INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

INFLAMMATORY LEUKOCYTE RECRUITMENT generally fulfills a crucial function for host defense; however, unrestrained it can cause considerable damage (49). For example, ischemia-reperfusion (I/R) injuries occur in myocardial infarction and stroke and cause inappropriate inflammatory leukocyte recruitment (9, 19, 24, 29, 42).

Nitric oxide (NO) is produced by three different NO synthase (NOS) enzymes and has a variety of functions. Neuronal NOS (nNOS) is primarily localized in nervous tissue, where it generates NO for neurotransmission (35, 36, 38). Inducible NOS (iNOS) can be found in macrophages and other tissues and is thought to generate large amounts of NO in response to tissue stimulation with various cytokines and proinflammatory agents (35, 36, 38). Little is known about the effects of nNOS- or iNOS-generated NO on leukocyte-endothelium interactions (20). NO released by endothelial NOS (eNOS) is a potent vasodilator that regulates mean arterial blood pressure and inhibits platelet and leukocyte adhesion and aggregation (7, 13-15, 31, 36). In I/R injury, the mechanism of inhibition of leukocyte-endothelium interactions by eNOS is not well understood. Our study focused on eNOS-deficient (eNOS^/) mice generated by gene targeting and homologous recombination (45). These mice show significant increases in blood pressure and plasma renin concentration and a significant decrease in heart rate. Leukocyte-endothelium interactions have not been investigated in these mice; however, experiments conducted in a different strain of eNOS^/ mice show slightly increased interactions under baseline conditions (23, 33).

Recently, we reported that estrogen can reduce I/R-mediated injury through stimulation of eNOS activity (46). The activation of eNOS by estrogen involves the interaction of the estrogen receptor with the regulatory subunit of phosphatidylinositol 3-kinase (PI3K). PI3K catalyzes the synthesis of second messenger lipid mediators (2, 39), which recruit proteins containing specific phosphatidylinositol (3,4,5)-trisphosphate-binding or Pleckstrin homology domains, such as phosphatidyl-dependent kinases (PDK)-1 and -2 (8, 48). The PDKs phosphorylate the serine-threonin protein kinase Akt (5). Akt mediates many of the downstream cellular effects of PI3K (11, 12), one of which is the phosphorylation and activation of eNOS (48). Interestingly, this effect does not involve gene transcription or translation.

To test whether the protective effect of estrogen depends on eNOS, we investigated a standardized I/R injury in untreated and estrogen-treated wild-type (WT) and eNOS^/ mice and quantified the leukocyte-endothelium interactions, namely leukocyte rolling velocity, flux, and adhesion, in the venules of the mouse cremaster muscle. Leukocyte rolling velocity is an important parameter regulating successful transition to firm adhesion, because slower rolling leukocytes have longer contact with inflamed endothelium (27) and hence a higher likelihood for arrest (32). The leukocyte recruitment observed after I/R has been shown to be primarily dependent on P-selectin because P-selectin-deficient mice showed no leukocyte-endothelium interactions before or after I/R, and the increase in leukocyte rolling observed in untreated WT mice after I/R could be abolished by administration of an anti-P-selectin antibody (28). Furthermore, several studies have suggested that the P-selectin upregulation caused by I/R in this model and other models of microvascular I/R is regulated by eNOS (10, 16, 22, 25). Therefore, we also investigated P-selectin expression at baseline conditions and after I/R.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. Intravital experiments were performed on a total of 29 male mice 6-14 wk old and weighing 20-40 g. Mice included WT C57BL/6 (Hilltop; Scottsdale, PA) and gene-targeted mice deficient in eNOS (45). eNOS knockout mice were backcrossed into the C57BL/6 background for at least seven generations. Data (red blood cell velocity and leukocyte rolling velocity and adhesion) from five of the eNOS^/ mice used in the control group also appear in a study by Hafezi-Moghadam et al. (18).

Reagents. Estrogen-treated animals were injected with conjugated estrogen (0.25 mg ip, Ayerst Laboratories; Philadelphia, PA) 1 h before the start of surgery. The cremasters of two estrogen-treated eNOS^/ mice were superfused with N^G-nitro-L-arginine methyl ester (L-NAME; 0.1 mM, Sigma; Louis, MO), a nonspecific NOS inhibitor (40), during the ischemia period. FITC-conjugated mAb RB40.34 (rat IgG₁, 30 µg/mouse iv, PharMingen; San Diego, CA), which binds to murine P-selectin, was used for P-selectin histology. Sodium azide was removed using an Amicon Centricon 30 Microconcentrator (Amicon; Danvers, MA).

Intravital microscopy. Mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride (100 mg/kg, Ketalar, Parke-Davis; Morris Plains, NJ), xylazine (0.05 mg/kg), and atropine (0.1 mg/kg, Elkins-Sinn; Cherry Hill, NJ). Animals were kept at 37°C throughout the experiment with a heating pad. The cremaster muscle was externalized over a Plexiglas observation platform and pinned in place as previously described (3). The preparation was superfused with a thermocontrolled 35°C bicarbonate-buffered saline saturated with 95% N₂-5% CO₂ throughout the experiment. Microscopic observations were made with the use of an Axioskope intravital microscope (Zeiss; Thornwood, NY) with a saline immersion objective (SW 40/0.75 numerical aperture). Between one and four venules per animal with diameters ranging from 20 to 35 µm were observed before the 30-min ischemia period and for 60 min of reperfusion after ischemia. Ischemia was achieved by tying off the supplying arteries with polyethylene-10 tubing and visually confirming that blood flow had ceased. Video recordings were made using a charge-coupled device camera system (model VE-1000CD, Dage-MTI; Michigan City, IN) on a Panasonic S-VHS recorder. Venules were recorded for ~1.5-min segments before ischemia and at 5-min intervals throughout the reperfusion period. Red blood cell centerline velocity was measured using an optical Doppler and cross-correlation system (Circusoft Instrumentation; Hockessin, DE). Centerline velocities were converted to mean blood flow velocities by multiplying by an empirical factor of 0.625 (34). Shear rates were calculated using the equation _w = 2.12 × 8V_b/d, where _w is the wall shear rate, V_b is the mean blood flow velocity, d is the in vivo diameter of the vessel, and 2.12 is an empirical correction factor for the shape of the velocity profile (41). All vessels had calculated wall shear rates between 400 and 3,700 s¹. Leukocyte rolling velocity was measured using Scion Image (Scion; Frederick, MD) software on a G4 Macintosh computer. Rolling velocity was measured before ischemia and at 10-min intervals starting at 5 min after the end of the ischemic period. Each rolling leukocyte passing a line perpendicular to the vessel wall was followed for 0.5-1 s. Adherent cells and rolling flux were counted before ischemia and at 5-min intervals starting at 2 min after the end of the ischemic period. A cell was counted as adherent if it was stationary for at least 30 s.

Immunostaining for P-selectin. Whole cremaster muscles subjected to either 30 min of ischemia and 10 min of reperfusion or no treatment were harvested from WT and eNOS^/ mice injected intravenously with FITC-conjugated mAb RB40.34 (30 µg/mouse, Pharmingen) 10 min before the end of the ischemic period and perfused with PBS to remove blood and unbound antibody after the 10-min reperfusion period (26). The cremaster muscle was laid flat on a gelatin-coated slide and allowed to air dry for ~5-10 min. Slides were then coverslipped using VectaShield (Vector Laboratories; Burlingame, CA) and viewed and photographed using a Nikon Microphot SA microscope with attached Nikon CoolPix 900 digital camera (Nikon; Melville, NY).

eNOS activity assay. Mouse cremasters were homogenized in ice-cold PBS containing 1 mM EDTA using a polytron. The homogenates were pelleted in a microfuge (2 min, 13,000 rpm, 4°C) to remove the insoluble material. Protein concentration was determined with the micro BCA assay kit (Pierce; Rockford, IL), and 5 µg of protein extracts from each sample were used for the eNOS assay. The eNOS activity was detected by measuring the conversion of L-[³H]arginine to L-[³H]citrulline at 37°C for 30 min with the eNOS assay kit (Calbiochem; La Jolla, CA) as described. Unlabeled L-arginine was added to L-[³H]arginine (specific activity 60 Ci/mmol) at a ratio of 3:1. Rat cerebellum extracts, containing elevated amounts of nNOS, were used as positive controls, whereas samples incubated in the presence of the competitive NOS inhibitor L-NAME (1 mM) were used to determine nonspecific activity. Nonspecific activity accounted for 20-35% of the total activity.

Statistical analysis. All data are presented as means ± SE. Data from all vessels from each animal were averaged and used as an individual data point. Statistical analyses were performed using each animal rather than each vessel as an entity. Two-group comparisons between animals before and after ischemia were performed using paired Student's t-test. Probabilities of 0.05 or less were considered statistically significant. All statistical analysis was performed using Microsoft Excel and NCSS (Kaysville, UT).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic data. Red blood cell rolling velocities, diameters and wall shear rates measured before the start of ischemia and 5 and 15 min into the reperfusion period are presented in Table 1. In individual vessels, we observed a positive correlation between leukocyte rolling velocity and red blood cell velocity, as shown in Fig. 1A (P < 0.05 for all groups), and a negative correlation between the number of adherent cells and red blood cell velocity, as shown in Fig. 1B. Rolling velocities were significantly higher (P < 0.05, one-tailed t-test) in eNOS^/ mice compared with WT mice, consistent with lower P-selectin expression in eNOS^/ than WT mice (Fig. 2). At low blood flow velocities (<3 mm/s), significantly more adherent leukocytes were observed in eNOS^/ mice. Because there were no systematic hemodynamic differences among the groups, all data are presented without adjustments for hemodynamics.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Untreated endothelial nitric oxide synthase (eNOS)-deficient (eNOS/; solid line) mice show a higher baseline leukocyte rolling velocity than untreated wild-type (WT; dotted line) mice. A: leukocyte rolling velocity versus red blood cell velocity. There was a positive correlation between rolling velocity and red blood cell velocity for both WT and eNOS/ mice (P < 0.01). B: leukocyte adhesion versus red blood cell velocity.

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Expression of endothelial P-selectin (infusion of mAb RB40.34) present on the luminal surface in WT and eNOS/ mice. Control, no ischemia; after ischemia-reperfusion (I/R), sample taken at 5 min of reperfusion.

Leukocyte rolling velocity. Leukocyte rolling velocities were measured at 10-min intervals, and a time course is shown in Fig. 3A. The numbers of mice and venules represented by each data point are presented in Table 2. The maximal decrease in rolling velocity occurred ~5 min into the reperfusion period except in estrogen-treated WT mice (no decrease). In WT mice, I/R caused a decrease of leukocyte rolling velocity from 37 to 26 µm/s (P < 0.05, one-tailed t-test) in untreated mice (Fig. 3B) but no change (33 µm/s before and after I/R, not significant) in estrogen-treated mice (Fig. 3C). In eNOS^/ mice, I/R caused a similar decrease of leukocyte rolling velocity from 61 to 42 µm/s (P < 0.05) as in untreated mice (Fig. 3D), which was not reversed in estrogen-treated mice (Fig. 3E). Application of L-NAME to estrogen-treated eNOS^/ mice produced no further reduction in rolling velocity, suggesting that eNOS indeed accounted for all NO production relevant to leukocyte rolling in this model. There was no significant decrease of rolling velocity at later times in any group, suggesting that the injury induced by 30 min of ischemia and reperfusion was transient and reversible.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   A: average leukocyte rolling velocities (means ± SE). B-E: cumulative histograms of leukocyte rolling velocities before and 5 min after reperfusion for untreated WT mice (B), estrogen (E)-treated WT mice (C), untreated eNOS/ mice (D), and estrogen-treated eNOS/ mice (E).

P-selectin expression. P-selectin expression was investigated under baseline conditions and after 30 min of ischemia and 10 min of reperfusion in WT and eNOS^/ mice. We observed an increase in P-selectin expression after I/R in WT mice (Fig. 2). In eNOS^/ mice, P-selectin expression was low, but present, and did not increase after I/R. These findings suggest that increased leukocyte adhesion and reduced rolling velocity may be caused by altered P-selectin expression in WT mice. The expression levels of P-selectin in eNOS^/ mice do not account for increased leukocyte adhesion, suggesting a different molecular mechanism.

Leukocyte adhesion. The number of adherent leukocytes per square millimeter of endothelial surface area was measured in each vessel. The time course is shown in Fig. 4. Maximal increase in adherent leukocytes occurred between 10 and 15 min into the reperfusion period except in the estrogen-treated wild type mice (no increase). The number of adherent leukocytes after I/R increased 62% (from 430 to 700 cells/mm²) in untreated WT mice (P < 0.01) but remained unchanged in WT mice treated with estrogen (390 cell/mm² before vs. 380 cell/mm² after). We observed a 130% increase (from 430 to 980 cells/mm², P < 0.01) in untreated eNOS^/ mice and a 50% increase (from 430 to 650 cells/mm², not significant) in eNOS^/ mice treated with estrogen. Estrogen treatment reduced the duration of increased leukocyte adhesion in eNOS^/ mice from 35 to 15 min.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Leukocyte adhesion before and after I/R (means ± SE).

/

/

View larger version (16K): [in this window] [in a new window]   Fig. 5.   A: rolling velocity for all treatment groups before and 5 min after reperfusion (means ± SE). B: leukocyte adhesion for all treatment groups before and 2-12 min after reperfusion (means ± SE). L-NAME, NG-nitro-L-arginine methyl ester. * Significant differences.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Leukocyte rolling velocity 5 min after reperfusion versus adherent leukocytes 2-12 min after reperfusion. There was a significant negative correlation [P < 0.05 for WT (dashed line); P < 0.01 for eNOS/ (solid line)] between rolling velocity and adhesion.

eNOS activity. eNOS activity was measured in cremaster muscles harvested from untreated and estrogen-treated WT and eNOS^/ mice used for intravital experiments (Fig. 7). eNOS activity in WT mice after ischemia was significantly increased in estrogen-treated cremasters (2.8 vs. 4.4 pmol · mg¹ · min¹, P < 0.05), whereas there was no difference in eNOS^/ mice (0.3 vs. 0.5 pmol · mg¹ · min¹). We found no significant correlation between eNOS activity and adherence or rolling velocities in individual mice.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Cremaster eNOS activity in untreated and estrogen-treated WT and eNOS/ mice. * Significant difference.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we examined the effect of pretreatment with estrogen on I/R injury in the microvasculature of WT and eNOS^/ mice. We found that, unlike WT mice, the eNOS^/ mice were not significantly protected by estrogen treatment. In eNOS^/ mice, I/R caused a 32% decrease of leukocyte rolling velocity in untreated mice that was similar in magnitude to that seen in untreated WT mice (30%) and that was not improved in estrogen-treated mice (decrease of 28%). We also observed increases in leukocyte adhesion of 130% and 50% in untreated and estrogen-treated eNOS^/ mice, respectively. The increase in adhesion in the untreated eNOS^/ mice (130%) was much greater than that seen in the untreated WT mice (62%). Also, eNOS activity was significantly increased by 57% in estrogen-treated WT mice compared with untreated WT mice after I/R.

In WT and eNOS^/ mice, the maximum decrease in rolling velocity occurred within 5 min after the onset of reperfusion. Likewise, the peak increase in leukocyte adhesion was observed ~10-15 min into the reperfusion period. This suggests that the injury created in this model of I/R is mild and transient. The lack of severity of the injury is supported by the absence of a significant reactive hyperemia at the onset of reperfusion. The rapid decrease in rolling velocity suggests that the adhesion molecules involved in this phenomenon are not synthesized de novo but are already present in the endothelial cells.

It was suggested by a previous study (46), and established in this study, that the protective effect of estrogen is partially mediated via an increase in production of NO by eNOS; however, the mechanism behind the protective effect of NO remains unknown. P-selectin, an adhesion molecule involved in the early stages of leukocyte-endothelium interactions, is stored in Weibel-Palade bodies and can be rapidly translocated to the endothelial surface upon cellular activation (47). Previous studies have indicated that P-selectin is the primary mediator of leukocyte adhesion and rolling in the cremaster I/R model (28), and the present study confirms a rapid increase in endothelial cell surface expression of P-selectin. Many studies have suggested a link between NO and P-selectin expression (1, 6, 16, 17, 22, 25, 33, 37, 43, 44). However, P-selectin expression was low in cremaster muscle venules of eNOS^/ mice and was not upregulated after I/R, so it is unlikely to be responsible for the increased amount of adhesion seen in this model. Upregulation of P-selectin may not be the only cause of increased adhesion seen in WT mice after I/R. Some evidence indicates that NO may also modulate expression of platelet activating factor (PAF), ICAM-1, and CD18 integrins (30, 31). PAF can activate CD18 integrins on leukocytes (21, 50). Further work will be necessary to elucidate the molecular mechanism of increased adhesion after I/R.

Other investigators have identified roles for iNOS and nNOS in other models of I/R injury (4, 20, 42). Generally, activation of nNOS and iNOS exacerbates the I/R-induced injury by producing toxic levels of NO; however, it is uncertain what role these enzymes may play in the complete absence of eNOS (4, 42). In this study, we superfused several of the cremasters from the estrogen-treated eNOS^/ mice with L-NAME during the ischemic period to discern whether other nNOS and iNOS play a role in this model. We did not observe any additional reperfusion injury in the L-NAME-treated mice, nor was there a protective effect, indicating that NO produced by eNOS is the only NO-producing system involved in this model.

Other observations from this study include elevated baseline leukocyte rolling velocity in eNOS^/ mice compared with WT mice. The reason for this increased rolling velocity could be related to decreased P-selectin expression in these mice. The interpretation of the present data is complicated by the lack of I/R-induced P-selectin expression in eNOS^/ mice. The mechanisms responsible for postischemic leukocyte recruitment are likely different in eNOS^/ and WT mice. Estrogen treatment has little effect on this recruitment mechanism, which may be the result of adaptive changes that occur during the development of eNOS^/ mice. We show that estrogen increases eNOS activity and reduces leukocyte rolling and adhesion, but our data stop short of demonstrating a causal relationship, because the molecular mechanisms underlying the modulation of leukocyte adhesion through eNOS products are essentially unknown.

In conclusion, the protective effect of estrogen as measured by leukocyte rolling and adhesion is abolished in eNOS^/ mice, suggesting that induction of eNOS activity is the major mechanism of vasoprotection by estrogen in this model. Furthermore, experiments conducted with the NOS inhibitor L-NAME suggest that other NOS isoforms do not significantly contribute to the inflammatory response in this model of I/R. However, the estrogen-treated eNOS^/ mice seemed somewhat protected because the number of adherent leukocytes was reduced and the duration of leukocyte accumulation was decreased. Therefore, estrogen may exert additional anti-inflammatory effects unrelated to NO production. Finally, leukocyte rolling velocity may be a predictor of leukocyte adhesion, not only in cytokine-induced inflammation, but also in I/R.