In Vivo Imaging of Leukocyte Recruitment to the Atheroprone Femoral Artery Reveals Anti-Inflammatory Effects of Rosuvastatin

Objective. To monitor the anti-inflammatory effect of rosuvastatin in leukocyte endothelial interactions in the atheroprone femoral artery in vivo. Methods and Results. Male Apolipoprotein E null mice (ApoE−/− mice, 6 weeks old) were fed a high-fat diet (20% fat, 1.25% cholesterol) with or without the HMG CoA reductase inhibitor rosuvastatin (10 mg/kg/day) for 6 weeks. Significant leukocyte adhesion was observed in the femoral artery of ApoE−/− mice, but not of wild type mice, in the absence of rosuvastatin. Interestingly, no obvious plaque formation was observed in the artery at this time point. The number of adherent leukocytes was dramatically diminished in ApoE−/− mice treated with rosuvastatin. DHE-associated oxidative stress and the expression of gp91-phox, a component of NADPH oxidase, were induced in ApoE−/− mice and were abolished by rosuvastatin treatment. Conclusion. Our data documented leukocyte recruitment prior to lipid accumulation and subsequent inhibition by rosuvastatin. The underlying mechanism seemed to involve oxidative stress and an anti-inflammatory effect on the endothelium of atheroprone vessels.


Introduction
In�ammatory cascades, such as leukocyte recruitment to the vascular wall, play an important role in the development of atherosclerosis [1][2][3]. Recent clinical studies have suggested a positive correlation between the serum level of in�ammatory markers and the rate of cardiovascular events [4,5]. Careful pathological examination of atherosclerosis specimens have revealed the presence of in�ammatory cells, such as monocytes, macrophages, and lymphocytes, at the lesion area, which are thought to be causatively involved in the development of atherosclerosis. Nevertheless, there is no direct evidence to con�rm that monocyte recruitment to the luminal surface of the artery occurs prior to the lipid deposition in vivo. As previously demonstrated, leukocyte endothelial interaction in vivo is heavily in�uenced by local shear stress created by blood �ow [6,7], and thus it is intriguing to note that leukocyte recruitment is occurring at the arterial wall in the presence of relatively high shear stress without mechanical injury.
To investigate this issue, we developed a novel imaging system to dynamically visualize leukocyte recruitment to the femoral artery in vivo. Using this technique, we previously documented that mechanical injury to the arterial intima signi�cantly induces leukocyte adhesion to the vascular wall [8].
In this study, we tried to demonstrate leukocyte adherence to the vascular wall in the absence of mechanical vascular injury in vivo and potential modulation by rosuvastatin treatment.
Statins are used clinically to reduce serum cholesterol levels leading to the reduction of cardiovascular events [9][10][11]. Several experimental studies, including ours, have indicated an anti-in�ammatory role of statins in vitro [12,13]. A recent clinical study has suggested a pleiotropic anti-in�ammatory property for rosuvastatin which may play a   role in reducing cardiovascular events among those with high serum CRP but normal cholesterol levels [14]. ough experimental animal studies have revealed that statins can inhibit or even diminish the development of atherosclerosis [15,16], their effect on leukocyte-endothelial interactions in the development of atherosclerosis has not been demonstrated in vivo. erefore, we sought to document recruitment of leukocytes at the vasculature of athero-prone animals in the absence of mechanical injury and the reversal of this recruitment by rosuvastatin.

Materials and Methods
2.1. Animals. C57BL/6 (6 weeks of age, male) mice were obtained from Charles River Laboratories Japan, Inc. and used as control (wild type: wt). Apolipoprotein E-de�cient mice (6 weeks of age, male; ApoE−/− mice) were used in this study. ey were provided with diet and water ad libitum. e experiments adhered to the APS Guiding Principles in the Care and Use of Animals and were approved by the Ethical Committee for Animal Experimentation of Tokyo Medical and Dental University.

Intravital Microscopy (IVM).
Intravital microscopy (IVM) of the femoral arteries was performed on ApoE−/− mice fed normal chow (NC; CE-2, CLEA Japan, Inc., Tokyo) or high fat diet (HF; 1.25% cholesterol, 20% fat in CE-2; CLEA Japan, Inc., Tokyo) for 0, 2, or 6 weeks and was compared with those performed on wt mice. In some of those experiments, rosuvastatin (rosuva; 10 mg kg −1 per day, AstraZeneca KK) or vehicle (water) was simultaneously administered to ApoE−/− mice fed HF and leukocyte adhesion was observed in the femoral artery as previously detailed [8]. In brief, mice were anesthetized with pentobarbital and mechanically ventilated so as to maintain a normal acid-base balance. Rectal temperature was maintained at 36.0-37.0 ∘ C with a heating pad and an infrared heat lamp. Aer injection of Rhodamine 6G chloride (Invitrogen, Carlsbad, CA, USA; 0.3 mg kg −1 in 300 L of phosphate buffered saline (−)) into the right femoral vein, the le femoral artery at the level of the epigastric branch was visualized with a �uorescent microscope (BX51WI, Olympus, Tokyo, Japan) equipped with a water immersion objective (×20). Epi�uorescence was illuminated by a 100-W �uorescent lamp source and images were directly captured with a PC via a CCD camera (CoolSnap HQ, Olympus). Adhesion of labeled leukocytes was clearly visualized on the anterior half of the vessels facing the objective. All images were recorded using a computer-assisted image analysis program (Meta Morph) according to the manufacturer's protocol. e parameters used to characterize the adhesive interactions of leukocytes have been described in detail previously [17]. e number of adherent leukocytes (i.e., those that did not move for ≥3 s during the 1 min recording period) was counted along a region of interest (ROI), a 10 4 m 2 segment of the vessel and expressed as the number of adherent cells per 10 4 m 2 of the vessel surface. Image analysis was carried out as previously described [8,[18][19][20][21][22]. IVM at 30 min aer antibody injection. e total number of leukocytes recruited to the vessel wall was derived from the sum of adherent and rolling cells.  Similarly, cross-sections of femoral artery in ApoE−/− mice fed NC at age of 52 weeks was staining.

Intravenous Injection of Externally Labeled MNCs.
In some experiments, recipient or donor mice were administered HF with rosuvastatin or vehicle for 6 weeks. Mononuclear cells (MNCs) were isolated from peripheral blood taken from two ApoE−/− mice treated with rosuvastatin or vehicle by gradient centrifugation using an LSM (Histopaque-1083, Sigma Aldrich Corp., St Louis, MO, USA). ey were labeled with Rhodamine 6G chloride and then 5 × 10 5 cells were injected intravenously into the mice, and intravital microscopy was performed at 5 min aer cell injection.

Statistical
Analysis . Data are expressed as the mean value ± s.e.m. One-way analysis of variance with a Tukey's post hoc test or two-tailed unpaired t-test was used to estimate statistical signi�cance, with a value of 5 considered to be statistically signi�cant.

Real-Time Observation of Leukocyte Recruitment to Femoral Artery in ApoE−/− Mice.
First we observed leukocyte recruitment to "noninjured" femoral arteries of wt or ApoE−/− mice fed NC. As shown in Figure 1(a), prominent leukocyte adhesion was observed as early as 6 weeks of age (0 weeks feeding) in ApoE−/− mice (31 67 ± 19 43/10 4 m 2 vessel surface, 3) and had increased at 12 weeks of age (6 weeks-feeding NC, 32 43 ± 3 91, versus wt , 3), whereas no adhesion was observed in femoral arteries of wt at any time points (2 w ± , 3; 6 w ± , 3, Figure 1(a)). e plasma levels of total cholesterol (TC) signi�cantly increased in ApoE−/− mice fed NC when compared to wt mice at any time. e level of triglyceride (TG) did not change between ApoE−/− mice and wt mice (Figure 1(b)). Body weight, plasma glucose level, and blood pressure did not change between ApoE−/− mice and wt (data not shown).
We examined the potential effect of a high-fat diet on leukocyte adhesion to the femoral artery. Since a high-fat diet alone failed to develop atherosclerosis in wild-type mice (data not shown), we utilized ApoE−/− mice and subjected them to a high-fat diet. Interestingly, the number of adherent cells in ApoE−/− fed HF did not increase statistically when compared with those obtained from ApoE−/− mice fed NC (HF2w, 38 ± 14 7, 3, versus NC2w 63; HF6w, 46 67 ± 14 19, 3, versus NC6w 21, Figure  1(a)). TC level signi�cantly increased in ApoE−/− mice fed HF when compared to NC feeding, whereas plasma TG did not signi�cantly increased in ApoE−/− mice fed HF when compared with NC. ( * , * * 1, # 1, 3, Figure 1(b)).

Adoptive Transfer of MNC Treated with Rosuvastatin.
To determine whether rosuvastatin affects the leukocytes or the vascular tissues, we performed adoptive transfer of peripheral MNCs. MNCs from mice treated with vehicle were harvested, labeled ex vivo with Rhodamine 6G, and administered intravenously into recipient mice treated with vehicle (24. ± 2.89/10 4 m 2 vessel surface, 3). As shown in Figure 4, the leukocytes from donor mice adhered to the recipient femoral artery in a similar way to the endogenous leukocytes. In contrast, when MNCs prepared from mice with vehicle were infused into recipient mice treated with rosuvastatin, MNC recruitment was slightly decreased (12.7 ± 3.2 , 4). Similarly, when MNCs prepared from mice with rosuvastatin were injected into recipient mice with vehicle, MNC recruitment was also slightly decreased (17.2 ± 8. 2, 7). However, the magnitude of antiadhesive effects was comparable among these two groups. When MNCs prepared from mice treated with rosuvastatin were injected into recipient mice treated with rosuvastatin, MNC recruitment in the recipient artery was signi�cantly inhibited ( .4 ± .2 , ).

Discussion
In this study, we were able to observe leukocyte recruitment to non-injured femoral arteries of ApoE−/− mice as early as 6 weeks of age. Interestingly, pathological examination of the specimen revealed that there was no atherosclerotic lesion formation at this age. Whereas prominent atherosclerotic lesions were found in the same vascular region of the femoral artery at 52 weeks (Figure 3(c)). us, observation of the femoral artery at an early time point may be suitable for studying vascular in�ammatory change in atherogenesis. To our knowledge, our data are the �rst to demonstrate that leukocyte recruitment to the luminal surface of the vasculature precedes atherosclerotic plaque formation in vivo. We also tried to examine a potential contribution of high-fat diet on early vascular in�ammation observed in the femoral artery of ApoE−/− mice. ough previous study examined that a high fat diet signi�cantly accelerate atherosclerosis lesion formation in ApoE−/− mice [26], we failed to detect a signi�cant increase in the number of adherent leukocytes at 6 weeks aer a high-fat diet. Potential qualitative differences of leukocyte adhesion such as distinct cell type recruited by NC and HF may affect these data observed in those treated with NC and high fat diet. ese phenotypic differences in adherent leukocytes in the femoral artery will be examined in our future studies. Next, we examined the expression level of adhesion molecules in these mice. As shown in Figure  1(c), VCAM-1, but not ICAM-1 was upregulated in ApoE−/− mice. Further, antibody against VCAM-1 signi�cantly blocks leukocyte adhesion to the femoral artery. ese data strongly suggest a contribution of VCAM-1 in leukocyte adhesion in ApoE−/− mice. In good agreement with our data, Nakashima et al. [27] also reported enhancement of VCAM-1 expression in the aortic arch of ApoE−/− mice. As we demonstrated in Figure 1(e), oxidative stress is also accumulated in the vasculature of ApoE−/− mice. Lee et al. reported that VCAM-1 expression is closely related with oxidative stress via Sp1dependent gene regulation [28]. Cayatte et al. found that inhibition of NADPH oxidase activity decreased atherosclerotic lesion areas in ApoE−/− mice via reduction of VCAM-1 expression [29]. Taking all together including ours, oxidative stress may play an important role to induce expression of VCAM-1 in ApoE−/− mice. In this study, expression levels of gp91-phox and p22-phox were increased in aortas of ApoE−/− mice. Sustained hyperlipidemia in ApoE−/− mice increases their systemic oxidative stress [30]. �ur �nding may point to a pivotal role of oxidative stress in connecting hyperlipidemia and vascular in�ammation in vivo. We also con�rmed an antiadhesive effect of rosuvastatin. e lipidindependent effect of statins has been focused in recent years and our groups con�rmed mechanistic insights of antiadhesive effect of statin in vitro using physiological �ow conditions [31]. Current data further strengthened our previous observation by using in vivo imaging system. Since serum cholesterol levels were not affected by rosuvastatin treatment (Figure 2(e)), our �nding is not due to the improvement of hyperlipidemia in ApoE−/− mice. Rather, inhibition of oxidative stress, primarily reduction of gp-91phox, may play an important role in this process. is observation is in good agreement with previous studies [32,33]. Adoptive transfer of MNC revealed that both vascular wall and leukocytes were target of rosuvastatin to reduce leukocyte recruitment, which exhibited comparison to own previous study using ARB in leukocyte adhesion in vivo, where leukocyte activation plays a dominant role [22].

Conclusion
We were able to document leukocyte adhesion to the endothelium of the femoral artery in mice with dyslipidemia.
e underlying mechanism seemed to involve oxidative stress and VCAM1 expression. Rosuvastatin abolished these phenomena via downregulation of gp91-phox, a component of NADPH oxidase. ese results indicate that rosuvastatin has a protective effect against vascular in�ammation and oxidative stress.
��n��c� �f �n�eres�s e authors declared that they have no con�ict of interests.