Figure 1.
Hepatic Stellate Cells express RGS5.
A. Rgs5LacZ/LacZ mouse liver with X-gal labeling. RGS5+ peri-sinusoidal cells are distributed throughout the liver. RGS5 is also expressed in a subset of SMC of the portal vein (yellow arrows). B–G. Immunofluorescence (IF) for cell-specific markers in the Rgs5LacZ/LacZ liver. B. SMA and α-β-gal show RGS5 expression in vascular SMCs, as expected (arrows). C. GFAP and α-β-gal IF in Rgs5LacZ/LacZ mouse liver. β-gal+ nuclei are visible within GFAP+ astrocyte-like HSC cells, localizing RGS5 expression to HSCs (arrows). D. CRBP1 and α-β-gal IF are co-localized in HSC of Rgs5LacZ/LacZ liver (arrows). E. VWF (a marker of endothelial cells) and α-β-gal are not co-localized. VWF extends through all sinusoids, while β-gal+ cells are sparsely distributed. F. F4/80 (a maker of macrophage/Kupffer cells) and β-gal+ cells represent distinct cell populations. G. Confocal image of α-GFAP IF showing co-localization of the nuclear α-β-gal (arrows). H. Confocal image of CD31 (a marker of endothelial cells) and α-β-gal. CD31+ cells have β-gal− nuclei, while β-gal+ nuclei are not associated with CD31+ endothelial cells. All scale bars are 100 µm.
Figure 2.
RGS5 expression is up-regulated in HCC and liver fibrosis.
Sections of liver from Tsc1fl/fl;AlbCre (A,C) and Ptenfl/fl;AlbCre (B,D) mice were stained with Masson's trichrome. A. Tsc1fl/fl;AlbCre non-tumor liver is histologically normal. B. Ptenfl/fl;AlbCre non-tumor liver tissue is steatotic and shows collagen deposition in sinusoids (blue). C. Tsc1fl/fl;AlbCre tumor tissue shows disorganized architecture and high levels of collagen deposition. D. Ptenfl/fl;AlbCre tumor tissue is glandular in appearance, with robust collagen deposition. Scale bars are 100 µm. E–F. RNA was isolated from wild-type normal tissue and matched tumor and non-tumor tissues of Tsc1fl/fl;AlbCre and Ptenfl/fl;AlbCre E. Tsc1fl/fl;AlbCre mice have normal RGS5, SMA, and Collagen expression in non-tumor parenchyma, and elevated expression in tumor tissue. F. Ptenfl/fl;AlbCre mice have elevated expression of RGS5 and collagen in non-tumor parenchyma and in tumors. Data is normalized to expression in wild-type liver tissue. n = 5, error bars = SEM, * = p<0.05.
Figure 3.
RGS5 expression is up-regulated in acute liver injury.
C57BL/6 mice were injected i.p. with 10 µl/g body weight 10% CCl4 diluted in olive oil 10% (v/v). Livers were collected at 24, 48, 72 hr post injection. RNA was isolated and expression of HSC activation markers was determined by qPCR. A. RGS5 expression is elevated from 24 to 72 hr post injury, peaking at 48 hr. Expression of B. SMA, C. Desmin, and D. PDGFRβ is up-regulated at 24 and 48 hours post injury. E. PDGFRα and F. Col 1a are up-regulated 48 hours post injury and remain high at 72 hr. Data is normalized to expression in untreated (ut) samples. n = 3–5, error bars = SEM, * = p<0.05.
Figure 4.
RGS5 expression is regulated by profibrotic cytokines in concert with ETB.
LX2 HSCs were treated with TNFα (5 ng/ml), TGFβ (5 ng/ml), PDGF-BB (10 µM), and ET-1 (100 nm) for 24 hr. RNA was collected for qPCR analysis of RGS5 and ETB expression. Both RGS5 and ETB are up-regulated by TNFα stimulation and down-regulated by TGFβ stimulation. RGS5 and ETB expression are correlated, responding similarly to the same stimuli. n = 3, error bars = SEM, * = p<0.05.
Figure 5.
RGS5+ HSCs participate in the response to acute hepatic injury.
Anti-GFAP and anti-β-gal immunofluorescence were used to localize HSCs in acutely injured liver tissue. A–F. Low magnification images. G–I. High magnification of Rgs5LacZ/LacZ A,D Uninjured liver tissue from Rgs5+/+ and Rgs5LacZ/LacZ mice show sparse HSCs distributed throughout the liver. High magnification in uninjured Rgs5LacZ/LacZ shows HSCs are GFAP+ and β-gal+. B,E. At 48 hours post injury, HSCs are concentrated in the necrotic foci surrounding the central veins. β-gal+ cells (E,H) are associated with GFAP+ cells. C,F At 96 hours post injury, HSCs are tightly clustered at the foci of injury. I. β-gal+ cells are GFAP+. All scale bars are 100 µm.
Figure 6.
Rgs5LacZ/LacZ mice have disrupted hepatocyte morphology after injury.
Acute CCl4-induced injury in Rgs5+/+ mice (A–C) and Rgs5LacZ/LacZ (D–F). A,D. Uninjured mice are histologically normal. B,E At 48 hr post CCl4 injection, foci of necrosis are visible central veins in both Rgs5+/+ and Rgs5LacZ/LacZ mice. C,F. At 96 hr post injury, clearance of necrotic hepatocytes is underway and infiltrating cells remain at the site of injury. F. In Rgs5LacZ/LacZ mice, hepatocytes throughout the liver have cleared cytoplasm. Scale bars are 100 µm.
Figure 7.
Rgs5LacZ/LacZ mice have increased liver injury and HSC activation following acute injury.
Livers from Rgs5+/+ and Rgs5LacZ/LacZ were collected at 96 hr following a single CCl4 injection. A. and B. H&E stain of Rgs5+/+ mice recover normally from acute injury. Foci of necrosis are centered on the central veins. B. Hepatocytes appear normal. C. and D. Rgs5LacZ/LacZ livers have extensive ballooning of hepatocytes throughout the liver. Foci of necrosis are present. D. Rgs5Lacz/LacZ hepatocytes show cleared cytoplasm and centralized nuclei in hepatocytes distant from necrotic foci. Scale bars are 100 µm. E. Quantification of damaged hepatocyte area reveals a significant increase in ballooning in Rgs5Lacz/LacZ mice compared to Rgs5+/+ mice (n = 8–10; error bars = SEM; * = p<0.05) F. mRNA expression of markers of HSC activation (Desmin, PDGFRβ, and ETB) are elevated in Rgs5Lacz/LacZ mice compared to Rgs5+/+ mice following acute CCl4 injury. n = 4–6, * = p<0.05 by Mann-Whitney U test.
Figure 8.
Knock-down of RGS5 expression enhances endothelin-1-mediated signaling in LX-2 HSCs.
LX2 cells were treated with Rgs5 siRNA or non-specific siRNA for 24 hours, then stimulated with 100 nM ET-1 for the indicated times. Whole cell protein extracts were isolated and analyzed by Western blot. A. A representative immunoblot against pERK1/2 demonstrates increased ET-1-mediated signaling in the absence of RGS5 expression; tERK serves as loading control. B. Quantitation of densitometry of (A) n = 7, error bars = SEM, * = p<0.05.
Figure 9.
RGS5 expression is up-regulated with HSC activation in chronic liver injury.
Rgs5LacZ/LacZ mice were chronically injected with CCl4 (or oil), twice weekly for 4 weeks. RNA was isolated from whole liver and analyzed by qPCR for expression of Rgs5 and HSC activation markers. A. Chronic CCl4 treated Rgs5LacZ/LacZ dab labeled with anti-SMA shows activated HSCs. B. Rgs5LacZ/LacZ liver immunolabeled with α-β-gal and α-GFAP antibodies. GFAP+ HSCs are visible along fibrotic septa bridging the portal veins. β-gal+ HSCs are visible in Rgs5LacZ/LacZ (B) around the fibrotic septa. Scale bars are 100 µm. C. qPCR of Rgs5+/+ liver RNA shows Rgs5 is up-regulated in chronic CCl4 injury. Collagen 1α expression is elevated and multiple HSC activation markers (PDGFRα, PDGFRβ, and SMA) are increased relative to oil-injected mice. Data is normalized to oil injected mice. n = 6; error bars = SEM; * = p<.05.
Figure 10.
Rgs5LacZ/LacZ mice have increased fibrosis during chronic liver injury.
Mice were injected with CCl4 (or oil), twice weekly for 4 weeks. Formalin fixed, paraffin embedded liver sections were stained with picrosirius red to assess fibrosis. A. Rgs5+/+ mice show periportal fibrosis and bridging fibrosis between portal veins. B. Rgs5LacZ/LacZ mice show severe periportal fibrosis bridging between portal veins and surrounding lobules. C. Rgs5+/+ and D. Rgs5LacZ/LacZ oil injected mice have minimal fibrosis. Scale bars are 500 µm. E. ImageJ quantitation of total picrosirius red staining in chronic CCl4 injury shows significantly more fibrosis in Rgs5LacZ/LacZ mice, compared to Rgs5+/+ mice. n = 6, * = p<0.05 by Mann-Whitney U test.