INTRODUCTION

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OCCLUSION OF BLOOD VESSELS supplying the heart, brain, kidneys, and mesentery often produces ischemia-reperfusion (I/R) injury that is characterized by cellular necrosis and tissue destruction. One of the earliest events in the pathogenesis of I/R injury is vascular dysfunction that is accompanied by a diminution in nitric oxide (NO) production by the endothelium (7, 13, 16, 17). This pronounced deficit in NO release has been implicated as a trigger for leukocyte-endothelial cell interactions resulting in cellular injury (16). Early experimental investigations by Tsao et al. (16) demonstrated that vascular reactivity of feline coronary arteries to acetylcholine (ACh) was significantly attenuated at 5 min of reperfusion. In addition, a number of other studies have reported postischemic impairment of endothelium-dependent vascular reactivity in a variety of species and organs.

It is now believed that there are at least two distinct mechanisms responsible for endothelial cell injury after I/R. Treatment with oxygen-derived free radical scavengers such as superoxide dismutase before reperfusion of ischemic myocardium has resulted in the preservation of coronary NO release and suggests that oxygen radicals may mediate postischemic vascular dysfunction (16). Oxygen free radicals such as superoxide anion are produced within minutes after reperfusion, and this rapid burst of toxic radicals coincides with the time course of endothelial dysfunction (20). Furthermore, the results of other studies (10-12, 14) have suggested that circulating leukocytes such as neutrophils [polymorphonuclear leukocytes (PMNs)] actively participate in the endothelial cell injury resulting from I/R. Studies employing blocking monoclonal antibodies directed against CD18 (11), intercellular adhesion molecule 1 (ICAM-1) (10), L-selectin (12), and P-selectin (8, 18) have reported preservation of endothelial NO release in addition to inhibition of PMN infiltration and tissue necrosis.

In the present study, we reexamined the role of leukocytes in mediating I/R-induced alterations in vascular reactivity using a novel experimental strategy, i.e., by assessing endothelium-dependent vasodilation in gene-targeted mice. To avoid potential nonspecific actions of monoclonal antibodies, we examined postischemic vascular reactivity in wild-type mice and in mice that are genetically deficient in either CD11/CD18, ICAM-1, or P-selectin.

	MATERIALS AND METHODS

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Gene-targeted mice. Male wild-type (C57BL/6) mice and CD11/CD18-deficient, ICAM-1-deficient, or P-selectin-deficient mice (C57BL/6J background) were utilized for the experimental studies (1, 15, 19). The gene-targeted mice were all prepared and provided by Pharmacia Upjohn Laboratories (Kalamazoo, MI). All mice were obtained at 4 wk of age and maintained on standard mouse chow before the experiments.

Animal care guidelines. These experimental procedures complied with federal and state regulations and with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 86-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892] approved by the Council of the American Physiological Society. The experimental protocol was approved by the Animal Care and Use Committee of Louisiana State University Medical Center (Shreveport, LA).

Superior mesenteric artery I/R. The mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) diluted 1:1 with 0.9% sodium chloride. All surgical procedures were performed using an Olympus model SZ240 dissecting microscope (Olympus Optical, Tokyo, Japan) with an ACE I series fiber-optic light system (Fostec, Auburn, NY). On complete anesthesia, the mice were secured to a surgical platform and the core body temperature was maintained at 37°C with an infrared heating lamp (Cole-Parmer, Chicago, IL). A midline abdominal incision was made, the skin and peritoneum were retracted laterally, and the intestines were mobilized to the right. The origin of the superior mesenteric artery (SMA) was isolated, and all connective tissue and lymphatics were dissected from the SMA. The intestines were then replaced into the abdomen and the mice were allowed to stabilize for 30 min. The origin of the SMA was then ligated with 7-0 monofilament suture for 45 min, at which time the suture was carefully removed and the artery was reperfused for 45 min.

Isolated SMA ring experiments. The SMA was then transected and gently retracted away from the underlying connective tissue, which typically yielded a segment of SMA 3-4 mm in length. The harvested SMA was then placed in a petri dish containing ice-cold Krebs buffer with the following composition (mmol/l): 120 NaCl, 5.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 20 NaHCO₃, 11 glucose, and 0.029 disodium EDTA. With the aid of a dissecting microscope, the terminal ends of the SMA were transected, yielding a 2-mm segment of SMA. Two pieces of tungsten wire (World Precision Instruments, Sarasota, FL) with a diameter of 0.077 mm were meticulously inserted into the SMA ring, and triangles were constructed with both of the wires. The SMA rings were then mounted onto force transducers (Radnoti Glass, Monrovia, CA) and submerged into glass organ chambers (Radnoti Glass) filled with 10 ml of Krebs buffer. The organ chamber solution was maintained at 37°C bubbled with a gas mixture of 95% O₂-5% CO₂. The rings were placed under an initial tension of 250 mg for 1 h, and the Krebs buffer was changed every 15 min. The SMA rings were then contracted with phenylephrine (Sigma Chemical, St. Louis, MO) at a concentration of 10⁵ M, followed by addition of increasing concentrations of ACh (Sigma Chemical) ranging from 10⁹ to 10⁶ M. The SMA rings were then washed with Krebs buffer and allowed to recover for 30 min, during which time the rings were washed with Krebs buffer every 5 min. The rings were contracted again with phenylephrine, and then sodium nitroprusside (SNP; Sigma Chemical) was added to the organ chambers at concentrations of 10¹⁰ to 10⁶ M. In additional experiments, N^G-nitro-L-arginine methyl ester (L-NAME, Sigma Chemical) was added at a final concentration of 10⁴ M to SMA rings 10 min before contraction with phenylephrine, and the vasorelaxation responses to ACh and SNP were then tested. Maximal relaxation was expressed as the percentage of the plateau tension induced by phenylephrine.

Statistical analysis. All values in the text and in Figs. 1-3 are presented as means ± SE of n independent experiments. Statistical tests were performed using StatView version 4.1 software (Abacus Concepts, Berkeley, CA) using a 95% confidence level to determine significance differences. Comparisons were made using Bonferroni's post hoc test or with the nonparametric Kruskal-Wallis test when standard deviations were not equal.

	RESULTS

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Vasorelaxation of SMA from wild-type mice. Initial studies examined the vasoreactivity of the SMA from C57BL/6 mice under control conditions as well as after either endothelial cell denudation or administration of the NO synthase inhibitor L-NAME. In addition, we studied the vasoreactivity of C57BL/6 SMA segments after 45 min of SMA occlusion (without reperfusion). These data are summarized in Fig. 1.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Vascular reactivity of isolated superior mesenteric artery (SMA) segments to ACh (106 M) and sodium nitroprusside (SNP; 106 M) from C57BL/6 mice under control conditions (n = 6), after SMA ischemia alone for 45 min (n = 5), after endothelial denudation (n = 4), and after treatment with NG-nitro-L-arginine methyl ester (L-NAME; n = 4). Vasorelaxation to ACh was dramatically impaired after denudation and administration of L-NAME. Bars represent means ± SE for 6-8 mice. ** P < 0.01 vs. control.

Effects of I/R on SMA vascular reactivity. In additional studies, we investigated the effects of occlusion of the SMA for 45 min followed by 45 min of reperfusion in wild-type (C57BL/6) and in CD11/CD18-, ICAM-1-, and P-selectin-deficient mice (Fig. 2). Vascular reactivity to ACh was normal in SMA subjected to sham I/R (83.5 ± 3.3%) but was significantly attenuated in SMA rings isolated from wild-type mice after I/R (51.3 ± 4.7%; P < 0.05 vs. sham). In contrast, vascular responses to ACh were normal in the SMA rings from mice deficient in CD11/CD18 (84.7 ± 2.3%), ICAM-1 (83.1 ± 2.9%), or P-selectin (78.1 ± 3.3%) after SMA occlusion and reperfusion (P = NS vs. sham or C57BL/6).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   ACh-induced vasorelaxation of the isolated SMA after 45 min of ischemia and 45 min of reperfusion from C57BL/6 wild-type mice (n = 9) and mice deficient in CD11/CD18 (n = 10), intercellular adhesion molecule 1 (ICAM-1; n = 10), or P-selectin (n = 8). Ischemia-reperfusion (I/R) of C57BL/6 wild-type mice SMA significantly reduced the vasorelaxation response to ACh compared with sham I/R (n = 8). SMA isolated from mice deficient in CD11/CD18, ICAM-1, and P-selectin displayed normal relaxation responses to ACh. Bars represent means ± SE. * P < 0.05 vs. sham.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   SNP-induced vasorelaxation of the isolated SMA after 45 min of ischemia and 45 min of reperfusion. Vasorelaxation to SNP was unaffected by I/R in C57BL/6 wild-type mice (n = 9) and in mice deficient in CD11/CD18 (n = 10), ICAM-1 (n = 10), and P-selectin (n = 8). Bars represent means ± SE.

	DISCUSSION

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In the present study, we provide strong evidence for the involvement of CD11/CD18, ICAM-1, and P-selectin in the vascular dysfunction elicited by I/R. Vascular reactivity to the endothelium-dependent vasodilator ACh was significantly attenuated in wild-type mice after I/R. Vascular reactivity to the endothelium-independent vasodilator SNP was unaffected by I/R, indicating that the vascular dysfunction was confined to the endothelium. Mice deficient in either CD11/CD18, ICAM-1, or P-selectin were completely protected from endothelial cell injury after I/R. Furthermore, this endothelial cell dysfunction was dependent on reperfusion of the occluded SMA because ischemia alone did not produce the deficit in vascular reactivity. Thus it appears that leukocyte-endothelial cell interactions mediated by both leukocyte and endothelial cell adhesion molecules contribute to the vascular dysfunction associated with I/R.

In the present study, we report a dramatic attenuation in endothelium-dependent vasorelaxation of isolated segments of SMA after occlusion and reperfusion. Lefer and Ma (7) previously reported that SMA occlusion for 90 min followed by reperfusion produced a dramatic reduction in the response of isolated rat SMA rings to ACh but not to acidified sodium nitrite. Furthermore, the degree of vascular dysfunction was more severe in the distal segments of the SMA compared with the proximal segments. This study also demonstrated that administration of human recombinant superoxide dismutase reversed endothelial dysfunction in the ischemic-reperfused SMA. Our demonstration of endothelial cell dysfunction in wild-type mice after I/R serves to confirm the results of Lefer and Ma (7). To our knowledge there have been no other studies investigating the effects of antileukocyte therapy on the postischemic vascular reactivity of SMA.

Occlusion and reperfusion of a vessel have been shown to trigger an inflammatory response that is characterized by neutrophil infiltration into ischemic-reperfused tissue (9). The accumulation of neutrophils within postischemic tissue occurs rapidly and is modulated by a number of distinct adhesion molecules that are expressed on the surface of endothelial cells and neutrophils (2). Initial interactions between circulating neutrophils and the inflamed endothelium are mediated by the selectin family of adhesion molecules that includes P-selectin, E-selectin, and L-selectin (6). P-selectin and E-selectin are expressed by endothelial cells after stimulation with cytokines, oxygen radicals, and a variety of other inflammatory mediators (6). L-selectin is primarily expressed on the surface of unactivated neutrophils and other leukocytes and is rapidly shed after leukocyte activation (6). The selectins have been shown to regulate the initial "rolling" response of neutrophils that occurs before firm adhesion of neutrophils to the endothelium (6). Previous studies have implicated both P-selectin (8, 18) and L-selectin (12) in the recruitment of neutrophils after I/R. The neutrophil ₂-integrins (LFA-1, MAC-1, and p150,95) interact with endothelial cell ICAM-1 to promote the firm adherence of neutrophils to the vascular endothelium after I/R (2).

Previous experimental investigations have suggested that neutrophils contribute to vascular injury after I/R (13, 16). Administration of monoclonal antibodies directed against CD18, ICAM-1, and P-selectin has provided insights into the role of leukocyte-endothelial cell interactions on impaired vascular reactivity and tissue injury after arterial occlusion and reperfusion. Ma et al. (11) previously reported that treatment with a monoclonal antibody directed against the common -chain of the ₂-integrins, CD18, resulted in the preservation of coronary artery vasorelaxation to ACh after myocardial I/R in the cat. Similarly, immunoneutralization with an anti-ICAM-1 monoclonal antibody (10) also blunted coronary artery endothelial dysfunction elicited by I/R. Studies of monoclonal antibodies that inhibit the function of P-selectin (8, 18) have also demonstrated vascular protection in the coronary circulation after coronary I/R.

Reductions in vascular responses to ACh indicate an inability of the endothelium to produce NO. It is well appreciated that NO is an important modulator of neutrophil adhesion to the endothelium and that reductions in endothelial NO can result in enhanced adhesion of neutrophils to the endothelium (4). It has been previously reported that the reductions in coronary NO production that occur very rapidly after reperfusion of the ischemic myocardium are a trigger for neutrophil accumulation and myocardial cell necrosis (13). In the present study, we did not measure neutrophil infiltration or tissue injury in the intestine, but it is conceivable that the inability of the SMA to release NO may have contributed to neutrophil-mediated injury to the mesentery after I/R. Furthermore, administration of an NO donor has been shown to reduce the extent of leukocyte-endothelial interactions and vascular injury in the mesentery after SMA occlusion and reperfusion (5).

One potential limitation of our study relates to the measurement of postischemic vascular reactivity in a large artery. It is well known that the majority of leukocyte-endothelial cell interactions occur in the microcirculation during acute inflammatory responses (6). Hence, for our data to be explained, leukocytes that are adherent in the mesenteric and intestinal microcirculation would have to elicit endothelial dysfunction in upstream arteries, i.e., in the SMA. This possibility appears tenable inasmuch as it has been shown that leukocytes that adhere in postcapillary venules can alter endothelial cell barrier function in upstream capillaries (3). These responses have been attributed to phenomena such as ascending propagation (via cell-to-cell communication).

A novel feature of the present study is the application of gene-targeted mice for the elucidation of mechanisms that underlie I/R-induced vascular dysfunction. The advent of techniques that allow for genetic manipulation of mice has led to the creation of a number of mutants that are particularly relevant to research on the problem of I/R-mediated alterations in vascular reactivity. In addition to adhesion molecule-deficient mice, mutants are now available that are either deficient in specific isoforms of NO synthase or overexpress antioxidant enzymes such as superoxide dismutase or catalase. Although our study represents the first effort to apply gene-targeted mice to studies of I/R-induced changes in vascular reactivity, the potential for extending this experimental strategy to other mutant mice models appears to hold much promise. One must be cautious, however, in the interpretation of results obtained with gene-targeted mice because it is possible that gene modification may result in the removal and/or the disruption of neighboring genes, resulting in other functional losses.

In summary, we have provided strong evidence using gene-targeted mice that the interaction of circulating leukocytes with the endothelium contributes to endothelial dysfunction of the SMA after occlusion and reperfusion. This vascular dysfunction is clearly dependent on reperfusion of the occluded artery because ischemia alone does not produce any vascular dysfunction. One potential mechanism for the protective effects of antileukocyte therapies may be the preservation of endothelial cell NO release. Our findings also suggest that therapeutic strategies aimed at the inhibition of leukocyte-endothelial cell interactions may prove beneficial in the treatment of ischemic syndromes in a variety of organ systems. Although these results demonstrate vasculoprotection in adhesion molecule-deficient mice after I/R, it should be pointed out that prolonged immune depression may actually be deleterious because of an increased susceptibility to infection.