Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma

Abstract

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as alphav-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

Quantitative and Qualitative Alterations of Heparan Sulfate in Fibrogenic Liver Diseases and Hepatocellular Cancer

“…HS labeling of non-sinusoidal surfaces of hepatocytes was very faint or undetectable. The following inferences are based on the earlier observations of others (Roskams et al 1995), as well as on our own previous (Kovalszky et al 1998;Tátrai et al 2006) and present results. Continuous sinusoidal HS labeling is thought to reflect the presence of syndecan-1 and perlecan.…”

“…For the immunodetection of agrin, sections were deparaffinized and rehydrated, immersed in boiling Target Retrieval Solution (Dako; Glostrup, Denmark) in a pressure cooker for 5 min, digested with 0.5 mg/ml Pronase E (Sigma-Aldrich) for 5 min, treated with 10% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase activity, and blocked with 5% BSA in PBS for 30 min at 37C. After samples were incubated overnight with primary antibody at 4C [mouse anti-HSPG, clone 7E12 (Invitrogen), previously proven to react specifically with agrin (Tátrai et al 2006)], biotinylated secondary antibody (Dako) was applied for 30 min, signal was amplified using a Tyramide Signal Amplification biotin system kit (PerkinElmer; Waltham, MA), and detected using a DAB substrate kit (Vector Laboratories; Burlingame, CA) as chromogen. For the immunodetection of glypican-3, deparaffinized and rehydrated slides were heated in citrate buffer, pH 6, at 98.5C for 30 min and blocked with 5% BSA in PBS.…”

“…As we had access to a well-established rodent model of liver fibrogenesis and hepatocarcinogenesis, we were able to compare the HS immunopatterns of the healthy and diseased rat liver with human counterparts. Changes in the amount and distribution of HS were interpreted in the context of altered expression of four major liver HSPGs: syndecan-1, perlecan, agrin, and glypican-3 (Roskams et al 1995;Zhu et al 2001;Tátrai et al 2006). HS samples from normal liver and HCC were also compared by using disaccharide analysis.…”

Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells

“…Agrin mediate cell adhesion and control the activities of numerous growth and motility factors and play a critical role in carcinogenesis and tumor progression [33,34].…”

Agrin Pathway is Controlled by Leukemia Inhibitory Factor (LIF) in Murine Implantation

“…Strong expression of agrin in the hepatocarcinoma indicates its involvement in angiogenesis [38]. Agrin may play roles in decidual angiogenesis for successful placentation as well as perlecan or collagen XVIII, whose terminal fragments have effects on angiogenesis [9][10][11].…”