Rationale Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein (LDL)-like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B (apoB). Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) selectively binds LDL, we hypothesized that it can also be associated with Lp(a) in plasma. Objective Characterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39-320 mg/dl) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via co-expression of human apo(a) and human apoB). Methods and Results We show that PCSK9 is physically associated with Lp(a) in vivo using three different approaches: (i) Analysis of Lp(a) fractions isolated by ultracentrifugation; (ii) Immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (iii) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) vs. LDL (1.7-fold increase) was seen in subjects with high Lp(a) and normal LDL. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels. Conclusions Our results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

Gene therapy is a potential approach for inhibiting many of the processes that occur during atherogenesis, particularly when targeting key molecular regulators that have multiple adverse downstream signaling pathways. Local expression of human-apoAI in the artery wall has been shown to protect against atherosclerosis in mice, without affecting systemic HDL levels. We hypothesized that focal gene delivery in arterial plaque is possible using ultrasound (US)-mediated cavitation of acoustically-active cationic microbubble gene carriers. Lipid-shelled cationic microbubbles (MB) with a decafluorobutane gas core were prepared. Bicistronic plasmids (luciferase with either GFP or apoAI) were charge coupled to the MB and injected intravenously in 12 LDLR -/- mice fed high-fat diet for 8 weeks. The thoracic aortic root was exposed to US at 1.6MHz, a pulsing interval of 1Hz, and a mechanical index of 1.3 in order to produce repetitive cavitation of MB. In vivo optical imaging showed a focal luciferase activity only at the site of UMGD starting from a day after the procedure and lasting for 7-10 days. Activity was subsequently localized to the aortic root on ex vivo optical imaging. Immunohistochemistry of the aortic root confirmed luciferase transfection of both endothelial cells of the aortic root and base of the aortic cusps, and intramural cells in atherosclerotic lesions, likely macrophages. We further show that gene therapy with the luciferase-apoAI plasmid, results in migration of cells to the thymus 6-8 days after transfection. These results indicate that local US-mediated gene therapy approach for controlling atherosclerosis is feasible in this pre-clinical model.