The Role of P38a MAPK in Atherosclerosis Figure 2. Comparable atherosclerotic plaque formation in p38aEC-KO/ApoE2/2 and ApoE2/2 mice after 10 weeks of HCD. (A) Analysis of deletion efficiency by immunoblotting in p38aEC-KO/ApoE2/2 and their ApoE2/2 littermates after 5 weeks of tamoxifen diet and 10 weeks of HCD, by MACS sorting of MLECs with CD146. CD146+ = MLECs, CD1462 = other cells. P38EC-KO/Apo2/2 females, n = 9; ApoE2/2 females, n = 6. (B) Quantification of lesion area on whole aorta from female p38aEC-KO/ApoE2/2 and ApoE2/2 mice. En face Sudan IV staining of plaques. P38aEC-KO/ApoE2/2 females, n = 14; ApoE2/2 females, n = 13. Scale bar, 2 mm. (C) Quantification of lesion area on atherosclerotic plaques at the aortic sinus of p38aEC-KO/ApoE2/2 and ApoE2/2 mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38aEC-KO/ApoE2/2 females, n = 14; ApoE2/2 females, n = 13; p38aEC-KO/ApoE2/2 males, n = 14; ApoE2/2 males, n = 15. Scale bar, 0.2 mm. doi:10.1371/journal.pone.0021055.g002 sclerosis. We therefore assessed the expression of a number of cytokines, chemokines and adhesion molecules in aortic roots from p38aEC-KO/ApoE2/2 and ApoE2/2 control mice at the end of the HCD treatment. These experiments did not show any significant differences in cytokine, chemokine or adhesion molecule expression between p38aEC-KO/ApoE2/2 and ApoE2/ 2 control mice (Figure 3D). Thus, our results show that endothelial cell-specific ablation of p38a does not affect the development of atherosclerosis in the ApoE2/2 model of the disease. Macrophage-specific function of p38a in atherosclerosis To study the macrophage-specific role of p38a in atheroscle- rosis, we crossed p38aFL/FL/ApoE2/2 mice with LysMCre mice expressing Cre recombinase in macrophages and neutrophils [25]. Southern blot and immunoblot analysis of DNA and protein extracts from Bone Marrow- Derived Macrophage (BMDM) extracts showed efficient ablation of p38a in macrophages of p38aMY-KO/ApoE2/2 mice (Figure 4A and B middle panel, respectively and figure S3A). LPS stimulation induced strong p38 phosphorylation in ApoE2/2 control cells, while low levels of p38 phosphorylation were detected in p38a-deficient macrophages (Figure 4B, top panel and S3B). To address the potential role of macrophage p38a in atherosclerosis, we placed groups of male and female p38MY-KO/ ApoE2/2 and their ApoE2/2 littermates on HCD for 10 weeks, starting from 6–8 weeks of age. Analysis of bodyweight and cholesterol levels, before and after the HCD treatment, revealed no differences between the two genotypes, which showed similarly increased cholesterol levels and body weight after HCD feeding (Figure S2B). After 10 weeks on HCD, mice were sacrificed, and atherosclerotic lesion development was assessed in the whole aorta by en face staining with Sudan IV (Figure 4C), but also at the aortic sinuses (Figure 4D). This analysis did not reveal differences in lesion size between p38aMY-KO/ApoE2/2 and their ApoE2/2 littermates, either in the whole aorta or in the aortic sinuses. Quantification of collagen content, foam cell formation and necrotic area also did not reveal any differences between the two genotypes (Figure 5A, B and C, respectively). Taken together, our experiments showed that macrophage p38a deficiency did not affect atherosclerotic plaque development in ApoE2/2 mice as assessed by quantification of lesion size, collagen and macrophage content and necrotic core area. These findings are in contrast to the results reported by Seimon and colleagues, who found increased lesional necrosis and decreased collagen content in atherosclerotic plaques from p38aMY-KO/ApoE2/2, compared to ApoE2/2 controls [8]. Lesional macrophages secrete a wide range of pro-inflammatory and pro-atherosclerotic genes and p38a has been shown to regulate the expression of many of these genes in this cell type [18], [26], [20]. Thus, we measured the expression of a panel of cytokines, chemokines and adhesion molecules on RNA isolated from the aortic arch of p38aMY-KO/ApoE2/2 and ApoE2/2 littermates after 10 week on HCD. QRT-PCR expression analysis failed to reveal any considerable differences in the expression of any of the genes tested (Figure 5D), suggesting that p38a macrophage deficiency does not seem to significantly affect the expression of several pro-inflammatory mediators in the aorta. Previous studies using chemical p38 inhibitors suggested that p38a activity in macrophages is required for oxLDL uptake and foam cell formation [12]. To address the potential role of p38a in oxLDL uptake we stimulated thioglycolate-elicited peritoneal macrophages (PMs) from p38aMY-KO/ApoE2/2 and ApoE2/2 mice with 50 mg/ml oxLDL and measured uptake by flow cytometric analysis (Figure S4A) and Oil Red O staining for lipids by microscopy (Figure S4B). Contrary to the findings of Zhao et al., our results revealed that p38a deficiency did not affect the capacity of macrophages to take up oxLDL. In conclusion, the results from our in vivo and in vitro analysis did not reveal differences in any of the examined parameters addressing atherosclerotic plaque formation in p38aMY-KO/ApoE2/2 mice compared to ApoE2/2 controls, suggesting that p38a signaling in macrophages does not play an important role in the development of atherosclerosis in the ApoE deficient mouse model. Discussion Inflammation is now recognized as a critical pathogenic component for the development and progression of atherosclerotic plaques. Modified lipids act on resident vascular cells and also infiltrating inflammatory cells to induce and sustain the constant recruitment of blood monocytes into the arterial intima. Recruited monocytes differentiate into lipid laden foam cells, which are trapped and eventually die within the lesions. Inefficient clearance of apoptotic foam cells gives rise to a necrotic core in advanced lesions [5]. Thus, the mechanisms that control the recruitment, differentiation, activation, death and clearance of macrophages in atherosclerotic plaques are crucial for the development and progression of atherosclerosis [3], [4]. Intracellular signaling pathways that are activated downstream of innate and cytokine receptors and coordinate the expression of adhesion molecules, chemokines and cytokines that control inflammatory responses, are believed to play an important role in atherosclerosis. The p38 MAPK pathway is activated by a multitude of inflammatory and danger signals and coordinates cellular responses including survival, differentiation, proliferation, and the activation of pro-inflammatory gene expression programs [27]. These properties strongly suggested that p38 signaling must be implicated in the pathogenesis of atherosclerosis. In addition, systemic deficiency of MK2, a downstream effector of p38 signaling, reduced the severity of atherosclerosis in Ldlr2/2 mice [28], suggesting that p38 signaling indeed is important for this disease. We therefore decided to address in vivo the potential role of p38 in the development of atherosclerotic plaques. Since systemic p38a deficiency leads to embryonic lethality, we used conditional targeting of p38a in the ApoE2/2 model of the disease. We chose to target p38a in endothelial cells and macrophages, two cell types with important functions in atherosclerosis. Endothelial cell activation and expression of pro-inflammatory mediators and adhesion molecules plays a crucial role in the development of atherosclerosis by recruiting monocytes into the PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21055