LRP1 Controls Vascular PI3K Figure 2. Increased expression and activation of PDGFRb and PI3K binding by PDGFRb in aortic extracts of smLRP12/2 mice. (A) Protein extracts (10 mg/lane) from mouse aortas of the indicated genotypes were analyzed by immunoblotting for PDGFRb, p-Erk1/2, Erk1/2, and LRP1. (B) 200 mg of aortic extracts of the indicated genotypes were immunoprecipitated with the designated antibodies (anti-PDGFRb and anti- phosphotyrosine) to semi-quantitatively determine the interaction between PI3K and PDGFRb. Precipitated proteins were analyzed by immunoblotting using the indicated antibodies (anti-phosphotyrosine, anti-PI3K-p85 and anti-PDGFRb). Actin served as a loading control. WB: Western blot; IP: immunoprecipitation. (C) Expression of PDGFRb relative to the loading control was quantified using Image J software (NIH). Data are expressed as mean6SD. ** p,0.01. doi:10.1371/journal.pone.0006922.g002 of Marfan syndrome. As a connective tissue disorder with autosomal dominant inheritance, Marfan syndrome is caused by loss-of-function mutations in fibrillin-1, a matrix component of extracellular microfibrils [21]. Fibrillin-1 regulates activation of the cytokine TGFb, and its deficiency results in enhanced TGFb signaling [22,23]. Recent studies have shown that abnormal activation of TGFb contributes to the pathogenesis of Marfan syndrome [22]. Paradoxically, loss of function mutations in TGFb receptor I or II also result in increased TGFb signaling and give rise to Marfan syndrome [24,25]. LRP1 is identical to the type V TGFb receptor (TbR-V), which co-expresses with other TGFb receptors (TbR-I, TbR-II and TbR-III) [13]. LRP1/TbR-V mediates TGFb induced growth inhibition in concert with TbR-II/TbR-I/Smad2/3/4 signaling [13,14] and TGFb signaling is increased in LRP1 deficient mouse aortas [15]. To evaluate the TGFb activation state in the aortas of the different genotypes, immunofluorescent staining and Western blotting of p-Smad2 were performed. Significantly increased Smad2 nuclear translocation was seen in smLRP12/2; LDLR2/2 aortas. Importantly, blockade of PI3K binding to PDGFRb did not suppress over-activation of TGFb signaling (Figure 6A). An approximately 2.5-fold increase of Smad2 phosphorylation at Ser 465/467 was also detected in LDLR- expressing, LRP1-deficient SMCs (Figure 6B, C). These data indicate that in the absence of LRP1, TGFb signaling is abnormally activated independent of the absence or presence of the LDL receptor, and this over-activation of TGFb is not suppressed by the PDGFRb F2 mutation. Taken together, our findings suggest that aorta elongation and elastic layer disruption, both hallmarks of Marfan syndrome, require the PDGFRb dependent activation of PI3K. Activation of the TGFb signaling pathway alone is not sufficient. Thus, LRP1 acts as a molecular switch that integrates TGFb and PDGFRb/ PI3K signals and this is essential for maintaining the integrity of the vascular wall architecture. Discussion In this study we have investigated the role of LRP1 for PI3K activation by PDGFRb in SMCs, and the impact this LRP1 ‘checkpoint’ has for preventing atherosclerotic lesion formation and progression, as well as for the maintenance of vascular wall integrity. We found that the selective genetic blockade of PI3K activation by PDGFRb substantially suppressed spontaneous atherosclerotic lesion development, which is prominent in smLRP12/2; LDLR2/2 mice. Furthermore, vascular wall elongation and medial thickening, due to SMC hyperproliferation, increased SMC migration and disruption of elastic layers are normalized throughout the entire aorta. Our findings suggest that PI3K is the main driving force that promotes SMC proliferation and migration, elastolysis, spontaneous atherosclerosis and lesion progression in the absence of LRP1. Prominent atherosclerotic lesions preexisted in smLRP12/2; LDLR2/2 mice maintained on standard, low-fat and cholesterol- free rodent chow, but not in smLRP12/2 animals of comparable age. These data suggest that in the presence of an intact endothelium and low plasma cholesterol levels, proliferative signals alone are not sufficient to initiate the pathogenic mechanisms that culminate in classic atherosclerotic plaques. By contrast, the aorta of LRP1+/+; LDLR2/2 mice appears PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e6922