LRP1 Controls Vascular PI3K Figure 1. Hypertrophic and hyperplastic SMCs and elongated aortas in smLRP12/2 mice. (A) Unopened (a–e) and Oil Red O stained (f–j) aortas from 11-month old mice of the indicated genotypes. Mice were maintained on standard rodent chow diet. Arrows indicate lipid-laden atherosclerotic lesions. (B) Histological analysis of thoracic aortas from 11-month old mice of the indicated genotypes. k–m: hematoxylin & eosin (HE); n–p: trichrome; q–s: elastin staining. Scale bar, 20 mm. (C) Thickness of the aortas of the indicated genotypes was quantified using Image J software (NIH). Data represent mean6SD from 3 mice per group. *** p,0.001. doi:10.1371/journal.pone.0006922.g001 shape (Figure 4a) and the expression of smooth muscle actin (Figure 4b). LRP1 expression was verified by both immunocyto- chemical staining (Figure 4c, d) and Western blotting (Figure 4e). Interestingly, the morphology of SMCs of different genotypes looked quite distinct from each other. Actin organization was disrupted in the SMCs lacking LRP1 expression and an ‘‘actin ring’’ was present below the plasma membrane (Figure 4B). However, in smLRP12/2; PDGFRb F2/F2 SMCs actin organi- zation was restored (Figure 4B), suggesting that LRP1 mediates actin remodeling through PI3K activation upon PDGFRb phosphorylation. Increased migration of LRP1-deficient SMCs is diminished by the PDGFRb F2 mutation Our in vivo experiments showed disarranged SMCs in the medial layer of the aorta (Figure 1m, 3h, 3k) and disrupted elastic laminas when LRP1 was deficient in the SMCs (Figure 1s, 3t, 3w). To investigate if these phenomena are caused by abnormal migration due to the absence of LRP1, we performed two different kinds of in vitro migration assays. Compared with wild type cells, SMCs lacking LRP1 showed markedly increased migratory activity, both in a Boyden chamber transmigration assay (Figure 5A, C) and in the commonly used scratch assay, in which the migration of the cells into a denuded area of a tissue culture dish is quantified (Figure 5B, D). Cell migration was significantly reduced in those cells containing the PDGFRb F2/F2 mutation (Figure 5A–D), and this correlated with the improved architecture of the elastic layers in the aortic wall of smLRP2/2; LDLR2/2; PDGFRb F2/F2 mice (Figure 3u, x). These findings thus confirm that SMC migration is regulated by LRP1 through the PDGFRb- dependent PI3K pathway. PDGF-BB-induced chemotaxis is inhibited by blocking PI3K activation through PDGFRb To investigate whether PDGF-BB-induced SMC migration involves the PDGFRb-PI3K pathway, 10 ng/ml PDGF-BB was administrated as a chemo-attractant in the bottom well of a Boyden chamber. As shown in Figure 5E, PDGF-BB induced the transmigration of SMCs independent of LRP1 expression. However, when the Y739/750 phosphorylation sites of PDGFRb were mutated, this PDGF-BB-induced chemotaxis was completely abolished. This finding suggests that the increased migratory propensity of LRP1-deficient SMCs, which is further enhanced by PDGF-BB activation of PDGFRb, is in its entirety dependent upon the activation of PI3K by PDGFRb. Abnormal activation of TGFb signaling is present in the absence of LRP1 Our in vivo findings indicate that lack of LRP1 in SMCs results in elongation of the aorta, thickening of the vascular wall, and disruption of the elastic layers. Tortuous aorta and elastic laminar disruption are also two key cardiovascular manifestations PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e6922