Hepatocyte Tissue Plasminogen Activator Limits Very-Low-Density Lipoprotein Production via Inhibiting ApoB-Microsomal Transfer Protein Interaction and ApoB Lipidation

Lower plasma tissue plasminogen activator (tPA), a key fibrinolytic enzyme, is associated with higher atherogenic cholesterol levels in humans, but the mechanism is unknown. Hepatocyte (HC) is the factory producing atherogenic apoB lipoproteins, also a major source of basal plasma tPA. Our hypothesis is that tPA limits apoB-VLDL production in HCs.Silencing tPA in HCs using AAV8-H1-sh-tPA in Ldlr -/- mice leads to increased plasma cholesterol, triglyceride, apoB, and exacerbated atherosclerosis compared with scramble-silenced controls. Fractionation of plasma lipoproteins by FPLC or ultracentrifugation shows increased cholesterol and apoB in VLDL and LDL fractions. Inhibiting VLDL hydrolysis by P407 leads to a faster plasma triglyceride-rising rate in HC-tPA-silenced wild type mice, suggesting higher hepatic VLDL production. In a pulse-chase assay, tPA-silenced human primary HC has higher apoB-associated radioactivity in cell medium and lysate, suggesting increased apoB production.Adequate apoB lipidation is required for its secretion. By electronic microscopy, the distribution of VLDL shifts to a larger diameter in plasma VLDL isolated from HC-tPA-silenced Ldlr -/- mice, suggesting higher lipid contents in VLDL. The endoplasmic reticulum (ER)-associated apoB is higher in tPA-silenced vs. control HCs, more prominent in the less denser density fractions. As density and lipidation are inversely related, these findings are consistent with the hypothesis that tPA limits apoB lipidation in ER. MTP is an ER chaperone incorporating neutral lipids onto apoB in HC. The tPA-silenced human primary HC has higher neutral lipid transfer activity than controls, despite similar MTP protein levels. Moreover, higher apoB is in the anti-MTP-precipitations from tPA-silenced vs. control HCs, suggesting that silencing tPA increases apoB-MTP interaction. Proximity ligation and immunoprecipitation assays show tPA interacts with apoB in HC. Solid-phase binding assay and native gel reveal purified human tPA binds to LDL, but not to MTP. tPA competes with MTP in binding to LDL. Moreover, tPA inhibits MTP-mediated neutral lipid transfer. These findings suggested tPA binds to apoB and inhibits MTP-dependent apoB lipidation and VLDL secretion.