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Role of angiotensin II in ischemia/reperfusion-induced leukocyte-endothelium interactions in the colon ¹

AMJID A. RIAZ^*, YUSHENG WANG^*, RENE SCHRAMM^*,, TOHRU SATO^*, MICHAEL D. MENGER, BENGT JEPPSSON^* and HENRIK THORLACIUS^*,2

^* Department of Surgery, Malmö University Hospital, Lund University, Malmö, Sweden; and
Department of Thoracic and Cardiovascular Surgery and
Institute for Clinical & Experimental Surgery, University of Saarland, Homburg/Saar, Germany

²Correspondence: Department of Surgery, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden. E-mail: henrikthorlacius{at}kir.mas.lu.se

SPECIFIC AIMS

Angiotensin II (Ang II) is an important regulator of blood pressure and fluid homeostasis and has recently been ascribed proinflammatory actions. The aims of the present study were to determine the effects and mechanisms of Ang II on leukocyte-endothelium interactions in the colonic microcirculation. Moreover, we wanted to define the role of Ang II in a novel model of colonic ischemia/reperfusion (I/R). For this purpose, we used inverted intravital fluorescence microscopy (IIVM) of the colonic microcirculation in C57BL/6 mice.

PRINCIPAL FINDINGS

1. Ang II exerts proinflammatory effects in the colon
We have recently developed a new model to study the molecular mechanisms of I/R-provoked leukocyte responses in the colonic microcirculation by use of IIVM, which permits detailed analysis of leukocyte-endothelium cell interactions in the colon. By use of IIVM, we found that intraperitoneal challenge with Ang II dose-dependently increased leukocyte rolling and adhesion in postcapillary venules in the colon. In fact, we found that 100 nM Ang II evoked the most prominent response; i.e., leukocyte rolling and adhesion increased by more than ninefold and 78-fold, respectively. We also analyzed the role of specific adhesion molecules in Ang II-induced leukocyte-endothelium interactions. It was found that leukocyte rolling was mainly mediated by P-selectin and firm adhesion by lymphocyte function antigen-1 (LFA-1). Thus, immunoneutralization of P-selectin reduced Ang II-induced leukocyte rolling by more than 90%, whereas inhibition of E-selectin had no effect. Moreover, leukocyte adhesion provoked by Ang II was reduced by 96% in LFA-1-deficient mice compared with wild-type mice. Next, we demonstrated that P-selectin-dependent rolling was a precondition for the subsequent firm adhesion of leukocytes provoked by Ang II. Identical adhesive pathways (P-selectin/LFA-1) described here for Ang II-induced leukocyte rolling and adhesion have been described in I/R-induced leukocyte recruitment in the colon. Activation and trafficking of leukocytes in inflamed tissues are coordinated by secreted chemokines. Indeed, we demonstrated that Ang II increased the colonic production of macrophage inflammatory protein- 2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), which belong to the CXC chemokine family. We have recently observed that both CXC chemokines mediate leukocyte adhesion in colonic I/R. In separate experiments, we also found that MIP-2 and KC were capable of stimulating neutrophil chemotaxis directly, whereas Ang II had no effect on neutrophil migration in vitro, suggesting that Ang II mediates its proinflammatory effect on leukocytes in vivo indirectly via CXC chemokine generation. Prior to examining the role of Ang II in colonic I/R-induced leukocyte-endothelial cell interactions, we wanted to determine whether the proinflammatory effects of Ang II may be mediated by reactive oxygen species (ROS). It was found that allopurinol, which is a potent inhibitor of xanthine oxidase-generated ROS, had no effect on Ang II-induced leukocyte rolling and adhesion. However, administration of the oxygen radical scavenger superoxide dismutase significantly attenuated reperfusion-induced leukocyte adhesion by 70%. Considered together, all these findings led us to hypothesize that Ang II may be involved in colonic I/R.

2. Ang II plays an important role in colonic I/R
To determine the role of Ang II in colonic I/R, we first examined the gene expression of angiotesin converting enzyme (ACE) in the colon by reverse transcriptase-polymerase chain reaction and found that ACE mRNA was only expressed at low levels in the colon of sham-operated mice. In contrast, I/R markedly increased the gene expression of ACE mRNA in the colon, suggesting that ACE is generated in colonic I/R. To confirm this at the protein level, we used a radioimmunoassay to detect the protein levels of Ang II in the plasma. Indeed, we observed that colonic I/R significantly increased the plasma levels of Ang II; i.e., the plasma concentration of Ang II was 9 ± 3 pg/ml in sham-operated mice and 35 ± 11 pg/ml in response to I/R (Fig. 1 ). Knowing that the expression of ACE is increased during I/R in the colon, we asked whether an ACE inhibitor such as captopril may protect against I/R-induced leukocyte responses in the colon. Notably, we found that treatment with captopril significantly attenuated I/R-provoked leukocyte adhesion by 64% (Fig. 2 ), whereas leukocyte rolling was insensitive to captopril treatment. Next, we determined the role of the AT₁ receptor in colonic I/R. For this purpose, we used losartan, which antagonizes the function of the AT₁ receptor and found that pretreatment with losartan had no effect on leukocyte rolling induced by I/R. Conversely, it was found that pretreatment with losartan significantly decreased the number of adherent leukocytes by 65% (Fig. 2) .

View larger version (13K): [in this window] [in a new window]   Figure 1. Plasma levels of Ang II after 30 min of ischemia and 120 min of reperfusion. Sham-operated animals served as negative controls. The plasma levels of Ang II were determined by use of a radioimmunoassay. Data represent mean ± SEM, and n = 5. *, P < 0.05, versus sham.

View larger version (16K): [in this window] [in a new window]   Figure 2. Venular leukocyte adhesion in the mouse colon after 30 min of ischemia and 120 min of reperfusion. Mice were pretreated intravenously with phosphate-buffered saline (PBS), an ACE inhibitor (captopril, 100 mg/kg), and an Ang II type 1 (AT1) receptor antagonist (losartan, 10 mg/kg). Sham-operated animals served as negative controls. Data represent mean ± SEM, and n = 5–8. *, P < 0.05, versus PBS + I/R; #, P < 0.05, versus PBS alone.

CONCLUSIONS

An accumulating body of experimental data suggests that Ang II is not only an important regulator of homeostatic functions, such as blood pressure and fluid balance, but also is involved in several aspects of pathological inflammation. Our work demonstrates that Ang II exerts potent proinflammatory effects in the colon. In fact, we found that Ang II dose-dependently increases leukocyte rolling and adhesion in colonic venules. Specific adhesion molecules support leukocyte-endothelium interactions, although the relative importance appears to be organ- and stimulus-dependent. Herein, we showed that P-selectin but not E-selectin supported Ang II-induced leukocyte rolling in the colon. The notion that Ang-II-provoked leukocyte rolling is mainly mediated by P-selectin is also corroborated by previous observations in the rat mesentery. Moreover, it was found that Ang II-provoked leukocyte adhesion was abolished in LFA-1-deficient mice. Notably, we also demonstrated that inhibition of the P-selectin-dependent rolling concomitantly abolished leukocyte adhesion. Combined, these findings suggest that Ang II-induced leukocyte recruitment in the colon is a multistep process mediated by an initial P-selectin-mediated rolling interaction, which is a precondition for the subsequent leukocyte adhesion supported by LFA-1 (Fig. 3 ). Secreted chemoattractant substances, such as chemokines, coordinate leukocyte activation and recruitment. Indeed, we found that Ang II had the capacity to up-regulate CXC chemokines (MIP-2 and KC) in the colon, which in turn can trigger an avidity shift in LFA-1 and increased, firm leukocyte adhesion to the microvascular endothelium in the colon (Fig. 3) . In this context, identical adhesive (P-selectin/LFA-1) and signaling (CXC chemokines) pathways are involved in the multistep process of leukocyte accumulation provoked by Ang II and I/R, which led us to hypothesize that Ang II may have a potential role in colonic I/R. It is widely held that Ang II is a potent inducer of ROS production, and in line with this, we showed herein that Ang-II-induced leukocyte adhesion was dependent on ROS generation. It is also interesting to note that we have recently established a link between ROS generation on one hand and CXC chemokine secretion on the other in this model of colonic I/R.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic model depicting the role of Ang II in colonic I/R and the inhibitory impact of captopril and losartan. This model stipulates that gastrointestinal I/R leads to the up-regulation of ACE and Ang II, which in turn stimulates P-selectin-dependent rolling and LFA-1-mediated, firm adhesion of leukocytes in postcapillary venules in the colon.

The gastrointestinal mucosa is particularly vulnerable to even mild hypovolemia and ischemia, and Ang II appears to be particularly important in the splanchnic circulation. For example, it has been shown that the splanchnic vasculature has extraordinarily high concentrations of Ang II receptors, which may help to ensure adequate allocation of blood supply to vital organs, such heart and brain during critical conditions. Indeed, we found that the gene expression of ACE was up-regulated in response to I/R in the colon. Moreover, we were able to demonstrate that the plasma levels of Ang II were markedly increased after I/R. Thus, it seemed logical to treat mice with the ACE inhibitor captopril, which greatly reduced I/R-induced leukocyte responses in the colon, suggesting that the I/R-induced expression of Ang II observed herein indeed plays a pathophysiological role in colonic I/R. These findings add the colon to the list of organs being protected against I/R by interfering with the renin-angiotensin system, including the heart, brain, and liver. Notably, we could also show a functional role of the AT₁ receptor in I/R-induced leukocyte responses in the colon. Thus, taken together, our novel data suggest that Ang II plays an important role in colonic I/R and that interference with the generation (ACE) or function (AT₁ receptor) of Ang II may constitute a pharmacological approach to control pathological inflammation in the gastrointestinal tract.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0502fje;

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