phosphorylated Smad2 (p-Smad2) and disruption of elastic layers in the vessel wall [15]. For the present study we have generated a new, genetically complex strain of compound mutant mice that are LDL receptor- deficient (LDLR2/2), lack LRP1 only in their vascular smooth muscle cells, and express an endogenous, crippled form of the PDGFRb that is incapable of activating PI3K. Our goal was to test, whether increased PDGFRb signaling through PI3K is the primary cause for the increased susceptibility to atherosclerotic lesion development in LDLR 2/2 mice lacking LRP1 in their SMCs, and whether PDGFRb-dependent PI3K signaling is required for the expression of the Marfan syndrome-like phenotype in smLRP1-deficient mice. Results Lack of LRP1 expression in the SMCs results in cell hypertrophy and vessel elongation Earlier data from our laboratory showed that smLRP12/2; LDLR2/2 mice are highly susceptible to atherosclerosis when fed a high-cholesterol diet [7]. To determine if this increased susceptibility to atherosclerosis is preserved in smLRP12/2 mice in the absence of hypercholesterolemia, smLRP12/2 mice either expressing or lacking LDLR were maintained on a standard low-fat rodent chow. Determination of plasma total cholesterol confirmed that smLRP12/2 mice did not develop hypercholesterolemia (129.965.3 mg/dl, Table 1). Whereas, smLRP12/2; LDLR2/2 mice had high total cholesterol levels of 246.5661.0 mg/dl with a major increase in LDL (Figure S1). Elongated aortas were present in the absence of hypercho- lesterolemia in smLRP12/2 mice (Figure 1c, h). However, atherosclerotic lesions were only visible in smLRP12/2; LDLR2/2 mice (Figure 1e, j). To study structural changes in the vascular wall of these elongated aortas, H&E, trichrome and elastin staining were performed. Thickened aortic walls with intima thickening, disarranged and hypertrophic SMCs, in- creased extracellular collagen accumulation, and elastic lamina disruption were observed in 11-month old smLRP12/2 mice (Figure 1B). Vascular wall thickening was also present in young mice at 7 weeks of age (Figure S2). Compared with wild type, the aortic wall of smLRP12/2 mice was significantly thicker (66.1463.32 mm vs. 39.3562.16 mm, Figure 1C). Our findings indicate that LRP1 expression in SMCs controls the architec- ture of the vascular wall in a plasma cholesterol-independent manner. Table 1. Total cholesterol and triglyceride levels of mouse plasma. WT smLRP12/2 LDLR2/2 Total Cholesterol 131.3616.8 129.965.3 a 294.2617.7 b 246.5661.0c,d Total Triglycerides 61.769.8 51.1620.0 a 125.0642.9 b 101.1628.4 c,d Total cholesterol and triglyceride levels were significantly higher in mice lacking LDLR, even though they were fed a standard rodent chow diet. LRP-deficient in smooth muscle cells did not change the lipid levels significantly. ap.0.05 (smLRP12/2 vs WT). bp,0.01 (LDLR2/2 vs WT). cp,0.05 (smLRP12/2; LDLR2/2 vs smLRP12/2). dp.0.05 (smLRP12/2; LDLR2/2 vs LDLR2/2). doi:10.1371/journal.pone.0006922.t001 smLRP12/2, LDLR 2/2 LRP1 Controls Vascular PI3K LRP1 controls PI3K binding and activation by PDGFRb in vivo PDGFRb signaling is crucial for regulating SMC responses and is an important contributing factor to atherogenesis. Previous work from our laboratory showed dramatically increased expression and activation of PDGFRb in aortas from smLRP12/2; LDLR2/2 mice on a high-cholesterol diet [7]. To investigate the basal expression and activation level of PDGFRb in mice maintained on a low-cholesterol diet, aortic extracts were prepared from mice expressing or lacking LRP1 and LDLR and analyzed by Western blotting. Because PDGFRb activation, through transphosphory- lation of tyrosine residues in its cytoplasmic domain, triggers a cascade of phosphorylation events which eventually lead to the activation of extracellular regulated-protein kinases (Erks), phos- phorylated-Erk1/2 was used as an indicator of PDGFRb activation [7]. About a two-fold increase of PDGFRb expression was detected in smLRP12/2 mice regardless of LDLR genotype (Figure 2A, C). Increased Erk1/2 phosphorylation was also observed in these aortas (Figure 2A). These data suggest that the expression and activation of PDGFRb is only regulated by LRP1, not LDLR. PI3K binding sites on PDGFRb are crucial for the receptor- mediated cell responses [16,17]. To explore the functional and biochemical interaction between LRP1 and the PDGFRb-PI3K signaling pathway, we performed a co-immunoprecipitation assay. Compared to wild type animals, smLRP12/2 mice showed increased tyrosine-phosphorylation of and PI3K binding to PDGFRb (Figure 2B), indicating that LRP1 regulates PDGFRb- dependent activation of PI3K by controlling PDGFRb phosphor- ylation. Disruption of PDGFRb-PI3K signaling in mice reduces atherosclerosis To investigate whether LRP1 regulates atherosclerosis through the PDGFRb-dependent PI3K pathway in vivo, we generated a compound mutant mouse model by crossing smLRP12/2; LDLR2/2 mice to PI3K binding-deficient PDGFRb F2/F2 mutant mice, in which tyrosine residues at position 739 and 750 are mutated to phenylalanines [18]. We found significantly decreased atherosclerotic lesions in the aortic arch and abdominal aorta of smLRP12/2; LDLR2/2; PDGFRb F2/F2 mice (Figure 3A, C). H&E, trichrome and elastin staining revealed well-arranged spindle-shaped SMCs, reduced extracellular matrix, and virtually normal elastic layers in smLRP12/2; LDLR2/2; PDGFRb F2/F2 aortas (Figure 3B). Vascular wall thickness, hypercellularity and length of the aortas in smLRP12/2; LDLR2/2; PDGFRb F2/F2 mutants were mark- edly reduced to approximately normal levels (Figure 3B, D, E, F). However, the prominent aneurysms of the mesenteric arteries, which are a hallmark of smLRP2/2 mice, were notably not abolished in smLRP12/2; LDLR2/2; PDGFRb F2/F2 mice (Figure 3A), suggesting that atherogenesis and aneurysm formation employ at least partially different molecular or regionally distinct mechanisms. Ablation of PI3K binding to PDGFRb reverses actin disorganization in LRP1-deficient SMCs To characterize SMCs lacking either LRP1 or LDLR and with crippled PI3K binding to PDGFRb in vitro, we generated primary cells from the aortas of these mice. To eliminate fibroblast contamination, LRP1-deficient SMCs were selected with Pseudo- monas exotoxin A (PEA) according to previous publications [19,20]. Primary SMCs were identified by their typical spindle PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e6922