Isolation of periportal, midlobular, and centrilobular rat liver sinusoidal endothelial cells enables study of zonated drug toxicity.
Many liver sinusoidal endothelial cell (LSEC)-dependent processes, including drug-induced liver injury, ischemia-reperfusion injury, acute and chronic rejection, fibrosis, and the HELLP (hemolytic anemia, elevated liver enzymes, low platelet count) syndrome, may have a lobular distribution. Studies of the mechanism of this distribution would benefit from a reliable method to isolate LSEC populations from different regions. We established and verified a simple method to isolate periportal, midlobular, and centrilobular LSEC. Three subpopulations of LSEC were isolated by immunomagnetic separation on the basis of CD45 expression. Flow cytometry showed that 78.2 ± 2.3% of LSEC were CD45 positive and that LSEC could be divided into CD45 bright (28.6 ± 2.7% of total population), dim (49.6 ± 1.0%), and negative populations (21.8 ± 2.3%). Immunohistochemistry confirmed that in vivo expression of CD45 in LSEC had a lobular distribution with enhanced CD45 staining in periportal LSEC. Cell diameter, fenestral diameter, number of fenestrae per sieve plate and per cell, porosity, and lectin uptake were significantly different in the subpopulations, consistent with the literature. Endocytosis of low concentrations of the LSEC-specific substrate, formaldehyde-treated serum albumin, was restricted to CD45 bright and dim LSEC. Acetaminophen was more toxic to the CD45 dim and negative populations than to the CD45 bright population. In conclusion, CD45 is highly expressed in periportal LSEC, low in midlobular LSEC, and negative in centrilobular LSEC, and this provides an easy separation method to isolate LSEC from the three different hepatic regions. The LSEC subpopulations obtained by this method are adequate for functional studies and drug toxicity testing.
Xie
Guanhua
G
Division of Gastrointestinal and Liver Diseases and the Research Center for Liver Diseases, University of Southern California Keck School of Medicine 90033, USA.
Wang
Lin
L
Wang
Xiangdong
X
Wang
Lei
L
DeLeve
Laurie D
LD
D000082:Acetaminophen / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000276:immunology; D004705:Endocytosis / Q000276:immunology; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D005434:Flow Cytometry; D007150:Immunohistochemistry; D017493:Leukocyte Common Antigens / Q000276:immunology*; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D008297:Male; D008855:Microscopy, Electron, Scanning; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000082:Acetaminophen; D017493:Leukocyte Common Antigens
J Pharmacol Exp Ther. 1994 Mar;268(3):1261-70
8138939
J Pathol. 1989 Jul;158(3):253-60
2769486
Hepatology. 1989 Sep;10(3):292-9
2668147
Biochem Pharmacol. 1997 May 9;53(9):1339-45
9214695
Hepatology. 2008 Sep;48(3):920-30
18613151
Am J Physiol Gastrointest Liver Physiol. 2006 Dec;291(6):G1187-9
16782698
Gastroenterology. 2009 Aug;137(2):704-12
19447108
Liver. 1994 Jun;14(3):113-23
8078390
Nat Immunol. 2003 Sep;4(9):815
12942076
Hepatology. 1985 Jul-Aug;5(4):683-92
3926620
Transplantation. 1993 Jul;56(1):69-75
8333070
Transplantation. 1988 Apr;45(4):834-7
3282369
Hepatology. 1992 Jul;16(1):191-203
1319950
Nat Med. 2006 Jun;12(6):642-9
16751767
Hepatology. 1996 Jun;23(6):1521-9
8675173
Gastroenterology. 2003 Sep;125(3):882-90
12949732
Hepatology. 1985 Mar-Apr;5(2):233-40
3979955
J Ultrastruct Res. 1972 Mar;38(5):528-62
4335119
Hepatology. 2001 Feb;33(2):363-78
11172338
Am J Physiol Gastrointest Liver Physiol. 2004 Oct;287(4):G757-63
15191879
J Ultrastruct Res. 1985 Feb;90(2):115-23
4068137
Hepatology. 1991 Jul;14(1):131-9
1906044
Am J Pathol. 1983 Dec;113(3):321-30
6650662
Microcirculation. 2003 Oct;10(5):391-400
14557822
Hepatology. 2003 Oct;38(4):900-8
14512877
Br J Obstet Gynaecol. 1986 Feb;93(2):145-55
3511956
Am J Physiol Gastrointest Liver Physiol. 2003 Jun;284(6):G1045-52
12584111