VHL and PTEN loss coordinate to promote mouse liver vascular lesions

Abstract

Von Hippel-Lindau (VHL) inactivation develops a tumor syndrome characterized by highly vascularized tumors as a result of hypoxia inducible factors (HIF) stabilization. The most common manifestation is the development of hemangioblastomas typically located in the central nervous system and other organs including the liver. PTEN (Phosphatase and tension homologue deleted on chromosome 10) inactivation also upregulates HIF-1alpha and may take part in promoting vascular lesions in tumors. The coordinate effect of loss of these tumor suppressors on HIF levels, and the subsequent effect on vascular lesion formation would elucidate the potential for mechanisms to modify HIF dosage supplementally and impact tumor phenotype. We therefore employed models of somatic conditional inactivation of Vhl, Pten, or both tumor suppressor genes in individual cells of the liver by Cre-loxP recombination to study the cooperativity of these two tumor suppressors in preventing tumor formation. Nine months after tumor suppressor inactivation, Vhl conditional deletion (Vhl (loxP/loxP)) mice showed no abnormalities, Pten conditional deletion (Pten (loxP/loxP)) mice developed liver steatosis and focal nodular expansion of hepatocytes containing lipid droplet and fat. Vhl and Pten conditional deletion (Vhl (loxP/loxP);Pten (loxP/loxP)) mice, however, developed multiple cavernous liver lesions reminiscent of hemangioblastoma. Liver hemangioblastomas in VHL disease may, therefore, require secondary mutation in addition to VHL loss of heterozygosity which is permissive for vascular lesion development or augments levels of HIF-1alpha.

Fig. 1

Generation of the scAAV-Cre vector and hepatocyte's transduction. a pHpa-trs-SK plasmid carrying GFP (scAAV-GFP); b scAAV-CRE, GFP was excised from the pHpa-trs-SK plasmid and Cre substituted for GFP; c Reporter gene expression in the liver 4 weeks after scAAV-GFP injected; d Frozen liver section X-gal stain 4 weeks after the scAAV-Cre vector injected into ROSA26 reporter mice showing diffuse β-galactosidase positive hepatocytes, expression of β-galactosidase indicated Cre-mediated excision of the loxP flanked stop signal and hepatocytes have undergone Cre-mediated recombination; e H&E stain showed no inflammatory cell infiltration in livers from scAAV-Cre injected mice. Magnification, ×100 (c through e)

Fig. 2

Comparison of external morphology and histological appearance of livers from AAV-Cre vector injected Vhl^loxP/loxP (a, d, g), Pten^loxP/loxP (b, e, h), and Vhl^loxP/loxP;Pten^loxP/loxP (c, f, i) mice. On gross examination, livers from AAV-Cre vector injected Vhl^loxP/loxP (a), Pten^loxP/loxP (b) appeared normal; livers from Vhl^loxP/loxP;Pten^loxP/loxP mice (c) showed irregular shape and multiple vascular lesions. Magnification, ×40 (d–f), ×100 (g–i)

Fig. 3

The highly vascular liver lesions from a Vhl^loxP/loxP;Pten^loxP/loxP mouse were isolated by using LCM. PCR analysis of recombination at the Vhl and Pten loci in the mice with recombination of AAV-Cre vector, showing Vhl and Pten floxed, recombined, and wild type alleles, respectively. (a) before dissection; (b) after dissection; (c) dissected tissue; (d) Vhl; (e) Pten

Fig. 4

PAS (a–c) and oil red O stain (d–f) of livers from AAV-Cre vector injected Vhl ^loxP/loxP (a, d), Pten ^loxP/loxP (b, e), and Vhl^loxP/loxP;Pten ^loxP/loxP (c, f) mice demonstrate the presence of glycogen (PAS) or lipid (oil red O stain). Magnification, ×200 (a through e) (Color figure online)

Fig. 5

Immunohistochemical analysis of livers from Vhl^loxP/loxP (a), Pten^loxP/loxP (b), and Vhl^loxP/loxP;Pten^loxP/loxP (c) mice using an antibody against the proliferation marker Ki-67. d Quantification of the number of positive cells per high power field of the liver (×400). One field per liver section was counted and the data represent mean ± standard deviation. * Statistically significant difference between Vhl^loxP/loxP;Pten^loxP/loxP and all other genotypes (P < 0.01, student's t test). Magnification, ×200 (a through c)

Fig. 6

Immunohistochemical analysis of livers from AAV-Cre vector injected Vhl^loxP/loxP (a), Pten^loxP/loxP (b), and Vhl^loxP/loxP;Pten^loxP/loxP (c) mice using an antibody against the phosphorylated AKT. Panel b showed that phosphorylated AKT was sporadically positive in livers from Pten^loxP/loxP mice; Panel c showed phosphorylated AKT presence in hepatocytes around the vascular lesions in Vhl^loxP/loxP;Pten^loxP/loxP mice. Magnification, ×200 (a through c)

Fig. 7

Immunohistochemistry stain of HIF-1α in livers from AAV-Cre vector injected Vhl^loxP/loxP (a, d), Pten^loxP/loxP (b, e), and Vhl^loxP/loxP;Pten^loxP/loxP (c, f) mice. Panel c showed HIF-1α expression upregulated in livers from Vhl^loxP/loxP;Pten^loxP/loxP mice. d–f showed the corresponding nuclear DAPI stain. Magnification, ×200 (a through f)

Fig. 8

Immunohistochemistry stain of HIF-2α in livers from AAV-Cre vector injected Vhl^loxP/loxP (a, d), Pten^loxP/loxP (b, e), and Vhl^loxP/loxP;Pten^loxP/loxP (c, f) mice. Panel c showed HIF-2α expression upregulated in livers from Vhl^loxP/loxP;Pten^loxP/loxP mice. d–f showed the corresponding nuclear DAPI stain. Magnification, ×200 (a through f)

Fig. 9

Immunohistochemistry stain of PECAM and VEGF in livers from AAV-Cre vector injected Vhl^loxP/loxP (a, d), Pten^loxP/loxP (b, e), and Vhl^loxP/loxP;Pten^loxP/loxP (c, f) mice. c PECAM expression upregulated in the liver vascular lesions from Vhl^loxP/loxP;Pten^loxP/loxP mice. f VEGF expression upregulated in the liver vascular lesions from Vhl^loxP/loxP;Pten^loxP/loxP mice. Magnification, ×200 (a through f)