Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

Table 1 References used in the definition of types of hematopoietic cells in the Kupffer cell fraction

No.	Reference
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8	Kiley SC, Parker PJ. Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation. J Cell Sci 1995; 108 (Pt 3): 1003-1016
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11	Thornton AJ, Ham J, Kunkel SL. Kupffer cell-derived cytokines induce the synthesis of a leukocyte chemotactic peptide, interleukin-8, in human hepatoma and primary hepatocyte cultures. Hepatology 1991; 14: 1112-1122
12	Manthey CL, Kossmann T, Allen JB, Corcoran ML, Brandes ME, Wahl SM. Role of Kupffer cells in developing streptococcal cell wall granulomas. Streptococcal cell wall induction of inflammatory cytokines and mediators. Am J Pathol 1992; 140: 1205-1214
13	Zhu XL, Zellweger R, Zhu XH, Ayala A, Chaudry IH. Cytokine gene expression in splenic macrophages and Kupffer cells following haemorrhage. Cytokine 1995; 7: 8-14
14	Loike JD, Kaback E, Silverstein SC, Steinberg TH. Lactate transport in macrophages. J Immunol 1993; 150: 1951-1958
15	Daberkow RL, White BR, Cederberg RA, Griffin JB, Zempleni J. Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells. J Nutr 2003; 133: 2703-2706
16	Merezhinskaya N, Ogunwuyi SA, Mullick FG, Fishbein WN. Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes. J Histochem Cytochem 2004; 52: 1483-1493
17	Marra F, Valente AJ, Pinzani M, Abboud HE. Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines. J Clin Invest 1993; 92: 1674-1680
18	Guc D, Gulati P, Lemercier C, Lappin D, Birnie GD, Whaley K. Expression of the components and regulatory proteins of the alternative complement pathway and the membrane attack complex in normal and diseased synovium. Rheumatol Int 1993; 13: 139-146
19	Schwaeble W, Huemer HP, Most J, Dierich MP, Strobel M, Claus C, Reid KB, Ziegler-Heitbrock HW. Expression of properdin in human monocytes. Eur J Biochem 1994; 219: 759-764
20	Yamanouchi S, Kuwahara K, Sakata A, Ezaki T, Matsuoka S, Miyazaki J, Hirose S, Tamura T, Nariuchi H, Sakaguchi N. A T cell activation antigen, Ly6C, induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses. Eur J Immunol 1998; 28: 696-707
21	Manoussaka MS, Smith RJ, Conlin V, Toomey JA, Brooks CG. Fetal mouse NK cell clones are deficient in Ly49 expression, share a common broad lytic specificity, and undergo continuous and extensive diversification in vitro. J Immunol 1998; 160: 2197-2206
22	Wrammert J, Kallberg E, Agace WW, Leanderson T. Ly6C expression differentiates plasma cells from other B cell subsets in mice. Eur J Immunol 2002; 32: 97-103
23	Katz P, Whalen G, Kehrl JH. Differential expression of a novel protein kinase in human B lymphocytes. Preferential localization in the germinal center. J Biol Chem 1994; 269: 16802-16809
24	Valent P, Ashman LK, Hinterberger W, Eckersberger F, Majdic O, Lechner K, Bettelheim P. Mast cell typing: demonstration of a distinct hematopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes. Blood 1989; 73: 1778-1785
25	Titos E, Claria J, Planaguma A, Lopez-Parra M, Villamor N, Parrizas M, Carrio A, Miquel R, Jimenez W, Arroyo V, Rivera F, Rodes J. Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis. FASEB J 2003; 17: 1745-1747
26	Titos E, Claria J, Planaguma A, Lopez-Parra M, Gonzalez-Periz A, Gaya J, Miquel R, Arroyo V, Rodes J. Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells. J Leukoc Biol 2005; 78: 871-878
27	Sawano A, Iwai S, Sakurai Y, Ito M, Shitara K, Nakahata T, Shibuya M. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood 2001; 97: 785-791
28	Coppola S, Narciso L, Feccia T, Bonci D, Calabro L, Morsilli O, Gabbianelli M, De Maria R, Testa U, Peschle C. Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line. Cell Death Differ 2006; 13: 61-74
29	Fernandez Pujol B, Lucibello FC, Zuzarte M, Lutjens P, Muller R, Havemann K. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol 2001; 80: 99-110
30	Bowling WM, Flye MW, Qiu YY, Callery MP. Inhibition of phosphatidylinositol-3'-kinase prevents induction of endotoxin tolerance in vitro. J Surg Res 1996; 63: 287-292
31	Capodici C, Hanft S, Feoktistov M, Pillinger MH. Phosphatidylinositol 3-kinase mediates chemoattractant-stimulated, CD11b/CD18-dependent cell-cell adhesion of human neutrophils: evidence for an ERK-independent pathway. J Immunol 1998; 160: 1901-1909
32	Bracke M, Nijhuis E, Lammers JW, Coffer PJ, Koenderman L. A critical role for PI 3-kinase in cytokine-induced Fcalpha-receptor activation. Blood 2000; 95: 2037-2043
33	Zweifel M, Breu K, Matozan K, Renner E, Welle M, Schaffner T, Clavien PA. Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy. Immunol Cell Biol 2005; 83: 587-595
34	Pemberton AD, Brown JK, Wright SH, Knight PA, Miller HR. The proteome of mouse mucosal mast cell homologues: the role of transforming growth factor beta1. Proteomics 2006; 6: 623-631
35	Schwartz LB. Analysis of MC(T) and MC(TC) mast cells in tissue. Methods Mol Biol 2006; 315: 53-62
36	Henningsson F, Yamamoto K, Saftig P, Reinheckel T, Peters C, Knight SD, Pejler G. A role for cathepsin E in the processing of mast-cell carboxypeptidase A. J Cell Sci 2005; 118: 2035-2042
37	Chen ZQ, He SH. Cloning and expression of human colon mast cell carboxypeptidase. World J Gastroenterol 2004; 10: 342-347
38	Lagasse E, Clerc RG. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation. Mol Cell Biol 1988; 8: 2402-2410
39	Bhardwaj RS, Zotz C, Zwadlo-Klarwasser G, Roth J, Goebeler M, Mahnke K, Falk M, Meinardus-Hager G, Sorg C. The calcium-binding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions. Eur J Immunol 1992; 22: 1891-1897
40	Ryckman C, Vandal K, Rouleau P, Talbot M, Tessier PA. Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion. J Immunol 2003; 170: 3233-3242
41	Yoshimoto T, Suzuki H, Yamamoto S, Takai T, Yokoyama C, Tanabe T. Cloning and expression of arachidonate 12-lipoxygenase cDNA from porcine leukocytes. Adv Prostaglandin Thromboxane Leukot Res 1991; 21A: 29-32
42	Gu JL, Natarajan R, Ben-Ezra J, Valente G, Scott S, Yoshimoto T, Yamamoto S, Rossi JJ, Nadler JL. Evidence that a leukocyte type of 12-lipoxygenase is expressed and regulated by angiotensin II in human adrenal glomerulosa cells. Endocrinology 1994; 134: 70-77
43	Nakamura M, Ueda N, Kishimoto K, Yoshimoto T, Yamamoto S, Ishimura K. Immunocytochemical localization of platelet-type arachidonate 12-lipoxygenase in mouse blood cells. J Histochem Cytochem 1995; 43: 237-244
44	Kaminski WE, Jendraschak E, Baumann K, Kiefl R, Fischer S, Marcus AJ, Broekman MJ, von Schacky C. Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes. Blood 1996; 87: 331-340
45	Takenaga K, Nakamura Y, Sakiyama S. Expression of a calcium binding protein pEL98 (mts1) during differentiation of human promyelocytic leukemia HL-60 cells. Biochem Biophys Res Commun 1994; 202: 94-101
46	Yancopoulos GD, Oltz EM, Rathbun G, Berman JE, Smith RK, Lansford RD, Rothman P, Okada A, Lee G, Morrow M. Isolation of coordinately regulated genes that are expressed in discrete stages of B-cell development. Proc Natl Acad Sci USA 1990; 87: 5759-5763
47	Quelo I, Jurdic P. Differential regulation of the carbonic anhydrase II gene expression by hormonal nuclear receptors in monocytic cells: identification of the retinoic acid response element. Biochem Biophys Res Commun 2000; 271: 481-491
48	Baron WF, Pan CQ, Spencer SA, Ryan AM, Lazarus RA, Baker KP. Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages. Gene 1998; 215: 291-301
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The reference numbers correspond to the numbers in Figure 5 in the supplementary data.

Table 2 Marker genes for hepatocytes that showed a strong relationship with fibrogenesis

Functional category	Cluster No.	Direction of change	Annotation	Symbol	Common
Amino acid metabolism	7	Increase	Glutamate oxaloacetate transaminase 1	Got1	J04171
	7	Increase	Glutamic-pyruvate transaminase (alanine aminotransferase)	Gpt	D10354
	9	Increase	Glutathione-S-transferase, alpha type2	Gsta2	AA945082
	3	Decrease	Cytosolic cysteine dioxygenase 1	Cdo1	AA942685
	10	Decrease	Cysteine-sulfinate decarboxylase	Csad	M64755
Cholesterol synthesis	3	Decrease	Nuclear receptor subfamily 0, group B, member 2	Nr0b2	D86745
Steroid hormone synthesis	7	Increase	Hydroxysteroid (17-beta) dehydrogenase 9	Hsd17b9	U89280
	7	Increase	(20-alpha-hydroxysteroid dehydrogenase)	---	AA866264
	1	Decrease	Hydroxysteroid (17-beta) dehydrogenase 2	Hsd17b2	X91234
	3	Decrease	Sulfotransferase, estrogen preferring	Ste	S76489
Lipid biosynthesis, metabolism, fatty acid & lipid transport	9	Increase	Carboxylesterase 2 (intestine, liver) (drug metabolism)	Ces2	AB010635
	1	Decease	2-hydroxyphytanoyl-CoA lyase(peroxisomal) alpha-oxidation	Hpcl2	AA893239
	3	Decease	Carboxylesterase 3	Ces3	L46791
	3	Decease	Hydroxyacid oxidase 2 (long chain)(peroxisomal)( alpha-oxidation)	Hao2	AI232087
	3	Decease	Fatty acid binding protein 7, brain (cytosolic)	Fabp7	U02096
	10	Decease	Alpha-methylacyl-CoA racemase Peroxisomal)	Amacr	U89905
Retinoid synthesis & metabolism	3	Decease	Retinol dehydrogenase type II (RODH II)	RoDHII	U33500
	10	Decease	Retinol dehydrogenase type III	Rdh3	U18762
Dlucolysis & gluconeogenesis	7	Increase	Glucokinase	Gck	X53588
	3	Decrease	Phosphoenolpyruvate carboxykinase 1(PEPCK1) (cytosolic)	Pck1	K03243
	3	Decrease	Pyruvate carboxylase	Pc	U32314
	3	Decrease	Solute carrier family 37 (glycerol-6-phosphate transporter), member 4	Slc37a4	AF080468
	3	Decrease	Ribonuclease, RNase A family 4	Rnase4	AF041066
	3	Decrease	Ectonucleotide pyrophosphatase/phosphodiesterase 2(lysophospholipaseD)	Enpp2	D28560
Drug-metabolism	7	Increase	Cytochrome P450, family 3, subfamily a, polypeptide 13	Cyp3a13	U46118
	9	Increase	Aldehyde dehydrogenase family 1, member A1	Aldh1a1	AF001898
	1	Decrease	Cytochrome P450, family 1, subfamily a, polypeptide 2	Cyp1a2	E01184
	1	Decrease	Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase)	Cyp2c	X79081
	1	Decrease	Flavin containing monooxygenase 1	Fmo1	M84719
	3	Decrease	Cytochrome P450, family 17, subfamily a, polypeptide 1	Cyp17a1	M21208
	3	Decrease	Cytochrome P450, family 3, subfamily a, polypeptide 11	Cyp3a11	M13646
	3	Decrease	Alcohol dehydrogenase 4 (class II), pi polypeptide	Adh4	X90710
	3	Decrease	3-hydroxybutyrate dehydrogenase (heart, mitochondrial)	Bdh	AA817846
	3	Decrease	Camello-like 1 N-acetyltransferase 8, NAT8)	Cml1	AA892382
	3	Decrease	(hydroxysteroid sulfotransferase )	---	M31363
	3	Decrease	(hydroxysteroid sulfotransferase subunit)	---	AA818122
Growth factors & their receptors	7	Increase	Lipopolysaccharide binding protein	Lbp	L32132
	9	Decrease	Insulin-like growth factor binding protein 2	Igfbp2	J04486
	3	Decrease	Activin beta E	Inhbe	AF089825
	10	Decrease	Growth hormone receptor	Ghr	S49003
	10	Decrease	Insulin-like growth factor binding protein 1	Igfbp1	M58634
	10	Decrease	Deiodinase, iodothyronine, type I	Dio1	X57999
Hepatic secretory proteins	7	Increase	Alpha-fetoprotein	Afp	X02361
	1	Decrease	Alpha-2u globulin PGCL4 /// alpha-2u globulin PGCL2 /// alpha-2u-globulin (L type)/// alpha-2u globulin PGCL1 /// alpha-2u globulin PGCL3	Obp3 /// LOC298109 /// LOC298116 /// LOC259246 /// LOC259244	M27434
	1	Decrease	Alpha-2u globulin PGCL4 (Ppp2r2a protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), alpha isoform)	Obp3	J00738
	10	Decrease	Metallothionein	Mt1a	AI102562
Protease & protease inhibitor	9	Increase	Lipocalin 2	Lcn2	AA946503
	1	Decrease	Elastase 1, pancreatic	Ela1	L00117
	10	Decrease	Esterase 2 (liver carboxylesterase)	Es2	M20629
	3	Decrease	Serine protease inhibitor	Spin2a	D00752
Cell surface proteins & structural proteins	7	Increase	Keratin complex 2, basic, gene 8 (cytokeratin-8)	Krt2-8	S76054
	7	Increase	Similar to cytokeratin(keratin complex 1, acidic, gene 18)	(Krt1-18)	AI072634
Signal transduction	3	Decrease	Regucalcin	Rgn	D31662
Transporters	7	Increase	solute carrier family 17 (sodium phosphate), member 1	Slc17a1	U28504
	7	Increase	Aquaporin 7	Aqp7	AB000507
	9	Increase	ATP-binding cassette, sub-family B (MDR/TAP), member 1	Abcb1	M81855
	3	Decrease	Solute carrier family 10 (sodium/bile acid cotransporter family), member 1	Slc10a1	M77479
Protein related vitamins	10	Decrease	L-gulono-gamma-lactone oxidase	Gulo	D14564
Others	7	Increase	Rabphilin 3A-like (without C2 domains)	Rph3al	AF022774
	1	Decrease	Carbonic anhydrase 3	Ca3	AF037072

Gene expression profiles in the chronic phase (d 7, 14, 21 and 28) were clustered into 10 patterns using K-means analysis. Clustered genes that showed a tendency to temporally decrease (clusters 1, 3 and 10) or increase (clusters 7 and 9) were selected, as shown in Table 1 in the supplemental data. Among these genes, those showing a strong relation with fibrogenesis were further selected based on a t-test statistical analysis of the rate of change in expression intensity and the number of days of fibrosis. Hepatocyte-specific gene markers showing a strong relation with fibrogenesis are listed.