Abstract Body: Background and Aim: Lipoprotein(a) [Lp(a)] is highly atherogenic but the mechanisms by which it causes disease remain uncertain. We hypothesized that circulating Lp(a) can cross the endothelium by receptor-mediated transcytosis leading to intimal accumulation.

Methods: We assessed transcytosis of fluorophore-labeled Lp(a) across primary human coronary artery endothelial cells (HCAEC) using Total Internal Reflection Fluorescence (TIRF) microscopy. Receptor involvement was tested by siRNA knockdown of Activin receptor-Like Kinase 1 (ALK1), Low Density Lipoprotein Receptor (LDLR), Scavenger receptor B1 (SR-BI) and caveolin-1 (CAV1), and via an antibody against the extracellular domain (ECD) of ALK1. To assess whether Lp(a) activates receptor signaling, we performed Western blots to evaluate downstream pathways. For in vivo studies, male mice (C57BL/6, 12 weeks) acutely received intravenous fluorophore-labeled Lp(a) and deposition of Lp(a) was quantified in atheroprone aortic regions. Statistical analysis was by ANOVA/t-tests, with significance set at p<0.05.

Results: Lp(a) underwent transcytosis across HCAECs in a dose-dependent manner. Competition with excess unlabelled Lp(a) and unlabelled LDL both attenuated Lp(a) transcytosis, suggesting potentially common receptors. Knockdown of ALK1 or CAV1 significantly attenuated Lp(a) transcytosis, whereas LDLR- and SR-BI-knockdown had no effect. The requirement for ALK1 was observed using Lp(a) from two different donors exhibiting different lengths of apolipoprotein(a). An antibody against the ALK1 ECD reduced Lp(a) transcytosis, compared to IgG-treated controls. Unexpectedly, Lp(a) (but not LDL) induced phosphorylation of SMAD1/5 (known to be downstream of ALK1) and this was inhibited by knockdown of ALK-1. Mutational analysis identified specific regions of ALK1 that are required for transcytosis. Finally, in mice, acute and dose-dependent deposition of fluorophore-tagged Lp(a) in the atheroprone region of murine aortas was observed, without deposition of equimolar amounts of dextran (a control for paracellular leakage).

Conclusions: Lp(a) crosses the arterial endothelium via ALK1- and CAV1-dependent transcytosis and unexpectedly activates downstream ALK1 signaling. This may contribute to Lp(a)-induced atherogenesis and residual cardiovascular risk.