title: Lipids promote survival, proliferation, and maintenance of differentiation of rat liver sinusoidal endothelial cells in vitro.
abstract: Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48-72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Ta-Chun
authors.initials: TC
authors.lastname: Hang
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Lauffenburger
authors.forename: Linda G
authors.initials: LG
authors.lastname: Griffith
authors.forename: Donna B
authors.initials: DB
authors.lastname: Stolz
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D008099:Liver / Q000166:cytology*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008297:Male; D008855:Microscopy, Electron, Scanning; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019301:Oleic Acid / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D017452:Receptors, IgG / Q000378:metabolism
chemical_list: D001549:Benzamides; D001973:Bromodeoxyuridine; D002867:Chromones; D016895:Culture Media, Serum-Free; D004159:Diphenylamine; D005230:Fatty Acids, Nonesterified; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D009025:Morpholines; D019301:Oleic Acid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D018809:Proliferating Cell Nuclear Antigen; D051057:Proto-Oncogene Proteins c-akt; D017452:Receptors, IgG; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C506614:mirdametinib; C535149:Mapk1 protein, rat
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