The Role of P38a MAPK in Atherosclerosis Figure 3. Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38aEC-KO/ApoE2/2 and ApoE2/2 mice. Staining and quantification at the aortic sinus of p38aEC–KO/ApoE2/2 mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows) (B) Foam cell content, MOMA2 staining (red, indicated by arrows). (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, chemokines and pro-inflammatory cytokines (from left to right) of aortal arches from p38aEC-KO/ApoE2/2 and ApoE2/2 mice after 10 weeks on HCD. P38aEC-KO/ApoE2/2 females, n = 9; ApoE2/2 females, n = 6. Scale bar 50 mm. Error bars represent SD. doi:10.1371/journal.pone.0021055.g003 arterial intima [7]. OxLDL, cytokines but also shear stress can activate intracellular signaling pathways that control endothelial cell responses important for atherosclerosis. P38a has been implicated in this process through regulating migration and proliferation of endothelial cells [14], [15], cell permeability [16], apoptosis [17], expression of adhesion molecules like VCAM1 [18] and of various pro-inflammatory cytokines [10], [18]. Using p38a- deficient primary endothelial cells, we could indeed show here that p38a plays a crucial role in the expression of VCAM1 and the chemokines MCP-1, IP-10 and Gro-KC upon oxLDL stimulation. However, our in vivo experiments showed that endothelial cell- specific ablation of p38a did not affect the development of atherosclerosis in ApoE2/2 mice, as assessed by measurement of lesion size, macrophage and collagen content and necrotic core area. In addition, endothelial p38a deficiency did not affect the expression of a number of proinflammatory mediators known to regulate atherosclerosis in aortic lesions from ApoE2/2 mice fed with HCD for 10 weeks. Considering our in vitro results and the large amount of literature claiming an important role for p38a in endothelial cells for the development of atherosclerosis, the finding that endothelial p38a deficiency did not affect atherosclerosis development in vivo is surprising. Likewise, myeloid cell specific p38a deficiency did not have a measurable effect in the development of atherosclerotic plaques in ApoE2/2 mice fed for 10 weeks with a HCD, as assessed by en face staining of whole aortas and histological analysis of aortic root sections. Moreover, quantification of plaque characteristics like macrophage and collagen content and of necrotic core areas in aortic root lesions also revealed no considerable differences between p38aMY-KO/ApoE2/2 and ApoE2/2 mice. Inflammatory gene expres- sion analysis in aortic lesions also failed to reveal an effect of macrophage p38a deficiency in the expression of mediators controlling the development of atherosclerosis. Thus, our results demonstrate that macrophage p38a deficiency did not affect the development of atherosclerotic plaques in the ApoE2/2 mouse model, suggesting that macrophage p38a does not have an important role in the pathogenesis of atherosclerosis. Seimon et al. also studied the role of macrophage p38a in atherosclerosis using a similar experimental system [8]. In agreement with our results, they showed that macrophage p38a deficiency did not affect lesion size and macrophage content in aortic root plaques. However, in contrast to our findings and the findings from Jagavelu et al. with the downstream p38 effector MK2 [28], they found that plaques in ApoE2/2 mice lacking p38a in macrophages showed increased necrotic areas, attributed to a function of p38a in protecting macrophages from ER- stress induced apoptosis. Since both studies were performed on mice in C57Bl/6 genetic background and using the same high cholesterol diet for a similar time period, currently, we cannot explain this difference between our study and that of Seimon et al. At this stage we cannot exclude that combined deficiency of p38a in both macrophages and in endothelial cells might not affect atherosclerosis development in vivo or that p38a might function in other cell types such as smooth muscle cells to control atheroscle- rotic lesion pathogenesis. However, our findings highlight the potential complexity and redundancy in the processes regulating the development of atherosclerotic plaques in vivo and suggest that multiple mechanisms that are controlled by different signaling cascades contribute to the pathogenesis of atherosclerosis. Taken together, our studies demonstrate that the macrophage and endothelial specific inhibition of p38a signaling does not protect against atherosclerosis. Without neglecting the fact that this molecule could be an important player in other cell types involved in atherosclerosis development (e.g. smooth muscle cells), these results suggest that p38 inhibitors may not be an appropriate therapeutic strategy for the prevention and treatment of atherosclerosis. Materials and Methods Ethics statement All animal procedures were approved by the governmental ethics committee and were performed in compliance with the licenses No. /50.203.2-K13, 12/06/ of July, 7 2006 (elongated May, 12 2009) and /8.87-50.10.37.09.242/ of October, 10 2009, granted by the State Office for Nature, Environment and Consumer Protection of North Rhine-Westphalia (Landesamt fu¨ r Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen), Germany. The investigation conforms to the ‘Guide for the Care and Use of Laboratory Animals’ published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Mice and diet Conditional male and female p38aFL/FL [29] mice were crossed with LysMCre [25] and Tie2ERT2Cre [23] transgenic mice, to generate myeloid and endothelial cell-specific knockouts, respec- tively. These mice were then crossed with ApoE2/2 mice [30], a widely accepted mouse model of atherosclerosis. For induction of Cre activity, mice carrying the Tie2ERT2Cre transgene and their Cre-negative littermates were fed a tamoxifen-containing diet (400 mg/kg tamoxifen citrate, 5% sucrose, 95% Teklad Global, 16% Rodent Diet) from Harland Teklad, as previously described [7], for 5 weeks. Atherosclerosis development was accelerated, in both macrophage- and endothelial-specific p38a knockout mice, by placing them on a high-cholesterol diet (HCD) from Harlan Teklad (Teklad Adjusted Calories 88137; 21% fat (WT/WT), 0.15% cholesterol (WT/WT) and 19.5% casein (WT/WT); no sodium cholate) for 10 weeks, starting at 6–8 weeks, or after tamoxifen feeding, respectively. All mice used in the experiments described here were backcrossed into the C57BL/6 genetic background for at least five generations. Genotyping and blood analysis Primers for the wild-type, floxed or deleted p38a alleles were: 59-CTACAGAATGCACCTCGGATG-39; 59-AGAAGGCTGGATTTG- CACAAG-39; 59-CCAGCACTTGGAAGGCTATTC-39 and for the wild-type or ApoE deleted alleles were: 59-GCCTAGCCGAGGGA- GAGCCG-39; 59-TGTGACTTGGGAGCTCTGCAGC-39; 59-GCCGC- CCCGACTGCATCT-39. Primers for the LysMCre transgene were: 59-CTTGGGCTGCCAGAATTTCTC-39; 59-TTACAGTCGGCCAG- GCTGAC-39; 59-CCCAGAAATGCCAGATTACG-39 and for the Tie2ERT2Cre transgene were: 59-GTCCAATTTACTGACCGTA- CAC-39; 59-CTGTCACTTGGTCGTGGCAGC-39. PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e21055