Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May;341(2):307-16.
doi: 10.1124/jpet.111.189340. Epub 2012 Jan 23.

Hypoxia-inducible factor activation in myeloid cells contributes to the development of liver fibrosis in cholestatic mice

Bryan L Copple  1 , Sophia Kaska, Callie Wentling
Affiliations

Hypoxia-inducible factor activation in myeloid cells contributes to the development of liver fibrosis in cholestatic mice

Bryan L Copple et al. J Pharmacol Exp Ther. 2012 May.

Abstract

Macrophages play an integral role in the development of liver fibrosis by releasing mediators, such as platelet-derived growth factor-B (PDGF-B) and transforming growth factor-β1, which stimulate hepatic stellate cell proliferation, chemotaxis, and collagen production. However, the mechanism by which chronic liver injury stimulates macrophages to release these mediators is not completely understood. We tested the hypothesis that chronic liver injury activates hypoxia-inducible factor (HIF) transcription factors in macrophages that regulate the production of mediators that promote fibrosis. To test this hypothesis, Cre/lox technology was used to generate myeloid cell-specific HIF-1α or HIF-1β knockout mice. When these mice were subjected to bile duct ligation (BDL), levels of α-smooth muscle actin and type I collagen in the liver were reduced compared with those of mice with normal levels of HIFs. The deficiency of HIFs in macrophages did not affect liver injury or inflammation after BDL but reduced PDGF-B mRNA and protein, suggesting that HIF activation in macrophages may promote fibrosis by regulating the production of PDGF-B. Consistent with a role for HIFs in liver fibrosis in cholestatic liver disease, nuclear HIF-1α protein was present in macrophages, hepatocytes, and fibroblasts in the livers from patients with primary biliary cirrhosis and primary sclerosing cholangitis. These studies demonstrate that HIFs are important regulators of profibrotic mediator production by macrophages during the development of liver fibrosis and suggest that HIFs may be a novel therapeutic target for the treatment of chronic liver disease in patients.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Decreased HIF-1β protein in Kupffer cells isolated from HIF-1βLysMCre+ mice. A, Kupffer cells were isolated from HIF-1βLysMCre− and HIF-1βLysMCre+ mice, and HIF-1β was detected by Western blot analysis. HIF-1βLysMCre+ mice were subjected to BDL. B–D, 10 days after surgery, immunohistochemistry was used to detect Cre recombinase (red staining in B and D) and macrophages (green staining in C and D). B, C, and D show the same field. Yellow staining in D demonstrates the colocalization of Cre recombinase and macrophages. Scale bar, 50 μm.
Fig. 2.
Fig. 2.
Liver injury in HIF-1βLysMCre− and HIF-1βLysMCre+ mice after BDL. HIF-1βLysMCre− and HIF-1βLysMCre+ mice were subjected to sham operation or BDL. Ten days after surgery, liver sections were stained with hematoxylin and eosin. A and B, representative photomicrographs from HIF-1βLysMCre− (A) and HIF-1βLysMCre+ (B) mice subjected to BDL. Arrows indicate areas of necrosis. Scale bar, 50 μm.
Fig. 3.
Fig. 3.
Levels of macrophages and neutrophils in HIF-1βLysMCre− and HIF-1βLysMCre+ mice after BDL. HIF-1βLysMCre− and HIF-1βLysMCre+ mice were subjected to sham operation or BDL. Ten days after surgery, macrophages and neutrophils were detected in the liver by immunohistochemistry. A and B, representative photomicrographs of immunohistochemistry for macrophages in liver sections from HIF-1βLysMCre− (A) and HIF-1βLysMCre+ (B) mice subjected to BDL. Positive staining appears black in the photomicrographs. Scale bar, 100 μm. C, the area of macrophage immunostaining was quantified using morphometric techniques. D, neutrophils were quantified in liver sections. Data are expressed as mean ± S.E.M.; n = 8. a, significantly different (p < 0.05) from sham-operated mice.
Fig. 4.
Fig. 4.
Liver fibrosis in HIF-1βLysMCre− and HIF-1βLysMCre+ mice after BDL. HIF-1βLysMCre− and HIF-1βLysMCre+ mice were subjected to sham operation or BDL. A and B, ten days after surgery, mRNA levels of α-SMA (A) and type I collagen (B) were quantified by real-time PCR. C and D, representative photomicrographs of type I collagen immunohistochemistry in liver sections from BDL HIF-1βLysMCre− (C) and HIF-1βLysMCre+ (D) mice. Positive staining appears dark gray in the photomicrographs. Scale bar, 50 μm. E, the area of type I collagen immunostaining was quantified in liver sections. Data are expressed as mean ± S.E.M.; n = 8. a, significantly different (p < 0.05) from sham-operated mice. b, significantly different (p < 0.05) from BDL HIF-1βLysMCre− mice.
Fig. 5.
Fig. 5.
TGF-β1 protein levels in the livers of HIF-1βLysMCre− and HIF-1βLysMCre+ mice after BDL. HIF-1βLysMCre− and HIF-1βLysMCre+ mice were subjected to sham operation or BDL. Ten days after surgery, Western blot analysis was used to detect TGF-β1 in liver homogenates.
Fig. 6.
Fig. 6.
Quantification of PDGF-B and MCP-1 protein in HIF-1βLysMCre− and HIF-1βLysMCre+ mice after BDL. HIF-1βLysMCre− and HIF-1βLysMCre+ mice were subjected to sham operation or BDL. A–F, 10 days after surgery, immunohistochemistry was used to detect macrophages (red staining in A, C, D, and F) and PDGF-B (green staining in B, C, E, and F) in liver sections from HIF-1βLysMCre− (A, B, and C) and HIF-1βLysMCre+ (D, E, and F) mice subjected to BDL. A, B, and C show the same field. D, E, and F show the same field. Arrows indicate PDGF-B-positive macrophages from HIF-1βLysMCre− mice. Dashed arrows indicate PDGF-B-negative macrophages from HIF-1βLysMCre+ mice. G, MCP-1 protein was quantified in serum. Data are expressed as mean ± S.E.M.; n = 8. a, significantly different (p < 0.05) from sham-operated mice.
Fig. 7.
Fig. 7.
Liver injury in HIF-1αLysMCre− and HIF-1αLysMCre+ mice after BDL. A, Kupffer cells were isolated from HIF-1αLysMCre− and HIF-1αLysMCre+ mice and exposed to 1% oxygen for 1 h. HIF-1α was detected in nuclear extracts by Western blot analysis. HIF-1αLysMCre− and HIF-1αLysMCre+ mice were subjected to sham operation or BDL. B, 10 days after surgery, ALT activity was measured in serum. Data are expressed as mean ± S.E.M.; n = 8. a, significantly different (p < 0.05) from sham-operated mice. C and D, representative photomicrographs from HIF-1αLysMCre− (C) and HIF-1αLysMCre+ (D) mice subjected to BDL. Arrows indicates area of necrosis. Scale bar, 50 μm.
Fig. 8.
Fig. 8.
Liver fibrosis in HIF-1αLysMCre− and HIF-1αLysMCre+ mice after BDL. HIF-1αLysMCre− and HIF-1αLysMCre+ mice were subjected to sham operation or BDL. A–C, 10 days after surgery, mRNA levels of α-SMA (A), type I collagen (B), and PDGF-B (C) were quantified by real-time PCR. Data are expressed as mean ± S.E.M.; n = 8. a, significantly different (p < 0.05) from sham-operated mice. b, significantly different (p < 0.05) from BDL HIF-1αLysMCre− mice.
Fig. 9.
Fig. 9.
HIF-1α activation in the livers of patients with PBC and PSC. A–H, sections of frozen liver from patients with PBC (A, B, G, and H), patients with PSC (C and D), and normal human livers (E and F) were stained for HIF-1α (green staining in A, C, E, and G) and either CD68 (red staining in A–F) or α-SMA (red staining in G and H). Nuclei were stained with 4,6-diamidino-2-phenylindole (blue staining in B, D, F, and H). The same fields in A, C, E, and G are shown in B, D, F, and H. Insets show higher-power fields. Arrows indicate HIF-1α-positive nuclei in macrophages (A and C) or α-SMA-positive cells (G). Arrowheads indicate HIF-1α-positive nuclei in hepatocytes. Dashed arrows indicate HIF-1α-negative macrophages (E and F).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini P. (1997) Focal adhesion kinase and phospholipase C gamma involvement in adhesion and migration of human hepatic stellate cells. Gastroenterology 112:522–531 - PubMed
    1. Cash TP, Pan Y, Simon MC. (2007) Reactive oxygen species and cellular oxygen sensing. Free Radic Biol Med 43:1219–1225 - PMC - PubMed
    1. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Förster I. (1999) Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8:265–277 - PubMed
    1. Coleman ML, Ratcliffe PJ. (2007) Oxygen sensing and hypoxia-induced responses. Essays Biochem 43:1–15 - PubMed
    1. Copple BL, Bai S, Burgoon LD, Moon JO. (2011) Hypoxia-inducible factor-1α regulates the expression of genes in hypoxic hepatic stellate cells important for collagen deposition and angiogenesis. Liver Int 31:230–244 - PMC - PubMed

Publication types

MeSH terms

-