of the fluorescent signal during each experimental period. Each circle was positioned such that the vessel wall was in its center. The same field of view was maintained throughout the experiment to ensure that measurements of DHR fluorescence were obtained in the same section of the venule under each experimental condition. Values for fluorescence during treatment periods were expressed relative to the value observed during the normoxic control period, which was defined as 100%.

Experimental Protocols

Effect of WEB-2086 on oxidant-induced increases in DHR fluorescence. The goal of these experiments was to determine whether WEB-2086 interferes with oxidant-induced DHR fluorescence. In these experiments, 0.15 ml of the oxidant t-butyl hydroperoxide (10-9, 10-6, or 10-3 M), with or without WEB-2086 (20 µg/ml), was added to a well containing 0.15 ml of the DHR solution. Fluorescence intensity was measured during ~15 s after the DHR solution was placed in the well and immediately after addition of the oxidant.

Effect of WEB-2086 on PAF-induced leukocyte adherence. After a control period, the fluid over the mesentery was carefully removed with the use of gauze, and the mesentery was then superfused with a solution containing PAF. Increasing concentrations of PAF (1, 10, and 100 nM) were given for 10 min each. Changes in leukocyte adherence and shear rate within mesenteric venules were determined in response to each PAF concentration. In separate experiments, an intravenous (iv) infusion of the PAF receptor antagonist WEB-2086 (250 µg · kg-1 · min-1) (6) was begun 20 min before administration of PAF.

Effect of iv administration of the LTB4 antagonist LTB4-DMA on LTB4-induced leukocyte adherence. In the first group of animals, the mesentery was superfused with 0 and 20 nM LTB4 for 10-min periods, with a 10-min recovery period between each concentration. During the recovery period, the superfusate was carefully removed from the mesentery, which was then rinsed with saline.

The ability of the LTB4 receptor antagonist LTB4-DMA to attenuate LTB4-induced leukocyte adherence was examined in a separate group of animals. The protocol of these experiments was the same as described above except that LTB4-DMA was infused iv (1 µM, 2 ml/h) throughout the experiment.

Effect of WEB-2086 on the hypoxia-induced increase in ROS levels. Animals were randomly assigned to either untreated or WEB-2086-treated groups. The experimental protocol consisted of a 10-min control period, a 20-min equilibration period after iv injection of DHR with iv infusion of saline or WEB-2086 (250 µg · kg-1 · min-1), a 10-min hypoxic period, and a 10-min normoxic recovery period. Recordings of DHR fluorescence were made for ~15 s at the end of each period. Therefore, the time from injection of DHR to the first fluorescence recording during normoxia was 20 min, which was followed by 10-min intervals between recordings made at the end of the hypoxia and normoxic recovery periods.

Effect of PAF receptor blockade on hypoxia-induced leukocyte adherence. The animals spontaneously breathed room air or hypoxic gas mixtures through a two-way valve (2384 series, Hans Rudolph, Kansas City, MO), which was attached to the tracheal tube before the experiment. After a 10-min control period in which animals breathed room air, an iv infusion of either saline or WEB-2086 (250 µg · kg-1 · min-1, 2 ml/h) was begun and continued for the remainder of the experiment. Twenty minutes later, systemic hypoxia was produced by having the rats breathe from a bag containing a mixture of 10% O2-90% N2 (PO2 ~70 Torr) for 10 min. The O2 concentration of the gas mixture was determined with an Applied Electrochemistry O2 analyzer. Finally, the rats breathed room air again during a 10-min recovery period.

Effect of WEB-2086, LTB4-DMA, and WEB-2086 + LTB4-DMA on hypoxia-induced leukocyte emigration and increased vascular permeability. Conscious rats that had previously been implanted with arterial and venous catheters were placed into a Plexiglas chamber where either room air or 10% O2-90% N2 was circulated for a period of 4 h. Saline was infused iv (2 ml/h) to the rats breathing room air throughout the experiment. The rats breathing 10% O2 were infused with either saline or WEB-2086 (250 µg · kg-1 · min-1) at a rate of 2 ml/min beginning at the onset of hypoxia. After 3.5 h, all rats were given an iv injection of FITC-labeled albumin (50 mg/kg). After 4 h, the animals were quickly anesthetized with an iv injection of urethane (1.5 g/kg) and prepared for intravital microscopy. All animals breathed room air during surgical procedures and subsequent observation of the mesenteric microcirculation. The time elapsed between the induction of anesthesia and completion of the experiment was 10-15 min. Therefore, the total time from injection of FITC-albumin to completion of fluorescence recordings was 40-45 min.

The same protocol was followed in the experiments studying the effects of LTB4 receptor blockade on leukocyte emigration and vascular permeability. In these experiments, LTB4-DMA was infused iv (1 µM) at a rate of 2 ml/min beginning at the onset of hypoxia.

In a separate group of animals following the same experimental protocol described above, WEB-2086 (250 µg · kg-1 · min-1) and LTB4-DMA (1 µM) were simultaneously infused iv at a rate of 2 ml/h beginning at the onset of hypoxia.

Drugs and Chemicals

The commercial sources for drugs used in this study were PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine), t-butyl hydroperoxide, BSA, PBS, and urethane, Sigma Chemical (St. Louis, MO); LTB4-DMA, Biomol (Plymouth Meeting, PA); LTB4, Cayman Chemical (Ann Arbor, MI); DHR, Molecular Probes (Portland, OR); and WEB-2086, Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT).

A stock solution of PAF (2 mg/ml in chloroform) was stored at -35°C until use. An aliquot of this PAF stock solution was evaporated to dryness under a gentle stream of air immediately before each experiment. PAF was resuspended in PBS (pH 7.4) containing 0.2% BSA to minimize its adherence to glass and plastic. The solution was ultrasonicated for 1 min to resolubilize PAF. All other solutions were prepared on the day of the experiment.

Statistical Analysis

Means and standard errors were calculated for all values from each treatment group. The statistical significance of the observed differences was evaluated by use of a statistical analysis program (Statistix 4.0, Analytical Software, St. Paul, MN). Analysis of variance with Bonferroni correction for multiple comparisons was used to compare groups. Values of P < 0.05 were considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of WEB-2086 on Oxidant-Induced Increases in DHR Fluorescence

Table 1 shows the effect of increasing concentrations of t-butyl hydroperoxide on DHR fluorescence intensity in the presence and absence of WEB-2086. The concentration of WEB-2086 utilized was approximately 10 times higher than the estimated plasma concentration attained during iv infusion to the rats. In the absence of WEB-2086, fluorescence intensity, as determined by image analysis, increased with higher concentrations of the oxidant. The presence of WEB-2086 did not influence oxidant-induced increases in DHR fluorescence intensity, indicating that WEB-2086 does not have detectable antioxidant activity at this concentration.

                              
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  		Table 1.   Effect of WEB-2086 on t-butyl hydroperoxide-induced DHR fluorescence intensity in vitro

Effect of WEB-2086 on PAF-Induced Leukocyte Adherence

These experiments verified the effectiveness of WEB-2086 in attenuating PAF-induced leukocyte adherence as shown previously by others (42). Superfusion of the mesentery with PAF produced statistically significant, dose-related increases in the number of adherent leukocytes within mesenteric venules (Table 2). In animals infused with WEB-2086, there were no significant increases in leukocyte adherence in response to the same concentrations of PAF (Table 2).

                              
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  		Table 2.   Effect of WEB-2086 on PAF-induced leukocyte adherence in vivo

Effect of IV Administration of the LTB4 Antagonist LTB4-DMA on LTB4-Induced Leukocyte Adherence

In a previous study, we demonstrated that topical administration of LTB4-DMA over the mesentery completely prevented LTB4-induced leukocyte adherence to mesenteric venules (37). In the present study, experiments were designed to evaluate whether combined administration of WEB-2086 plus LTB4-DMA resulted in significantly greater attenuation of hypoxia-induced leukocyte emigration and increased vascular permeability compared with the effect of either antagonist given alone. Because these experiments involved a 4-h period of systemic hypoxia in conscious rats, it was not possible to utilize topical administration of LTB4-DMA as we had done previously. We evaluated the effect of iv administration of the LTB4 antagonist on responses to LTB4. As expected on the basis of our previous results (37), LTB4 significantly increased the number of adherent leukocytes in mesenteric venules (Table 3). Intravenous administration of LTB4-DMA completely blocked the increase in leukocyte adherence in response to 20 nM LTB4.

                              
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  		Table 3.   Efficacy of intravenous administration of LTB4-DMA on LTB4-induced leukocyte adherence in vivo

Effect of WEB-2086 on the Hypoxia-Induced Increase in ROS Levels

Figure 1 shows the average values of DHR fluorescence intensity observed during the normoxic control period, after 10 min of hypoxia, and 10 min after recovery from hypoxia in untreated rats and in rats pretreated with WEB-2086. As expected from our previous results (37, 38, 47), hypoxia was accompanied by an increase in DHR fluorescence intensity (P < 0.05, hypoxia vs. control, n = 6). Normoxic recovery resulted in a significant reduction of DHR fluorescence activity (P < 0.05 hypoxia vs. recovery, n = 6). Pretreatment with WEB-2086 completely blocked the increase in ROS during hypoxia (P > 0.05, hypoxia plus WEB-2086 vs. normoxia plus WEB-2086, n = 6).


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  		Fig. 1.   Reactive oxygen species (ROS)-dependent fluorescence in mesenteric venules in untreated (n = 6) and WEB-2086-treated rats (n = 5). Hypoxia significantly increased ROS levels in untreated rats but not in animals given the platelet-activating factor (PAF) receptor antagonist. During the normoxic recovery period, there was no significant difference in ROS levels in either group compared with baseline values. * P < 0.05 vs. normoxic control. + P < 0.05 vs. untreated hypoxia.

Effect of PAF Receptor Blockade on Hypoxia-Induced Leukocyte Adherence

Figure 2 shows the average values of leukocyte adherence (A) and shear rate for six untreated rats and six rats pretreated with WEB-2086. During the normoxic control period, the number of adherent leukocytes was not significantly different from zero in either group (A). Hypoxia produced a rapid increase in leukocyte adherence within mesenteric venules of the untreated rats. Compared with baseline values, the number of adherent leukocytes was significantly greater at 5 and 10 min after the onset of hypoxia. This response was accompanied by a significant decrease in shear rate compared with normoxia control values (B). No significant change in leukocyte adherence was observed during the normoxic recovery period compared with the value at the end of hypoxia.


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  		Fig. 2.   Number of adherent leukocytes per 100 µm of mesenteric venule (A) and venular shear rate (B) during the normoxic control (room air), 10 min of hypoxia (10% O2), and the normoxic recovery periods. * P < 0.05 untreated vs. the WEB-2086-treated group.

In contrast, Fig. 2 illustrates no significant increase in the number of adherent leukocytes during hypoxia in animals given WEB-2086. Values for leukocyte adherence were significantly different between the untreated vs. WEB-2086-treated animals at 5 and 10 min of the hypoxia period. Shear rate also decreased significantly during hypoxia compared with the normoxia period in animals given WEB-2086 (Fig. 2B). There were no significant differences in shear rate between the untreated and WEB-2086-treated groups at any time during the experiment.

Effect of WEB-2086, LTB4-DMA, and WEB-2086 Plus LTB4-DMA on Hypoxia-Induced Leukocyte Emigration and Increased Vascular Permeability

Figure 3 shows representative photomicrographs of mesenteric venules of untreated normoxic rats and from rats exposed to hypoxia for 4 h with either no treatment or administration of receptor antagonists to PAF and LTB4 alone and in combination. Figure 4 shows the average values obtained in these groups. Virtually no emigrated leukocytes were observed in the normoxic rats. In contrast, the number of emigrated leukocytes was significantly greater in the untreated hypoxia group than in the normoxia group. Administration of WEB-2086 significantly, although not completely, attenuated hypoxia-induced leukocyte emigration (P < 0.05, hypoxia plus WEB-2086 vs. untreated hypoxia). Administration of LTB4-DMA during hypoxia reduced leukocyte emigration to values not significantly different from those obtained with WEB-2086. Furthermore, combined administration of WEB-2086 plus LTB4-DMA did not produce a larger decrease in hypoxia-induced leukocyte emigration: whereas the number of emigrated leukocytes in this case was lower than that seen in the untreated hypoxia group, it was not significantly different from that seen after individual administration of either WEB-2086 or LTB4-DMA.


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  		Fig. 3.   Representative photomicrographs showing the effects of WEB-2086 and leukotriene B4 (LTB4)-dimethyl amide (DMA), alone or in combination, on leukocyte emigration in the mesenteric microcirculation of rats exposed to either normoxia or hypoxia for 4 h in the conscious state. Arrows indicate emigrated leukocytes. The 2 large black dots are used to align the Doppler velocimeter. The distance between the white lines represents a distance of 100 µm.



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  		Fig. 4.   Leukocyte emigration in normoxic controls (n = 5), untreated conscious rats exposed to hypoxia for 4 h (n = 6), and conscious rats exposed to hypoxia for 4 h and treated with either WEB-2086 (n = 6), LTB4-DMA (n = 5), or WEB-2086 plus LTB4-DMA (n = 5). * P < 0.05 vs. normoxia; + P < 0.05 vs. untreated hypoxia.

Figure 5 presents representative photographs showing FITC-albumin fluorescence in a rat from each experimental group. The nonuniform appearance of fluorescence intensity along the vessel wall during hypoxia is typical of the heterogeneous distribution of the vascular leakage of macromolecules observed in response to numerous agents (4, 29, 43). The photograph from the untreated hypoxic rat shows a nonuniform appearance in the perivascular tissue; a similar pattern has been observed after extensive leakage and attributed to accumulation of FITC-albumin in the collagen interstitial matrix of the mesentery (17).


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  		Fig. 5.   Representative photomicrographs showing the effects of WEB-2086 and LTB4-DMA, alone or in combination, on vascular permeability to FITC-albumin in the mesenteric microcirculation of rats exposed to either normoxia or hypoxia for 4 h in the conscious state.

Figure 6 presents the average data for vascular permeability, estimated as the ratio of fluorescent intensity outside the vessel to the fluorescent intensity inside the vessel. In the normoxia group, the vascular permeability index was 0.09 ± 0.03. After 4 h of hypoxia, the extra- to intravascular ratio of FITC albumin was increased to 0.93 ± 0.12, indicating a marked increase in vascular permeability (P < 0.05, untreated hypoxia vs. normoxia). Administration of WEB-2086 significantly attenuated the increase in vascular permeability during hypoxia: the vascular permeability index was 0.66 ± 0.11 (P < 0.05, untreated hypoxia vs. hypoxia plus WEB-2086; P < 0.05, hypoxia plus WEB-2086 vs. normoxia). Administration of LTB4-DMA had an effect almost identical to that of WEB-2086 on the hypoxia-induced increase in vascular permeability, with the permeability index being 0.69 ± 0.08 (P < 0.05, untreated hypoxia vs. hypoxia plus LTB4-DMA; P < 0.05, hypoxia plus LTB4-DMA vs. normoxia). In addition, combined administration of WEB-2086 and LTB4-DMA during hypoxia did not attenuate the increase in vascular permeability to a greater extent than that seen when either agent was given alone; the vascular permeability index in this case was 0.73 ± 0.04 (P > 0.05, hypoxia plus WEB-2086 vs. hypoxia plus WEB-2086 and LTB4-DMA; P > 0.05, hypoxia plus LTB4-DMA vs. hypoxia plus WEB-2086 and LTB4-DMA). When the extensive leakage of FITC albumin during hypoxia was attenuated by the treatments applied, the pattern of extravascular fluorescence intensity became more homogeneous, as shown in Figure 5.


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  		Fig. 6.   Vascular permeability in normoxic controls (n = 5), untreated conscious rats exposed to hypoxia for 4 h (n = 6), and conscious rats exposed to hypoxia for 4 h and treated with either WEB-2086 (n = 6), LTB4-DMA (n = 5), or WEB-2086 plus LTB4-DMA (n = 5). The vascular permeability index is the ratio of extra- to intravascular FITC-albumin fluorescence intensity. Fluorescence intensity was determined by image analysis. * P < 0.05 vs. normoxia; + P < 0.05 vs. untreated hypoxia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of this study are 1) the PAF receptor antagonist WEB-2086 blocked the hypoxia-induced ROS increase and leukocyte adherence to mesenteric venules of anesthetized rats during a brief period of systemic hypoxia; 2) WEB-2086 significantly attenuated, although did not completely prevent, leukocyte emigration and the increased vascular permeability in conscious rats during 4 h of hypoxia; 3) the LTB4 receptor antagonist LTB4-DMA attenuated hypoxia-induced leukocyte emigration and increased vascular permeability to the same degree as did WEB-2086; and 4) simultaneous administration of WEB-2086 and LTB4-DMA did not result in further attenuation of hypoxia-induced leukocyte emigration or increased vascular permeability than observed with either antagonist given alone.

These experiments were carried out by using two basic experimental protocols: anesthetized animals to study early responses to hypoxia and conscious animals to study slowly developing events. As with all experimental approaches, each has its advantages and limitations. To directly observe ROS levels and leukocyte adherence in the mesenteric microcirculation, the small intestine must be exteriorized, which necessitates the use of anesthesia. Because increased leukocyte-endothelial adherence and ROS generation occur within minutes of the onset of systemic hypoxia, the anesthetized preparation is best suited for documenting these responses. On the other hand, leukocyte emigration and extravasation of FITC-albumin due to increased vascular permeability are slowly developing phenomena. To study the effect of hypoxia on these events, we elected to use a conscious animal model. Because indwelling catheters had been previously implanted to administer anesthetic iv, the animal could be rapidly prepared for intravital microscopy, such that experimental observations could be completed within 10-15 min after induction of anesthesia. During this time in which the animal was anesthetized and breathing room air, it is theoretically possible that vascular permeability and leukocyte-endothelial interactions may have changed from those occurring during the conscious hypoxic state. However, the short time elapsed before measurements were completed makes it extremely unlikely that these possible changes would have substantially altered either the number of leukocytes present in the interstitium or the extravascular concentration of FITC-albumin. In the present experiments, we elected to use hypoxic exposure times of 10 min and 4 h, bearing in mind that microvascular inflammatory responses may evolve in the intervening time as different factors may come into play. Nevertheless, although other factors may have contributed, the results presented here, in conjunction with our previous study (37), clearly show a role for PAF and LTB4 in both the rapid as well as in the slowly developing microvascular inflammatory responses to hypoxia.

Our first observation is consistent with local generation of PAF as an important event involved in the early microvascular inflammatory response to systemic hypoxia. The tendency for leukocytes to adhere to the venular wall depends on the balance between proadhesive forces vs. the hydrodynamic dispersal forces (16). The latter represent the tendency of flowing blood to push leukocytes along the endothelial surface and can be estimated from the venular wall shear rate. Because there were no significant differences in shear rate between untreated- and WEB-2086-treated animals at corresponding times throughout the experiments, the ability of the PAF antagonist to attenuate hypoxia-induced leukocyte adherence reflects decreased proadhesive forces, rather than increased hydrodynamic dispersal forces.

The fact that WEB-2086 completely prevented the hypoxia-induced ROS increase and leukocyte adherence indicates that PAF plays an important role in the rapidly developing microvascular inflammatory responses during systemic hypoxia. Hypoxia has been shown to stimulate PAF release from endothelial cells in vitro (12) and to promote PAF-dependent neutrophil adherence to cultured endothelial cells (2). PAF has also been shown to increase ROS generation in isolated neutrophils (8, 10). The present study extends these observations and provides the first demonstration of the involvement of this inflammatory mediator in ROS generation and leukocyte-endothelial adherence during the early stages of hypoxia in vivo.

Both PAF and LTB4 have been shown to participate in the microvascular injury after ischemia and reperfusion, as evidenced by the fact that administration of PAF and LTB4 receptor antagonists attenuated the microvascular damage that occurs during reperfusion after a period of ischemia (16, 22, 33). Although these mediators also have an important role in systemic hypoxia, the different patterns of responses show that the mechanisms underlying the microvascular inflammation of systemic hypoxia and of ischemia-reperfusion are fundamentally different. Although in ischemia-reperfusion ROS levels and leukocyte-endothelial adhesive interactions occur only during the reperfusion period after O2 delivery is restored (16), in systemic hypoxia these phenomena occur only when PO2 is reduced and actually subside during the normoxic recovery period (37, 38, 47). Although the mechanism of ROS generation differs in ischemia-reperfusion and hypoxia, it is possible that in both cases increases in ROS lead to formation of PAF and LTB4, which contribute to microvascular inflammatory responses.

Formation of PAF during hypoxia could potentially occur through several pathways, including activation of phospholipase A2 through calcium-dependent and -independent routes (21, 26, 27, 30). A nonenzymatic route, secondary to ROS generation, has also been demonstrated (34). ROS generation is known to increase rapidly after the onset of hypoxia both in vitro (13, 15) and in vivo (37, 38, 47).

In a previous study (37), our group observed that LTB4-DMA substantially reduced the hypoxia-induced increase in ROS and leukocyte adherence in mesenteric venules. The present study shows that WEB-2086 also completely prevented these responses. The ability of antagonists to either inflammatory mediator to markedly attenuate hypoxia-induced increases in ROS and leukocyte adherence could indicate that subthreshold increases in tissue levels of these inflammatory mediators occur during hypoxia. According to this view, ROS generation and leukocyte adherence would not occur in response to either mediator acting alone, yet LTB4 and PAF acting in concert could elicit these responses. In support of this possibility, similar effects have been reported by other investigators (22, 35). An alternative interpretation of these data is that one mediator stimulates generation of the other. For example, PAF has been reported to increase LTB4 release from blood vessels, leukocytes, and mast cells (3, 7, 20), although conflicting results have also been reported (5). In addition, both mediators stimulate generation of ROS, which, in turn, are known to promote formation of both LTB4 and PAF. Regardless of the mechanism underlying the interaction between PAF and LTB4, our data presented here, along with our previous study (37), clearly demonstrate the involvement of both mediators in the early response to hypoxia as well as during prolonged hypoxia. A role for PAF and LTB4 in the early phase of the inflammatory response to hypoxia is consistent with evidence showing that these inflammatory mediators can be rapidly synthesized, as discussed previously (18, 25, 30, 34).

The second major finding of this study is that WEB-2086 attenuated, but did not totally prevent, the leukocyte emigration and increased vascular permeability that occurred in conscious rats during 4 h of hypoxia. Another finding is the observation that the LTB4 receptor antagonist LTB4-DMA blocked the hypoxia-induced leukocyte emigration and the increase in vascular permeability to the same extent as did WEB-2086. Even though both WEB-2086 and LTB4-DMA (37) completely blocked leukocyte adherence during the first 10 min of exposure to hypoxia, some leukocyte emigration was observed after 4 h of hypoxia in the treated animals. Because adherence is a requisite step in leukocyte emigration, one possible interpretation of this finding is that factors other than PAF and LTB4 contribute to leukocyte adherence in the period between 10 min and 4 h of hypoxia. The elucidation of these factors should be the subject of further research.

The attenuation of the hypoxia-induced increase in vascular permeability by WEB-2086 and LTB4-DMA indicates that both PAF and LTB4 contribute to the increase in vascular permeability during hypoxia. The effects of PAF and LTB4 on vascular permeability are well known (9, 36, 44). The pattern of hypoxia-induced increases in vascular permeability to FITC-albumin is similar to that observed by other investigators with albumin and other macromolecules in various conditions (4, 29, 43): increased permeability occurs at discrete sites along the venular wall, resulting in a heterogeneous pattern of vascular leakage (Fig. 5). Nonuniform accumulation of interstitial FITC-albumin fluorescence, which may be observed during untreated hypoxia (Fig. 5), has also been reported in the mesentery after ischemia/reperfusion (17); these investigators indicated that the patchy distribution of extravascular FITC-albumin after extensive vascular leakage corresponds to the organization of the collagen interstitial matrix.

PAF and LTB4 have been reported to increase vascular permeability in several organs through leukocyte-dependent (41) and -independent pathways (11, 45). In addition, evidence indicates that ROS are involved in the mechanism of PAF- and LTB4-induced alterations in vascular integrity (16, 24). The latter is consistent with our previous observation that antioxidant administration prevents increased vascular permeability in the mesenteric circulation during systemic hypoxia (46).

The partial attenuation of hypoxia-induced leukocyte emigration and increased vascular permeability produced by WEB-2086 and by LTB4-DMA are consistent with several interpretations, including an incomplete receptor blockade. We feel that this is unlikely, however, because the doses of the antagonists used in these experiments completely prevented the leukocyte adherence produced by agonist concentrations that, in the absence of the blocker, resulted in greater leukocyte adherence than observed during systemic hypoxia (Tables 2 and 3). A more likely explanation for the effects observed with WEB-2086 and LTB4-DMA is that agents other than PAF and LTB4 contribute to the responses observed after 4 h of hypoxia. In previous experiments (46), our laboratory observed that continuous administration of antioxidants during 4 h of exposure of conscious rats to hypoxia reduced leukocyte emigration and the increase in vascular permeability to larger extents than those observed in the present experiments. This supports the notion that during this time of exposure to hypoxia, factors other than LTB4 and PAF, but dependent on oxidant generation, participate in these microvascular inflammatory responses. Possible candidates include interleukin-6 (1) and TNF-alpha (13), which are increased only after several hours of hypoxia.

Administration of WEB-2086 and LTB4-DMA attenuated hypoxia-induced increases in leukocyte emigration and vascular permeability to approximately the same degree. On the basis of previous studies, it is not likely that these results can be attributed to a nonspecific action of WEB-2086 to inhibit LTB4 receptors or of LTB4-DMA on PAF receptors (28, 31, 32, 42). Our present results could indicate that PAF and LTB4 act through the same pathway to produce these responses; alternatively, one mediator may stimulate the generation of the other. Simultaneous administration of antagonists to PAF and LTB4 did not result in further attenuation of the leukocyte emigration and vascular permeability responses to hypoxia than those produced by either antagonist given alone. This finding suggests that, if both lipid inflammatory mediators independently act through a common pathway, this pathway must be fully activated by either mediator. The fact that the effects of the individual antagonists were not additive is also consistent with the idea that one mediator generates the other. The documented effects of PAF on LTB4 generation (3, 7, 20) have been referred to above. Regardless of the mechanism underlying the interaction between PAF and LTB4, the data presented here clearly show the involvement of these inflammatory mediators as well as other factors in hypoxia-induced leukocyte emigration and increased vascular permeability.

Although administration of WEB-2086 or LTB4-DMA significantly attenuated hypoxia-induced leukocyte emigration and vascular permeability, both agents had a stronger effect on leukocyte emigration than on vascular permeability. This pattern implies that mediators other than PAF and LTB4 are largely responsible for the effect of hypoxia in vascular permeability and suggest that such permeability changes may result from leukocyte-dependent and leukocyte-independent mechanisms. These results also show that a relatively large increase in vascular permeability may exist without a proportionally elevated leukocyte emigration, indicating a dissociation between these microvascular alterations.

In conclusion, this study provides new information regarding the role of lipid inflammatory mediators in hypoxia-induced microvascular inflammatory responses. Our results show that PAF promotes ROS generation and leukocyte-endothelial adhesive interactions in rat mesenteric venules during systemic hypoxia. In addition, this study presents the first evidence of the involvement of any lipid inflammatory mediator in microvascular inflammatory responses to sustained hypoxia, as shown by the contribution of PAF and LTB4 to leukocyte emigration and increased vascular permeability within the mesenteric microcirculation of conscious animals during 4 h of hypoxia.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-64195 (to J. G. Wood) and HL-39443 (to N. C. Gonzalez) as well as by the American Heart Association Heartland Affiliate Grant 9951289Z (to J. G. Wood).


    FOOTNOTES

Address for reprint requests and other correspondence: J. G. Wood, Dept. of Molecular and Integrative Physiology, Univ. of Kansas Medical Center, Kansas City, KS 66160 (E-mail: jwood2{at}kumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 21, 2003;10.1152/japplphysiol.00047.2002

Received 17 January 2002; accepted in final form 31 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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