LRP1 Controls Vascular PI3K Figure 6. Increased Smad2 phosphorylation at Ser 465/467 in smLRP12/2; LDLR2/2 and smLRP12/2; LDLR2/2; PDGFRb F2/F2 aortas. (A) Immunofluorescent staining of p-Smad2 (Ser 465/467, green) in aortas of atherosclerosis-free 1-month old mice. Scale bar, 20 mm. (B) Immunoblotting of aortic extracts for Smad2 phosphorylation at Ser 465/467. b-actin served as a loading control. (C) The density of p-Smad2 signals was normalized to the corresponding b-actin signals from the same blot and quantified using Image J software (NIH). Densitometric scanning from three independent experiments revealed a statistically significant average of 250%613% in smLRP12/2 mice and 260%629% in smLRP12/2; PDGFRb F2/F2 mice, compared with wild type controls (92%617%). Relative ratio of p-Smad2 in mouse aortas from the indicated genotypes was plotted. Data are presented as mean6SD. * p,0.001, n = 3. doi:10.1371/journal.pone.0006922.g006 histoanatomically normal despite increased plasma cholesterol levels on the same chow, and extensive atherosclerotic lesions develop only after feeding of a high-cholesterol diet for several months [26]. Thus, LRP1 in SMCs functions cell autonomously in the maintenance of vascular wall integrity and protection from cholesterol-induced atherosclerosis. In the absence of smLRP1, the mouse aorta undergoes hyperplastic and hypertrophic changes that were apparent in young (7 weeks) as well as older (11 months old) mice indicating that they are not the result of aging, but the manifestation of an intrinsic change of smooth muscle phenotype. This is most likely caused by the increased expression and activation of PDGFRb in smLRP12/2 mice and an accompanying increase in PI3K association with PDGFRb. Disruption of an obligatory proathero- genic proliferative pathway, involving PI3K and PDGFRb, prevents or greatly reduces lesion development at sites of high shear stress, such as the aortic arch and the abdominal aorta, where endothelial integrity is easily compromised. Thus, by selectively controlling SMC proliferation and migration indepen- dent of endothelial integrity and plasma cholesterol levels in a novel genetically complex animal model, we have been able to isolate and demonstrate the pivotal and interdependent roles of two central mechanisms of atherosclerotic lesion development. Activation of the PDGFRb results in actin reorganization in the forms of membrane ruffling and chemotaxis [11,27,28,29,30] and thus provides an excellent functional assay for the physiological activation of PDGFRb through other genetic manipulations, such as the disruption of LRP1. PI3K binding to the cytoplasmic domain of activated PDGFRb receptors requires phosphorylation at residues 739 and 750 and this interaction in turn activates the kinase [18,31]. Replacement of these tyrosines by non-phosphor- ylatable phenylalanines prevents binding of PI3K and fails to mediate membrane ruffling and cell migration [28,29]. As a result, the pronounced edge ruffling and circular membrane ruffling as well as greatly enhanced SMC migration that were observed in the absence of LRP1 were virtually normalized in mice in which PLoS ONE | www.plosone.org 8 September 2009 | Volume 4 | Issue 9 | e6922