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Am J Physiol Gastrointest Liver Physiol 290: G1203-G1210, 2006. First published February 9, 2006; doi:10.1152/ajpgi.00578.2005
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INFLAMMATION/IMMUNITY/MEDIATORS

Angiotensin II type 1 receptors and the intestinal microvascular dysfunction induced by ischemia and reperfusion

Thomas Petnehazy,1,2 Dianne Cooper,1 Karen Y. Stokes,1 Janice Russell,1 Katherine C. Wood,1 and D. Neil Granger1

1Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana; and 2University Clinic of Pediatric Surgery, Medical University of Graz, Graz, Austria

Submitted 21 December 2005 ; accepted in final form 6 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The acute phase of intestinal ischemia-reperfusion (I/R) injury is mediated by leukocytes and is characterized by oxidative stress and blood cell recruitment. Upregulation of angiotensin II type 1 receptors (AT1-R) has been implicated in the pathogenesis of conditions associated with oxidative stress. The AT1-R-antagonist Losartan (Los) attenuates leukocyte recruitment following I/R. However, the role of AT1-R in intestinal I/R injury and the associated platelet-leukocyte interactions remains unclear. The objective of this study was to define the contribution of AT1-R to I/R-induced blood cell recruitment in intestinal venules. Leukocyte and platelet adhesion were quantified by intravital microscopy in the small bowel of C57Bl/6 [wild-type (WT)] mice exposed to sham operation or 45 min of ischemia and 4 h of reperfusion. A separate WT group received Los for 7 days before gut I/R (WT-I/R + Los). AT1-R bone marrow chimeras that express AT1-R on the vessel wall but not blood cells also underwent I/R. Platelet and leukocyte adhesion as well as AT1-R expression in the gut microvasculature were significantly elevated after I/R. All of these responses were attenuated in the WT-I/R + Los group, compared with untreated I/R mice. A comparable abrogation of I/R-induced blood cell adhesion was noted in AT1-R bone marrow chimeras. I/R-induced platelet adhesion was unaltered in mice overexpressing Cu,Zn-SOD or mice deficient in NAD(P)H oxidase. These data suggest that although gut I/R upregulates endothelial expression of AT1-R, engagement of these angiotensin II receptors on blood cells is more important in eliciting the prothrombogenic and proinflammatory state observed in postischemic gut venules, through a superoxide-independent pathway.

leukocytes; platelets; endothelium; microcirculation; angiotensin II type 1 receptor expression

ISCHEMIA-REPERFUSION (I/R) injury has been implicated in a number of disease states such as sickle cell anemia (33) and arthritis (3) and often arises in the gut as a result of shock, trauma, or cardiovascular surgery. The adhesion of neutrophils (PMNs) to venular endothelial cells is a rate-limiting event in the pathogenesis of I/R injury (22). This has led to large numbers of studies that focus on the nature, kinetics, and mechanisms underlying the recruitment of adherent PMNs in the microcirculation after I/R. These studies have revealed that venules assume both an inflammatory and prothrombogenic phenotype after I/R, which is manifested as the recruitment of adherent leukocytes and platelets. Recent studies have demonstrated that a large proportion of leukocytes that adhere in postischemic venules are platelet bearing (8). It is estimated that 26% of platelets that adhere in postischemic intestinal venules are attached directly to venular endothelium, whereas the remaining 74% are bound to already adherent leukocytes. Nonetheless, the mechanisms that regulate these populations of platelets after I/R remain poorly understood.

The renin-angiotensin-aldosterone system (RAS) has recently been implicated in a variety of inflammatory conditions including I/R (6). Engagement of the angiotensin II type 1 receptors (AT1-R) by angiotensin II appears to play a critical role in the RAS-mediated inflammatory responses. Mesenteric venules exposed to exogenous angiotensin II exhibit leukocyte-endothelial adhesion that can be prevented by AT1-R antagonism (25). This finding is consistent with reports observing enhanced endothelial cell adhesion molecule expression both in vitro (9, 13) and in vivo (1) following AT1-R activation. However, it remains unclear whether angiotensin II-mediated adhesion molecule expression (via an AT1-R-dependent mechanism) will also result in the adhesion of platelets to endothelial cells and/or adherent leukocytes.

The splanchnic vasculature normally expresses high levels of angiotensin receptor compared with other vascular beds (16). In response to hypovolemia, blood levels of angiotensin II are higher in the splanchnic circulation compared with systemic blood (35). Interestingly, Riaz et al. (28) demonstrated that I/R of the gut not only results in increased systemic angiotensin II levels, but also increases mRNA for angiotensin-converting enzyme (ACE) in postischemic gut tissue. These changes in angiotensin II production and concentration were accompanied by the recruitment of adherent leukocytes in postcapillary venules, which was blocked in animals treated with either an ACE inhibitor or AT1-R antagonist. Although angiotensin II and the AT1-R were implicated in the inflammatory response to gut I/R, the study did not address whether I/R-induced platelet accumulation is similarly mediated by this mechanism nor did it address the contribution of endothelial cell vs. blood cell AT1-R to this response. Furthermore, one of the primary pathways initiated by the engagement of AT1-R on endothelium is activation of the superoxide-generating enzyme NAD(P)H oxidase (15). In studies employing a peptide inhibitor of NAD(P)H oxidase, Korthuis et al. (19) implicated this enzyme in the oxidative stress and leukocyte adhesion elicited by I/R. Whether NAD(P)H oxidase-derived superoxide also contributes to the thrombogenic phenotype assumed by the microvasculature of the postischemic intestine also remains unclear.

The overall objectives of this study were to determine whether 1) the I/R-induced platelet-endothelial cell and platelet-leukocyte interactions are linked to AT1-R activation; 2) AT1-R on circulating blood cells contribute to this phenomenon; 3) I/R alters the expression of AT1-R in the microvasculature; and 4) NAD(P)H oxidase-derived superoxide contributes to the platelet recruitment elicited by gut I/R and modulated by AT1-R. The first objective was addressed by assessing the influence of the AT1-R antagonist Losartan (Los) on I/R-induced platelet-endothelial cell and platelet-leukocyte interactions in postcapillary venules using intravital microscopy. To define the contribution of AT1-R on circulating and endothelial cells to the inflammatory responses elicited by I/R, we transplanted bone marrow from AT1-R–/– mice into WT mice, creating AT1-R chimeras whose circulating blood cells were deficient in AT1-R. The third objective was addressed by using the dual-radiolabeled monoclonal antibody technique (DRL) to quantify the expression of AT1-R in normal and postischemic gut microvasculature. Finally, Cu,Zn-SOD transgenic mice and NAD(P)H oxidase (gp91phox)-deficient mice were studied to assess the contribution of NAD(P)H oxidase-derived superoxide to the platelet adhesion response elicited by gut I/R.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male WT C57BL/6J, B6.129P2-Agtr1tm1Unc/J (AT1-R–/–), B6.129S6-Cybbtm1Din/J (gp91phox–/–), and breeder stocks for C57BL/6-Tg(SOD1)3Cje/J (SOD-TgN) mice on a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). The SOD-TgN mice were identified by qualitative demonstration of Cu,Zn-SOD using nondenaturing gel electrophoresis followed by nitroblue tetrazolium staining. In addition, chimeras were produced by the transfer of bone marrow from AT1-R–/– into WT recipients (AT1ch) at 10–12 wk of age. A separate group of WT mice was treated with the AT1-R antagonist Los (25 mg·kg–1·day–1) (23) in drinking water for 7 days before induction of ischemia (WT-I/R + Los group).

To make chimeric mice with circulating cells deficient in AT1-R but vascular cells expressing normal levels of AT1-R, bone marrow cells were isolated from the femurs and tibias of AT1-R–/– donor mice. These cells were resuspended at 4 x 107 cells/ml in PBS. Recipient (CD45 congenic WT) mice were irradiated with 2 doses of 500–525 Rad, 3 h apart, after which 8 x 106 donor bone marrow cells in 200 µl PBS were injected into the femoral vein. The chimeras were kept in autoclaved cages, with 0.2% neomycin drinking water for 2 wk, after which normal drinking water was used. Flow cytometry was used to verify chimera reconstitution (usually requiring 6–8 wk) by staining for CD45.1 and CD45.2 expression on circulating leukocytes with an FITC-labeled anti-CD45.1 antibody and a biotinylated anti-CD45.2 antibody with a streptavidin-PerCP secondary antibody (BD Biosciences, San Diego, CA). This procedure yielded 91.6 ± 0.23% penetrance of the transferred marrow at 6 wk following the transplant, as expected from previous studies (24).

I/R. Mice were anesthetized with ketamine hydrochloride (150 mg/kg body wt ip) and xylazine (7.5 mg/kg body wt ip). Ischemia was induced by clamping the superior mesenteric artery (SMA) for 45 min. The clamp was removed, and the gut was reperfused for 4 h. Sham-treated animals were exposed to the same procedure except for occlusion of the SMA. After SMA occlusion and reperfusion (I/R), the mice were prepared for either intravital microscopy or measurement of AT1-R expression.

In vivo measurement of AT1-R expression. The expression of AT1-R on vascular endothelial cells was measured in WT-sham, WT-I/R, and WT-I/R + Los mice at 4 h reperfusion using the DRL MAb technique (12). The N-10 rabbit polyclonal anti-AT1-R antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was labeled with 125I (DuPont New England Nuclear, Boston, MA) using the iodogen method. The nonbinding antibody (P23), a murine anti-human P-selectin MAb (Pharmacia-Upjohn, Kalamazoo, MI), was labeled with 131I (DuPont NEN) using the iodogen method. Receptor levels were expressed as nanograms MAb per gram tissue as described previously (12).

Platelets. Platelets were collected, isolated, and labeled as previously described (32). Mice in all groups received platelets from matching donors.

Surgical protocol. All platelet recipients were fasted for 24 h before intravital microscopy. The right jugular vein was cannulated for administration of fluorescently labeled platelets and rhodamine 6G, and the left carotid artery was cannulated for measurement of systemic arterial pressure. A midline laparotomy was performed, the animal was placed in a supine position, and a loop of small bowel was exteriorized and superfused with warm bicarbonate-buffered saline. Core body temperature was maintained at 35 ± 0.5°C. All animal procedures (intestinal I/R model, intravital microscopy, DRL monoclonal antibody technique, and bone marrow transfer) were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and were in accordance with the guidelines of the American Physiological Society.

Postcapillary venules, 20–40 µm in diameter, were selected for observation after a 30-min stabilization period. Platelets (100 x 106 in a volume of 120 µl) were infused via the jugular vein over 5 min and allowed to circulate for an additional 5 min. This was followed by intravenous administration of rhodamine 6G (0.02%) over 5 min to visualize leukocytes. One-minute recordings of the leukocytes and platelets were obtained under fluorescent microscopy for each of five randomly selected intestinal venules. The mean value of each variable was calculated, and comparisons were made between the experimental groups (for all parameters, n = 5 for WT-sham, WT-I/R, and WT-I/R + Los groups; n = 4 for AT1ch-I/R and SOD-TgN-I/R groups; and n = 6 for SOD-non-TgN-I/R and gp91phox–/–-I/R groups).

The number of adherent leukocytes and platelets was quantified during playback of videotaped images. Platelets (number/mm2) were considered saltating (transient platelet adhesion) if they arrested for ≥2 s and adherent if they remained stationary for ≥30 s. Total platelet adhesion was defined as the sum of saltation and adherence. Leukocytes were considered rolling if they moved at a velocity less than the red blood cell velocity (expressed as number·min–1·mm–1) or adherent if they remained stationary for ≥30 s (number/mm2) and were measured throughout the observation period. Leukocytes were further distinguished as platelet bearing (were associated with ≥1 fluorescent platelet) or platelet free (no fluorescent platelets were adherent to the leukocyte surface). Platelet interactions were characterized as endothelium dependent (interacting directly with the vessel wall) or leukocyte dependent (interacting with leukocytes that adhered to the vessel wall).

Statistical analysis. All values are reported as means ± SE. ANOVA with Bonferroni post hoc test was used for statistical comparison of experimental groups, with statistical significance set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
AT1-R expression in the microvasculature. As previously reported (23), significant constitutive expression of AT1-R is detected in several regional vascular beds using the DRL monoclonal antibody technique. Figure 1 demonstrates that SMA occlusion and reperfusion elicit an upregulation of AT1-R in the vasculature of several tissues perfused by the splanchnic vasculature including the small bowel, colon, liver (data not shown), and mesentery. AT1-R expression in the lung was also increased ~100% above sham-operated controls (data not shown). Although the stomach, cecum, pancreas, and kidneys exhibited a tendency for AT1-R upregulation after gut I/R, these changes were not statistically significant. Treatment with the AT1-R antagonist Los for 7 days before I/R largely prevented the increased endothelial expression (or reduced expression below the sham level) of AT1-R in all organs examined.


Figure 1
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  		Fig. 1. Angiotensin II type 1 receptor (AT1-R) expression in several organs (small bowel, colon and mesentery) of wild-type (WT) mice was upregulated by ischemia-reperfusion (I/R) of the superior mesenteric artery (SMA) when compared with sham-operated mice (WT-Sham). Treatment with 25 mg·kg–1·day–1 Losartan (Los) prevented the I/R-induced upregulation of AT1-R (WT-I/R + Los). *P < 0.05 vs. WT-Sham; #P < 0.005 vs. WT-I/R. (n = 6 for WT-Sham; n = 4 per group for WT-I/R, WT-I/R + Los).

 
I/R-induced platelet recruitment. I/R induced the recruitment of platelets in intestinal venules of WT mice, with comparable increases in both the saltation (Fig. 2) and firm adhesion (Fig. 3) of platelets. The majority of saltating platelets occurred in a leukocyte-dependent manner, where platelet-leukocyte aggregates briefly stopped on the vessel wall or platelets transiently interacted with already adherent leukocytes (Fig. 4). Most of the firmly adherent platelets accumulated in venules by binding to adherent leukocytes (Fig. 4). Mice treated with Los exhibited an attenuated increase in I/R-induced platelet saltation on both endothelial cells and leukocytes. The firm adhesion of platelets (both endothelium and leukocyte dependent) was completely abrogated by Los pretreatment, reducing the number of adherent platelets to levels detected in sham-operated controls (Figs. 2 and 3). Chimeric mice with AT1-R-deficient blood cells exhibited an attenuation of platelet adhesion (saltating and firmly adherent) that was comparable with the responses observed in the Los-treated group. The percentage of platelet firm adhesion that was a result of direct interaction with the endothelium was reduced from 21% in untreated I/R mice to 1% in the Los-treated group, whereas it remained unaltered in the AT1-R chimera group (Fig. 4).


Figure 2
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  		Fig. 2. Endothelium- and leukocyte-dependent platelet saltation (stationary for 2–29 s) was elevated in WT mice exposed to intestinal (WT-I/R) vs. sham-operated controls (WT-Sham). Treatment with the AT1-R antagonist Los (WT-I/R + Los) or absence of blood cell-associated AT1-R expression in bone marrow chimeras (AT1ch-I/R) attenuated platelet saltation. *P < 0.001 vs. WT-Sham; #P < 0.005 vs. WT-I/R.

 

Figure 3
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  		Fig. 3. Endothelium- and leukocyte-dependent platelet adhesion (stationary for ≥30 s) was increased in WT-I/R compared with WT-Sham. Treatment with the AT1-R antagonist losartan (WT-I/R + Los) or deficiency of blood cell-associated AT1-R expression in bone marrow chimeras (AT1ch-I/R) attenuated platelet adhesion comparably. *P < 0.0005 vs. WT-Sham; #P < 0.005 vs. WT-I/R.

 

Figure 4
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  		Fig. 4. The percentage of saltating platelets that was dependent on leukocytes vs. endothelium was comparable among all groups exposed to I/R. However, the proportion of platelet firm adhesion that occurred through direct interaction with the endothelium was reduced in Los-treated I/R animals when compared with untreated WT mice or mice deficient in AT1-R on blood cells only.

 
I/R-induced leukocyte rolling. Leukocyte rolling was significantly elevated in intestinal venules after I/R (Fig. 5). Although this response was associated with increases in the rolling of both platelet-free and platelet-bearing leukocytes, the proportion of leukocytes associated with exogenous platelets increased from 6.4% in sham mice to 47.5% in the I/R group (Fig. 6). Pretreatment with Los failed to reduce the total number of rolling leukocytes after I/R; however, the number of rolling platelet-bearing leukocytes was significantly reduced by the AT1-R antagonist. AT1ch mice exposed to gut I/R exhibited a slight attenuation of the number of rolling platelet-free leukocyte; however, the level of platelet-bearing leukocyte rolling was significantly reduced compared with the WT animals (Figs. 5 and 6).


Figure 5
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  		Fig. 5. The levels of rolling leukocytes [both associated (platelet bearing) or not associated (platelet free) with exogenous platelets] were elevated in WT-I/R vs. WT-Sham. Treatment with the AT1-R antagonist losartan (WT-I/R + Los) reduced the rolling of leukocytes bearing exogenous platelets, but platelet-free leukocyte rolling was slightly elevated. The absence of blood cell-associated AT1-R expression in bone marrow chimeras (AT1ch-I/R) led to a partial reduction of leukocyte rolling, primarily of those associated with exogenous platelets. *P < 0.01 vs. WT-Sham; #P < 0.005 vs. WT-I/R.

 

Figure 6
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  		Fig. 6. The percentage of total rolling leukocytes that formed aggregates with exogenous platelets (platelet bearing) was elevated in WT-I/R vs. WT-Sham. WT-I/R + Los or AT1ch-I/R mice exhibited significantly lower fractions of rolling cells that were associated with exogenous platelets. *P < 0.0001 vs. WT-Sham; #P < 0.005 vs. WT-I/R.

 
I/R-induced leukocyte adhesion. Figure 7 illustrates that I/R elicited an 82-fold increase in the number of firmly adherent leukocytes in intestinal venules. Approximately one-half of these adherent leukocytes was platelet bearing in the I/R mice. Pretreatment with Los abrogated the leukocyte adhesion response to I/R, with comparable reductions in both platelet-bearing and platelet-free leukocytes. A similar attenuation of I/R-induced leukocyte adhesion was noted in the AT1-R–/– chimeric mice; however, only 26.8% of the total adherent population was platelet bearing following I/R.


Figure 7
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  		Fig. 7. Adhesion of leukocytes that were either associated (platelet bearing) or not associated (platelet free) with exogenous platelets was significantly increased by I/R of the intestine (WT-I/R) when compared with shams (WT-Sham). Administration of the AT1-R antagonist Los (WT-I/R + Los) or deficiency of blood cell-associated AT1-R expression in bone marrow chimeras (AT1ch-I/R) attenuated the adhesion of these 2 leukocyte populations toward sham levels. *P < 0.0001 vs. WT-Sham; #P < 0.0005 vs. WT-I/R.

 
Role of superoxide and NAD(P)H oxidase in I/R-induced platelet recruitment. The level of total platelet adhesion achieved in SOD-TgN mice exposed to I/R (203 ± 72.2 cells/mm2) was comparable with SOD-non-TgN mice (194 ± 44.2 cells/mm2), i.e., their corresponding controls. Furthermore, deficiency of gp91phox, a subunit of NAD(P)H oxidase (294 ± 36.9 cells/mm2), did not confer significant protection against I/R-induced platelet recruitment in the postischemic intestine when compared with WT mice (394 ± 20.2 cells/mm2).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is now recognized that both leukocytes and platelets contribute to the inflammatory and tissue injury responses to I/R (31). In the intestinal microvasculature, I/R results in the accumulation of rolling and adherent platelets, and this is associated with the adhesion of leukocytes, many of which have platelets bound to their surface (8). It is now well appreciated that the renin-angiotensin system (RAS) contributes to I/R-induced microvascular alterations, including the oxidative stress, increased expression of endothelial cell adhesion molecules, and recruitment of adherent leukocytes (1, 2, 9, 13, 25, 28). However, little is known about the role of the RAS and AT1-R in the recruitment of platelets in postischemic microvessels and their contribution to I/R-induced platelet-leukocyte interactions. The results of this study confirm the importance of AT1-R in the leukocyte-endothelial cell adhesion induced by gut I/R. Our findings also provide novel insights regarding the influence of gut I/R on the expression of AT1-R on endothelial cells in the splanchnic vasculature and reveal a major role for blood cell-associated AT1-R in both the prothrombogenic and proinflammatory processes that are elicited by I/R in gut venules.

The DRL MAb technique has been extensively used to quantify the expression of adhesion molecules on endothelial cells in different regional vascular beds (14). In the present study, we applied this method to measure the expression of AT1-R on vascular endothelial cells in the gut and other tissues after I/R. We provide the first evidence for an increased surface expression of AT1-R in the microvasculature after gut I/R, with a twofold increase in the density of AT1-R in the intestinal microvasculature. Also of interest is our observation that AT1-R expression is significantly elevated in the microvasculature of distant organs, including the lung. This observation suggests that soluble circulating factors [e.g., TNF-{alpha} (34)] may be responsible for inducing the expression of AT1-R in distant organs. Because lung inflammation and injury frequently result from gut I/R (5), it is plausible that AT1-R activation also contributes to the inflammatory/injury responses to gut I/R in distant organs. An interesting and potentially important finding was that Los, an AT1-R antagonist, inhibited the I/R-induced upregulation of AT1-R in several organs. Whereas the exact mechanism underlying this Los-mediated inhibition of AT1-R upregulation after gut I/R is unknown, it is possible that the antagonist competes with the antibody for the same binding site on the AT1-R. This might explain why the Los-treated gut I/R group exhibited lower AT1-R expression in several organs, compared with sham controls. Alternatively, it has been demonstrated previously that AT1-R engagement leads to NF-{kappa}B (11) and AP-1 activation (4) and cytokine production (29) and that cytokines, in turn, are capable of upregulating AT1-R through the activation of NF-{kappa}B and alteration of mRNA splicing (10). Therefore, by preventing the initial signaling through AT1-R, Los may block this pathway of agonist-induced AT1-R upregulation. The ability of Los treatment to reduce ATR-1 expression levels below baseline values in several tissues is also consistent with such a mechanism and would suggest that the basal level of AT1-R expression may be governed by the local endothelial cell production of angiotensin II by the enzyme ACE.

Several reports (8, 17, 18) have described the accumulation of platelets in the microvasculature of postischemic tissues including intestine, brain, and liver. The present study confirms these findings and indicates that a majority of the firmly adherent platelets that accumulate in intestinal venules does so by binding to already adherent leukocytes. We report for the first time that AT1-R antagonism with Los exerts a profound inhibitory effect on the I/R-induced recruitment of platelets in the gut microvasculature. These findings are consistent with a recent study from our laboratory that describes an attenuation of hypercholesterolemia-induced platelet adhesion in skeletal muscle venules by Los (23). Hence, there is a growing body of evidence that AT1-R activation is an important determinant of the prothrombogenic phenotype that is assumed by the microvasculature in different pathological conditions. In the I/R model, we noted that the AT1-R antagonist was capable of blocking both the brief interactions between platelets and endothelial cells and leukocyte-dependent saltation. However, endothelium-dependent firm adhesion of platelets was more effectively inhibited vs. leukocyte-dependent adhesion after Los treatment. Perhaps Los first reduces endothelium-dependent platelet adhesion by reducing transient interactions with the vessel wall and second by targeting the conversion of saltation to firm adhesion. Nonetheless, these observations suggest that Los may be targeting both platelets (and perhaps leukocytes) as well as the endothelium to inhibit platelet accumulation in gut venules. To clarify the contribution of endothelial cell- vs. blood cell-associated AT1-R in mediating the prothrombogenic phenotype after I/R, we produced bone marrow chimeras that involved the transfer of marrow from AT1-R–/– donor mice to WT recipients. This procedure yielded mice that are devoid of AT1-R on circulating leukocytes and platelets, whereas the endothelial cells expressed normal levels of AT1-R. Because the platelet adhesion responses to gut I/R in the AT1-R–/– chimeras were nearly identical to the responses noted in Los-treated mice, it appears likely that blood cell-associated (platelet and/or leukocyte) AT1-R mediates the prothrombogenic phenotype induced by gut I/R. Interestingly, the lack of AT1-R on blood cells was more effective at abrogating leukocyte-dependent vs. endothelium-dependent platelet adhesion. Whereas the mechanism underlying the AT1-R-mediated thrombogenic response remains undefined, one possibility is that AT1-R engagement on the platelet leads to an increased expression of platelet P-selectin (20, 30), which has been shown to be a major contributor to gut I/R-induced platelet recruitment (7).

It is now known that I/R leads to the adhesion of a population of leukocytes that bears platelets in intestinal venules (8). Furthermore, I/R promotes leukocyte adhesion in the colon through an AT1-R-dependent pathway (28). Because we show in the present study that AT1-R also plays a major role in gut I/R-induced platelet adhesion, we examined the relative contributions of AT1-R to the accumulation of leukocytes that were associated with exogenous platelets (platelet bearing) and free of exogenous platelets (platelet free) in the postischemic gut microvasculature. As expected, leukocyte rolling was elevated in gut venules following I/R, and this recruitment of rolling leukocytes was more weighted toward platelet-leukocyte aggregates (which represented 47.5% of rolling leukocyte population) than in sham controls (where only 6.4% of the rolling cells were platelet-leukocyte aggregates). Because angiotensin II is known to elicit leukocyte rolling through an endothelial P-selectin-dependent pathway (1) and intestinal I/R is associated with elevated angiotensin II levels in the splanchnic circulation (28), one would expect Los to abrogate this response. However, the total number of rolling leukocytes was unaltered by Los treatment, which is in agreement with the findings of Riaz et al. (28) in colonic venules. Nonetheless, the proportion of rolling leukocytes that was associated with platelets was reduced from 47.5% in untreated mice exposed to I/R down toward a control level of 15.3% in Los-treated mice, whereas platelet-free rolling leukocytes were slightly increased. It must be noted that the technique employed does not allow for observation of endogenous platelets; therefore, the level of platelet-bearing leukocytes may be underestimated. Nevertheless, these observations suggest that although Los does not target the rolling step of leukocyte recruitment, the AT1-R antagonist may interfere with the formation of platelet-leukocyte aggregates, which is consistent with our findings on I/R-induced platelet adhesion. In contrast to Los-treated mice, AT1-R–/– chimeras exhibited a slight reduction in total rolling leukocytes, with levels of both platelet-associated and platelet-free rolling leukocytes partially decreased toward sham control levels. These data further underscore the contribution of platelet- and/or leukocyte-associated AT1-R in the formation of platelet-leukocyte aggregates following I/R. In addition, the results from the AT1-R–/– chimeras reveal a small role for AT1-R in the leukocyte rolling process that was not indicated by findings from Los-treated mice. Although angiotensin II is known to upregulate endothelial P-selectin through an AT1-R-dependent pathway, our findings in the AT1-R–/– chimeras suggest an alternate mechanism for the influence of AT1-R on leukocyte rolling behavior following I/R. It is possible that engagement of leukocyte-associated AT1-R results in leukocyte activation and L-selectin-dependent rolling. This possibility is supported by a report describing an ability of Los to attenuate leukocyte activation and modulate L-selectin on circulating leukocytes in patients with coronary artery disease (27). Alternatively, the observed differences between the Los and AT1-R–/– chimeras may simply reflect an incomplete blockade of AT1-R on leukocytes and platelets.

I/R is also associated with the adhesion of both platelet-bearing and platelet-free leukocytes in the intestinal microvasculature, with the majority of the adherent leukocytes being platelet free, as previously reported (8), although as mentioned previously, this does not take endogenous platelets into consideration. Treatment with Los dramatically reduced the number of leukocytes bearing exogenous platelets and those that were free of exogenous platelets that accumulated in postischemic venules. Interestingly, Los exerted a similar inhibitory influence on platelet-bearing leukocytes (79%) and platelet-free leukocytes (80%), suggesting that perhaps Los primarily acts directly on AT1-R expressed on leukocytes, rather than on platelets, to reduce leukocyte adhesion. The results obtained from the AT1-R–/– chimeras indicate that the absence of AT1-R on circulating blood cells is as effective as Los treatment in blocking the adhesion of leukocytes in the postischemic intestinal microvasculature, although there appeared to be a propensity to more effectively reduce the adhesion of leukocytes that were associated with exogenous platelets. Similar to platelets, the AT1-R may be linked to adhesion molecule expression/activation on leukocytes exposed to I/R. Such an association has been suggested for leukocyte beta2-integrin (CD11/CD18), a major mediator of I/R-induced leukocyte adhesion, and the AT1-R (21).

Oxidative stress has been implicated in the microvascular dysfunction and tissue injury induced by I/R in many organs. Angiotensin II, acting through AT1-R, is a potent activator of the superoxide-producing enzyme NAD(P)H oxidase (15), which has been implicated in the leukocyte adhesion and endothelial dysfunction in mesenteric venules following I/R (19). Angiotensin II stimulates NAD(P)H oxidase to produce superoxide in both neutrophils (11) and platelets (26) via an AT1-R-dependent mechanism. Whereas these observations suggest that Los may exert its beneficial effect on platelet recruitment via reduction of NAD(P)H oxidase-derived superoxide, our findings in SOD transgenic mice and gp91phox-deficient mice do not support this possibility. The disparities between our findings and those of others (19) may result from differences in the I/R model (duration of I/R), animal species (rat vs. mouse), and/or approach used to inactive NAD(P)H oxidase (peptide vs. genetic).

In conclusion, our findings reveal that AT1-R contributes to both the inflammatory and the thrombogenic phenotype assumed by the microvasculature following intestinal I/R. We showed that part of this response involves an increased expression of the AT1-R on vascular endothelial cells in the postischemic gut and in distant tissues. Whereas it is well established that angiotensin II initiates several inflammatory pathways by engaging AT1-R expressed on vascular endothelium, our results from AT1-R–/– bone marrow chimeras invoke a role for AT1-R expressed on leukocytes and platelets in mediating the recruitment of these blood cells in postischemic venules, via a superoxide-independent mechanism.


   GRANTS
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 MATERIALS AND METHODS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant P01 DK-43785.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Neil Granger, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1501 E. Kings Highway, Shreveport, LA 71130–3932 (e-mail: dgrang{at}lsuhsc.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.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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