A TRAK2-KIF5B Complex Mediates Microtubule-Based LDL Vesicle Transport During Endothelial Transcytosis

Transendothelial transport of LDL into the vascular intima is an early step in atherogenesis, yet the intracellular machinery governing endothelial LDL transcytosis remains incompletely understood. We sought regulators of LDL-containing vesicle trafficking and their mechanisms. Transcriptomic profiles of human abdominal aortic aneurysm specimens and normal aortas were analyzed to nominate candidates. Among 3,641 differentially expressed genes, trafficking kinesin protein 2 (TRAK2) was prioritized. In human umbilical vein endothelial cells, TRAK2 was silenced by small interfering RNA (siRNA) or overexpressed by plasmid transfection. LDL transcytosis was quantified by apical-to-basolateral flux in transwell assays and by basolateral exocytic events using total internal reflection fluorescence microscopy. TRAK2 knockdown reduced transcytosis in both assays (p<0.05), whereas overexpression did not enhance transcytosis (p=0.61). Findings were consistent across independent experiments and assay platforms. Fluorescent LDL uptake was unchanged, indicating a post-internalization role and suggesting TRAK2 promotes basolateral transport. Live-cell confocal imaging showed LDL vesicles colocalized with TRAK2 and moved directionally along microtubules. TRAK2 knockdown abolished LDL-induced responses of kinesin family member 13A (KIF13A) and further reduced KIF13B expression, while KIF5B remained LDL-responsive. Coimmunoprecipitation identified a TRAK2-KIF5B complex, but not interactions with KIF13A or KIF13B. Consistently, KIF5B depletion reduced LDL transcytosis (p<0.05), and LDL vesicles frequently colocalized with KIF5B during microtubule-based transport. These findings identify a TRAK2-KIF5B motor-adaptor axis that mediates microtubule-dependent trafficking of LDL-containing vesicles during endothelial transcytosis, defining an intracellular control point for LDL delivery to the intima and a potential target to modulate early atherogenic processes.