Monoclonal Antibody to Very Late Antigen-4 (VLA-4) Protects Skin Flaps against Ischemia-Reperfusion Injury: An Experimental Study in Rats

In recent years, various adhesion molecules have been discovered. Very late antigen-4 (VLA-4), an adhesion molecule belonging to the integrin family, plays an important role in lymphocyte migration and infiltration into sites of in ammation. In the present study, we investigated whether administration of antiVLA-4 monoclonal antibody (mAb) reduces in ammation in ischemia-reperfusion injury using a skin flap model in Sprague-Dawley rats. A superficial epigastric arteriovenous pedicle ap, sized 45×30 mm, was elevated in the right inguinal area. The vessel was clamped to induce ischemia and the clamp was removed after 9 hours. Fifteen minutes prior to clamp release, the rats were administered antiVLA-4 mAb (0.2 mg / kg i.v., n= 10) or saline (n= 10). At the same time, a sham group (n= 5) was established with a similar operation but the vessel was only clamped to induce ischemia for 5 minutes. Anti-VLA-4 mAb significantly improved the survival area of the skin ap to 92.4% (median), when compared to 12.5% (median) in the control group. Severe in ammatory cell in ltration and edema was consistently observed in the skin aps of the control group, while antiVLA-4 reduced these parameters to a level similar to the sham group. These results indicate that anti-VLA-4 mAb may be used as an anti-in ammation agent, however the underlying mechanism still remains to be elucidated.


Introduction
In recent years, various adhesion molecules have been discovered. Such molecules are closely associated with cellular interactions and play crucial roles in in ammation and the regulation of immunoreaction 1 . They play an important role in the progress of in ammation by activating leucocyte adherence to vascular endothelial cells. The tissue in ltration process of an in ammatory reaction can be classi ed into the following three steps : rolling step, strong adhesion step, and transmigration step. In each step, different adhesion molecules are involved.
To whom corresponding should be addressed.
The phenomenon called ischemia-reperfusion injury is known to play an important role in the mechanism of skin ap survival and necrosis. In particular, it has been suggested that changes in vascular endothelial cells during ischemia-reperfusion injury are involved in preventing skin ap survival and causing necrosis [2][3][4][5][6] . To elucidate the mechanisms involved in skin ap survival and necrosis, a rat model of skin ap ischemia-reperfusion injury has been developed. Thus, we used this rat model to understand the causative agents responsible for in ammation 6,7 .
Integrin, one of the adhesion molecules, is known to be highly involved in the regulation of intercellular and extracellular matrix adhesion and intracellular signal transduction via adhesion 8 . Very late antigen-4 VLA-4 , a member of the integrin family, is an adhesion molecule that plays an important role in the migration and in ltration of lymphocytes to sites of in ammation 9 . Therefore, we investigated whether the administration of anti-VLA-4 monoclonal antibody mAb reduces the in ammatory reaction in ischemia-reperfusion injury using the rat skin ap model. Here we report the results, along with a review of the current literature.

Skin ap ischemia-reperfusion injury model
Twenty-five male Sprague-Dawley rats 8-10 weeks of age, 225-250 grams, Saitama Experimental Animals Supply Co., Ltd., Saitama, Japan were used in this experiment. The animals were housed in wire-mesh cages and given free access to a pelleted diet Nosan Corporation, Kanagawa, Japan and drinking water tap water .
Ketamine hydrochloride 25 mg / kg was administered intraperitoneally as anesthesia. While anesthetised, the abdomen was shaved and a 45 35 mm super cial epigastric pedicled skin ap was designed and elevated from the right inguinal region. The ap pedicle was then clamped Fig. 1 6 . The animal experiment was conducted ethically according to the Showa University animal experiment guidelines.

Ischemic interval
The duration of ischemia was 9 hours for the anti-VLA-4 mAb and control groups, or 5 minutes for the sham group.

Antibody administration
Anti-rat VLA-4 mAb 0.20 mg / kg ; CD49d ; TA-2, Associates of Cape Cod Incorporated, Falmouth, MA was administered through the caudal vein 15 minutes before releasing the clamp. Sterilized saline was administered to the control group.

Experimental groups
The anti-VLA-4 mAb experimental group n 10 and the physiological saline control group n 10 were observed for 7 days. The same skin ap was elevated in the sham group n 5 , however the duration of ischemia was only 5 minutes. All rats were euthanized after 7 days of observation by an overdose of anesthesia, and histological examination of the skin aps was VLA-4 mAb Protects Skin Flap against I/R Injury performed Fig. 2 .

Method of evaluation
Skin aps were evaluated by macroscopic observation and histological analysis. 1 Skin ap survival area ratio : the areas of the survival region and necrotic region of the skin ap were calculated by tracing the areas, based on the template method of Morris et al 10 , and the survival area ratio was calculated as survival area / survival area + necrotic area x100.
2 Histological examination : samples were prepared by hematoxylin-eosin staining. Tissue samples were then examined to assess the extent of in ammation, and in ammation within blood vessels.

Statistical analysis
The data are expressed as median, with 25 th and 75 th percentiles. A Mann-Whitney U test was applied to evaluate the difference in survival area ratio between control and treatment groups. Differences of P 0.05 were considered to be statistically signi cant.

Skin ap survival area ratio
A nonparametric analysis of the experimental group and the control group was performed. Macroscopically, skin ap survival in the experimental group was good, and skin ap morphology and color were close to the sham group. However in the control group, skin aps showed severe in ammation accompanied by partial necrosis. The mean skin ap survival area ratio after 7 days observation was 92.4% in the experiemental group, which was signi cantly improved compared to 12.5% in the control group P 0.05 ; Fig. 3 and Table 1 .

Histological examination
In the experimental group, although in ammation was present and a disorganized structure was observed on part of the skin surface, the level of in ammatory cell in ltration and edema was relatively mild, and the vascular lumen was preserved, with a tissue structure similar to that of the sham group. In the control group, in ammatory cell in ltration, edema and necrosis were observed. In addition, the vascular lumen was lled with erythrocytes, indicating the formation of blood clots, which was dramatically different from the condition of the vascular lumen in the experimental group Fig. 4 .
Activated VLA-4 attaches onto vascular endothelial cells, and is a bronectin receptor. Unlike typical bronectin receptors, such as VLA-5, the binding of VLA-4 with bronectin is not via the RGD sequence 11,12 . VLA-4 induces migration of lymphocytes and monocytes to the site of in ammation by binding with in ammation-induced vascular cell adhesion molecule-1 VCAM-1 on vascular endothelial cells 13 . Then, the binding of VLA-4 with VCAM-1 leads to both rolling and adhesion of leucocytes. In short, it is believed that activated lymphocytes and macrophages expressing VLA-4 partially facilitate extravascular migration by binding with VCAM-1 which is expressed on the vascular endothelial cell by in ammatory cytokines 14 .
Various adhesion molecules expressed on activated leucocytes and vascular endothelial cells are involved in ischemia-reperfusion injury. Previously, in an experimental skin ap model of ischemia-reperfusion injury 15 , in ammation was signi cantly suppressed by the administration of antiintercellular adhesion molecule-1 mAb 6 . In the present study, we found that the administration of anti-VLA-4 mAb significantly suppressed the inflammation induced by ischemia-reperfusion injury. VLA-4 is involved with the rolling, adhesion and extravascular migration of leucocytes by binding with VCAM-1, which is expressed on vascular endothelial cells during in ammation. It is suggested that anti-VLA-4 antibody blocks the adhesion binding of VLA-4 with activated VCAM-1 on vascular endothelial cells and leucocytes during in ammation, consequently the rolling step is blocked, resulting in suppression of in ammation.
Kerrigan and Stotland 16 suggested that in ischemia-reperfusion injury, platelet activating factor, interleukin-6, and tumor necrosis factor TNF are induced after reperfusion to promote tissue damage. Moreover, there are several studies reporting that the administration of platelet activating factor antagonist or tacrolimus signi cantly reduces ischemia-reperfusion injury 7 . Inhibition of neutrophil-induced superoxide radical formation, inhibition of neutrophil-induced mediators such as TNF or leukotriene B4, suppression of reperfusion-induced TNF or interleukin-6, and attenuation of the neutrophil-vascular endothelial cell binding interaction by suppression of CD11 / CD18 activity have been mentioned as possible mechanisms involved in ischemiareperfusion injury.
Experimentally, it has been revealed that VLA-4 is related to the development of multiple sclerosis, based on the mouse model of autoimmune encephalomyelitis 17 . Additionally, it has been suggested that VLA-4 plays a role in cellular differentiation in bone marrow, as well as an important involvement in infiltration and metastasis of kidney cancer and acute myeloid leukemia 18,19 . Consequently, there has been a clinical study demonstrating the suppression of in ammation by administration of the anti-VLA-4 human mAb, natalizumab 20 .

Conclusion
In this study, we found in ammation was reduced by the administration of anti-VLA-4 mAb in a rat inguinal skin flap ischemia-reperfusion injury model. Anti-VLA-4 mAb significantly improved the survival area of the skin ap up to 92.4%, compared to 12.5% in control group. These results suggest that anti-VLA-4 mAb can be used as an anti-in ammation agent to prevent ischemia-reperfusion injury.

Con ict of interest disclosure
There are no conflicts of interest to declare concerning this study.