Abstract Body (Do not enter title and authors here): Background: Current lipid-lowering drugs rely on the LDL receptor (LDLR) pathway to lower LDL cholesterol. This leaves patients with null mutations in the LDLR, such as those with homozygous familial hypercholesterolemia (FH), potentially without an effective treatment. Remarkably, inhibitors of angiopoietin-like3 were found to reduce LDL-cholesterol in FH patients in an endothelial lipase (EL)-dependent mechanism. Recently, we found that EL overexpression enhances the cellular uptake of LDL in LDLR-knockout (KO) hepatocytes; however, the mechanism remains unclear. Aim: to investigate the mechanisms by which EL mediates LDLR-independent uptake of LDL in hepatocytes. Methods: CRISPR/Cas9-generated control and LDLR-KO HepG2 cells were transfected by lipofection with an empty plasmid or a plasmid containing the human LIPG gene (EL). Cellular uptake of fluorescent human LDL was measured by FACS. To test the contribution of heparan sulfate proteoglycans (HSPG), the cellular uptake of LDL was assessed with and without the pre-incubation with 5 U/ml heparin or a cocktail of heparinases I, II, and II. Furthermore, to analyze the role of EL enzymatic activity, LDL uptake was measured after incubating the cells with 25 µg/ml tetrahydrolipstatin (THL). Finally, we measured the expression of potential receptors by RT-qPCR to explore alternative mechanisms of internalization. Results: As previously shown, LIPG-transfected LDLR-KO hepatocytes showed a 2-fold increase in LDL uptake compared to LDLR-KO cells non-overexpressing EL (p<0.001). LDL uptake was completely blocked by heparinases and heparin in LDLR-KO cells regardless of EL over-expression. Treatment with THL partially reduced the cellular LDL uptake in LDLR-KO cells over-expressing EL (p=0.08). In addition, LDLR-KO cells showed a 2-fold increase in the mRNA levels of cluster of differentiation 36 (CD36) (p<0.01), a known scavenger receptor. Conclusions: Our findings demonstrate that, in LDLR deficiency, EL mediates the uptake of LDL by a combination of enzymatic and non-enzymatic mechanisms. Furthermore, our data shows that HSPG play a crucial role in the LDLR-independent LDL uptake in hepatocytes, possibly acting as a bridge facilitating LDL internalization. Further studies will determine the role of scavenger receptors in the EL-mediated uptake. These mechanisms may represent a potential druggable target to treat homozygous FH patients.