[12], control of the expression of the monocytic chemokine receptor CXCR2 that is induced in response to oxLDL [13] and the migration, proliferation [14], [15] permeability [16], apoptosis [17] and adhesion molecule expression (e.g. VCAM1) [18] of endothelial cells. Finally, p38a is critical for the production of multiple pro- inflammatory cytokines like tumor necrosis factor (TNF) and interleukins IL-1b, IL-6 and IL-8 in most of the cell types that participate in atherosclerosis development [18], [19], [20]. Thus, p38a has been suggested to have a crucial role in the pathogenesis of atherosclerosis by acting in cells that play a crucial role in atherogenesis, like endothelial cells, smooth muscle cells and macrophages. Here, we used conditional gene targeting in mice to address in vivo, for the first time, the endothelial cell-specific function of p38a in atherosclerosis, in the apolipoprotein E deficient (ApoE2/2) mouse model. Using the same model, we also studied the macrophage-specific role of p38a in this disease. We generated ApoE2/2 mice lacking p38a in vascular endothelial cells or macrophages and analyzed the development of atherosclerotic plaques, after feeding a high cholesterol diet (HCD). Our results show that specific ablation of p38a in macrophages or endothelial cells did not affect the development of atherosclerotic plaques in ApoE2/2 mice, suggesting that p38a activity in these cell types does not play a crucial role for the pathogenesis of atherosclerosis. Results P38a-deficiency inhibits oxLDL-induced adhesion molecule and chemokine expression in primary endothelial cells Vascular endothelial cells have an important role in the pathogenesis of atherosclerosis by expressing adhesion molecules and chemokines that facilitate the recruitment of inflammatory cells into the developing plaque. To address the role of p38a in endothelial cells in atherosclerosis, we studied the contribution of p38a activation in the expression of adhesion molecules and chemokines by endothelial cells, upon stimulation with oxLDL. For these experiments, we used primary mouse lung endothelial cells (MLECs) isolated by Dynabead-mediated negative (LEAF) and positive (CD102) selection from dissociated lung tissue from p38aFL/FL/ApoE2/2 or ApoE2/2 control mice. To delete p38a in these cells, we used His-TAT-NLS-Cre (HTNC), a transducible Cre recombinase that can be used efficiently to mediate recombination of loxP flanked alleles in culture [21]. Purity of endothelial cell cultures after Cre recombination was assessed by flow cytometric analysis after staining with CD146 (Figure 1A). Immunoblot analysis with p38a specific antibodies revealed a strong reduction of p38a expression in HTNC-treated p38aFL/FL/ ApoE2/2 MLECs, indicating efficient ablation of p38a (Figure 1B, middle panel). Furthermore, immunoblot with antibodies recog- nizing phosphorylated p38 revealed that stimulation with 100 mg/ ml oxLDL induced strong p38 activation in control (ApoE2/2) cells, while p38a-deficient MLECs showed weak p38 activation. As the phospho-p38 antibody used is not isoform-specific, the weak p38 phosphorylation detected most likely corresponds to phosphorylation of the residual p38a and also other p38 isoforms, such as p38b, which is the other major p38 isoform expressed in endothelial cells [22], (Figure 1B, top panel). We then assessed the oxLDL-induced expression of vascular adhesion molecule 1 (VCAM1) and the chemokines IP-10, MCP-1 and Gro-KC, known to be involved in the recruitment of monocytes into the arterial intima. Quantitative real-time PCR (qRT-PCR) analysis on RNA, isolated at different time points after oxLDL stimulation, revealed induction in expression of VCAM1, IP-10, MCP-1 and The Role of P38a MAPK in Atherosclerosis Gro-KC in control MLECs, which was strongly reduced in MLECs lacking p38a (Figure 1C). Endothelial cell p38a deficiency does not affect atherosclerosis development in ApoE2/2 mice The results of the above described experiments suggested that p38a activity in endothelial cells might play an important role for the development of atherosclerotic plaques and encouraged us to study in vivo the effect of endothelial cell specific p38a ablation, in the development of atherosclerosis. For this purpose we used Cre- loxP-mediated conditional targeting of p38a in ApoE-deficient mice, which spontaneously develop atherosclerotic plaques due to elevated blood cholesterol levels, a pathology that is further aggravated upon feeding with a HCD. Thus, we crossed mice carrying loxP-flanked p38a alleles with Tie2ERT2Cre transgenics, which mediate tamoxifen-inducible Cre recombination specifically in endothelial cells [23] and subsequently with ApoE2/2 mice. To induce Cre-mediated excision of the loxP-flanked p38a allele in endothelial cells, we fed groups of 6–8 week old p38aEC-KO/ApoE2/2 and their p38aFL/FL/ApoE2/2 littermates that did not carry the Tie2ERT2Cre transgene with a diet containing tamoxifen (400 mg/ kg tamoxifen citrate, 5% sucrose in phytoestrogen-free chow) for 5 consecutive weeks [24]. The mice were subsequently placed on HCD for 10 weeks to facilitate the development of atherosclerotic plaques. To assess the efficiency of p38a ablation, we measured p38a expression in primary endothelial cells isolated by CD146- mediated magnetic cell sorting (MACS) from the lungs of mice, at the end of the HCD feeding. Flow cytometric analysis of cell fractions collected during MACS sorting showed efficient separation of CD146+ and CD1462 fractions (Figure S1). Immunoblot analysis of protein lysates from whole lung, CD146+ and CD1462 cell fractions showed efficient ablation of p38a in endothelial cell isolates from p38aEC-KO/ApoE2/2 mice, indicating that tamoxifen-treat- ment induced efficient ablation of p38a in the vascular endothelium that persisted during the period of HCD feeding (Figure 2A). Measurement of bodyweight and cholesterol levels before and after the HCD treatment revealed no differences between p38aEC-KO/ ApoE2/2 and their ApoE2/2 littermate controls, which showed similarly increased cholesterol levels and body weight after HCD feeding (Figure S2A). 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 2B), but also at the aortic sinus by histological analysis of consecutive sections followed by cross-sectional plaque area quantification (Figure 2C). This analysis did not reveal differences in lesion size between p38aEC-KO/ApoE2/2 and their ApoE2/2 littermates, either in the whole aorta or at the aortic sinuses. To further characterize the lesions, we assessed collagen and foam cell content and necrotic core formation. Quantification of collagen on sections stained with Masson Trichrome did not reveal differences in collagen content in plaques from p38aEC-KO/ ApoE2/2 mice compared to ApoE2/2 controls (Figure 3A). In addition, staining with a MOMA2 antibody to detect macro- phages showed a similar foam cell content in plaques from p38aEC-KO/ApoE2/2 mice compared to ApoE2/2 controls (Figure 3B). Finally, quantification of necrotic core area in sections from the aortic sinus did not reveal differences between p38aEC-KO/ApoE2/2 and ApoE2/2 controls (Figure 3C). Taken together, our experiments showed that endothelial p38a deficiency does not affect atherosclerotic plaque size or composition in ApoE2/2 mice. These results were surprising in light of our in vitro findings, showing that p38a was essential for oxLDL-induced expression of adhesion molecules and chemo- kines that are important for monocyte recruitment in athero- PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21055