The mannose receptor on murine liver sinusoidal endothelial cells is the main denatured collagen clearance receptor.

UNLABELLED: The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. CONCLUSION: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs.

Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells.

Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen alpha-chain receptor and the Fc-gamma receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding.

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.