Hepatic microenvironment affects oval cell localization in albumin-urokinase-type plasminogen activator transgenic mice

Abstract

Mice carrying an albumin-urokinase type plasminogen activator transgene (AL-uPA) develop liver disease secondary to uPA expression in hepatocytes. Transgene-expressing parenchyma is replaced gradually by clones of cells that have deleted transgene DNA and therefore are not subject to uPA-mediated damage. Diseased liver displays several abnormalities, including hepatocyte vacuolation and changes in nonparenchymal tissue. The latter includes increases in laminin protein within parenchyma and the appearance of cytokeratin 19-positive bile ductule-like cells (oval cells) both in portal regions and extending into the hepatic parenchyma. In this study, we subjected AL-uPA mice to two-thirds partial hepatectomy to identify the response of these livers to additional growth stimulation. We observed several changes in hepatic morphology. First, the oval cells increased in number and often formed ductules in the parenchyma. Second, this cellular change was accompanied by a further increase in laminin associated with single or clusters of oval cells. Third, desmin-positive Ito cells increased in number and maintained close association with oval cells. Fourth, these changes were localized precisely to uPA-expressing areas of liver. Regenerating clones of uPA-deficient cells appeared to be unaffected both by stromal and cellular alterations. Thus, additional growth stimulation of diseased uPA-expressing liver induces an oval cell-like response, as observed in other models of severe hepatic injury, but the localization of this response seems to be highly regulated by the hepatic microenvironment.

Figure 1.

Oval cell response and associated hepatic changes in AL-uPA mice. A–I: Each column displays neighboring liver sections that were stained immunohistochemically with antibodies against cytokeratin 19 (A–C), laminin, (D–F), or desmin (G–I). A, D, and G: Liver from a 27-day-old nontransgenic mouse. In normal parenchyma, cytokeratin 19-positive cells (biliary epithelial cells) (A) and prominent laminin staining (D) are restricted to portal regions. Cytoplasmic processes of desmin-positive Ito cells (arrow) occasionally are visible in the perisinusoidal spaces (G). B, E, and H: Liver from a 27-day-old AL-uPA mouse. Arrowheads mark the edge of a focus of regenerating, transgene-deficient hepatocytes. Staining for cytokeratin 19-positive cells (B) and laminin protein (E) is present in diseased hepatic parenchyma. Increased numbers of desmin-positive Ito cells (H) also are present in diseased parenchyma; the arrow marks a desmin-positive Ito cell in the regenerative focus. Regenerating parenchyma displays reduced or no staining with each antibody. C, F, and I: Liver from a 42-day-old AL-uPA mouse. Arrowheads mark the edge of a regenerative focus. Staining for cytokeratin 19-positive cells (C), laminin protein (F), and Ito cells (I) is further increased exclusively in diseased parenchyma of older AL-uPA transgenic mice. The arrow marks a desmin-positive Ito cell (I) in the regenerative focus. J, K, and L: Appearance of oval cells in AL-uPA mice after partial hepatectomy. Liver sections were stained immunohistochemically for cytokeratin 19. Arrowheads mark the edges of regenerative foci. J: Liver morphology in an AL-uPA transgenic mouse at 7 days after hepatectomy. Cytokeratin 19-positive cells are present in diseased hepatic parenchyma, occasionally forming ducts. K: Liver morphology in an AL-uPA transgenic mouse at 9 days after hepatectomy. Extensive numbers of cytokeratin 19-positive cells are present in diseased parenchyma, frequently forming ducts. L: Liver morphology in a nontransgenic mouse at 11 days after hepatectomy. Only biliary epithelial cells in the portal region stain for cytokeratin 19. Original magnifications, ×200.

Figure 2.

Neighboring liver sections from an AL-uPA transgenic mouse at 11 days after hepatectomy. Arrowheads mark the edge of a regenerative focus. Sections were stained with H&E (A) or immunohistochemically with anti-cytokeratin 19 (B), anti-laminin (C), or anti-desmin (D). Double immunohistochemistry shows co-localization of cytokeratin 19-positive cells (red stain) and laminin protein (brown stain) in diseased parenchyma (E). A similar association was seen between cytokeratin 19-positive cells (red) and desmin-positive Ito cells (brown) (F). G: Section stained immunohistochemically with anti-cytokeratin 19 (red stain) and anti-BrdU (brown stain) to identify oval cells and hepatocytes undergoing DNA synthesis. BrdU injected 1 to 2 hours before euthanasia was incorporated into DNA of cells undergoing DNA synthesis. Original magnifications, ×400.