Quantitative and qualitative alterations of heparan sulfate in fibrogenic liver diseases and hepatocellular cancer

Abstract

Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC.

Figure 1

Immunoreactions of sulfation-specific anti–heparan sulfate (HS) antibodies in the normal and diseased human liver. Rows A–D, liver parenchyma; rows E,F: nonparenchymal structures. (A) normal liver tissue; (B) cirrhotic liver; (C) focal nodular hyperplasia; (D) hepatocellular carcinoma; (E) blood vessels; (F) bile ducts. Arrowhead in D, HS4C3 indicates a blood vessel belonging to the tumoral stroma. Bars: A–D = 100 μm; E,F = 50 μm.

Figure 2

Immunoreactions of sulfation-specific anti-HS antibodies in normal and diseased rat liver. Left column shows healthy liver (A,D,G,J,M); middle column shows cirrhosis with hepatocellular carcinoma (HCC) nodules (B,E,H,K,N); right column shows nonparenchymal structures (C,F,I,L,O). Thin arrows, blood vessels; thick arrows, bile ducts. Bars: A,B = 250 μm (left and middle column main panels); C = 50 μm (right column main panels); H,K,L insets = 50 μm.

Figure 3

Immunoreactions for syndecan-1 (A–C), perlecan (D–F), agrin (J–L), and glypican-3 (M–O) in normal and diseased human liver. G–I show merged images of A–C and D–F. Left column shows normal liver tissue; middle column shows cirrhotic tissue; and right column shows HCC tissue. Thin arrows, blood vessels; thick arrows, bile ducts; arrowheads, HCC microvessels. Bars: A–O = 200 μm; insets = 50 μm. Bar in A applies to A–I; bar in J applies to J–O.

Figure 4

mRNA expression of selected HS-modifying enzymes in non-fibrotic liver, fibrogenic diseases, and HCC tissues. (A,B) Relative expressions of the nine enzymes in the three sample groups (reference, eukaryotic 18S rRNA) are shown in arbitrary expression units (2^−ΔCt × 10⁶). Isozyme pairs are compared in A; enzymes with only a single investigated isoform are presented in B. Median expression differences and minimum and maximum expression values of the nine enzymes in the fibrotic and HCC groups relative to the median of the non-fibrotic group are shown in C. *, p<0.05; **, p<0.01; ***, p<0.001, Mann-Whitney U tests.