Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats - PubMed (original) (raw)

Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats

Guanhua Xie et al. Gastroenterology. 2012 Apr.

Abstract

Background & aims: Capillarization, characterized by loss of differentiation of liver sinusoidal endothelial cells (LSECs), precedes the onset of hepatic fibrosis. We investigated whether restoration of LSEC differentiation would normalize crosstalk with activated hepatic stellate cells (HSC) and thereby promote quiescence of HSC and regression of fibrosis.

Methods: Rat LSECs were cultured with inhibitors and/or agonists and examined by scanning electron microscopy for fenestrae in sieve plates. Cirrhosis was induced in rats using thioacetamide, followed by administration of BAY 60-2770, an activator of soluble guanylate cyclase (sGC). Fibrosis was assessed by Sirius red staining; expression of α-smooth muscle actin was measured by immunoblot analysis.

Results: Maintenance of LSEC differentiation requires vascular endothelial growth factor-A stimulation of nitric oxide-dependent signaling (via sGC and cyclic guanosine monophosphate) and nitric oxide-independent signaling. In rats with thioacetamide-induced cirrhosis, BAY 60-2770 accelerated the complete reversal of capillarization (restored differentiation of LSECs) without directly affecting activation of HSCs or fibrosis. Restoration of differentiation to LSECs led to quiescence of HSCs and regression of fibrosis in the absence of further exposure to BAY 60-2770. Activation of sGC with BAY 60-2770 prevented progression of cirrhosis, despite continued administration of thioacetamide.

Conclusions: The state of LSEC differentiation plays a pivotal role in HSC activation and the fibrotic process.

Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Disclosures: None of the authors have a financial conflict of interest with the work in this manuscript.

Figures

Figure 1

Figure 1. VEGF is required to maintain LSEC phenotype both in vitro and in vivo

(A) Representative SEM of LSEC cultured with (left panel) and without (right panel) VEGF for 2 days show loss of fenestrae in sieve plates in vitro in the absence of VEGF. Scale bar, 5μm. (B) Hepatic expression of VEGF on immunoblot with densitometry and (C) representative SEM of hepatic sinusoids from rats treated with VEGF ASO or control oligonucleotides. * p<0.05. Scale bar, 2μm. All figures represent n ≥ 3.

Figure 2

Figure 2. VEGF-stimulated cGMP is necessary but not sufficient to maintain LSEC phenotype in vitro

Either ODQ (sGC inhibitor) or Rp-8-pCPT-PET-cGMPS (PKG inhibitor) completely blocks VEGF-stimulated (A) fenestration and (B) porosity in LSEC cultured for 2 days. Scale bar: 5μm. * p<0.001 versus VEGF control, n=3. (C) Either YC-1 (sGC activator) or 8-pCPT-cGMP (cGMP analog) without VEGF fails to normalize fenestration. However, VEGF+L-NAME (eNOS inhibitor) +YC-1 or VEGF+ODQ+8-pCPT-cGMP normalizes LSEC fenestration; thus both VEGF independent of NO plus the VEGF-stimulated cGMP pathway are required, whereas the experiment with VEGF + L-NAME + YC-1 also demonstrates that protein S-nitrosylation is not necessary. Scale bar: 5μm. (D) Porosity measurement of experiments described in (C). * p<0.001 versus VEGF control; NS, not significant; n = 3.

Figure 3

Figure 3. Both VEGF-independent of NO plus VEGF-stimulated NO are required to maintain normal LSEC fenestration

LSEC cultured with VEGF+L-NAME (top left panel) or DETA-NONOate (NO donor; top right panel) for 2 days lack fenestration, while LSEC cultured with VEGF+L-NAME+DETA-NONOate (bottom left panel) for 2 days demonstrate normal fenestration. Porosity is shown in the bottom right panel. Scale bar, 5μm. * p<0.001versus VEGF control; NS, not significant; n = 3.

Figure 4

Figure 4. In vivo sGC activation restores LSEC phenotype in thioacetamide (TAA) -induced capillarization

(A) Representative SEM of hepatic sinusoids. Top left panel: normal hepatic sinusoid with fenestrae grouped into sieve plates. Top right panel: capillarization after 3 weeks of TAA. Bottom left panel: minimal reversal of capillarization 1 week after discontinuing TAA. Bottom right panel: complete reversal of capillarization by 1-week of daily sGC activator after discontinuing TAA. Scale bar: 2μm. (B) Porosity from rats treated as in (A). * P<0. 001. (C) Cellular cGMP level in LSEC isolated from rats treated as indicated in (A). * P<0.001. n = 3.

Figure 5

Figure 5. Restoration of the differentiated LSEC phenotype accelerates regression of thioacetamide (TAA)-induced early cirrhosis

(A) Protocol for 5 treatment groups (n = 9–12). (B) Immunoblot with densitometry and (C) immunohistochemistry show elevated α-SMA expression after 3 weeks of TAA treatment. 1 week of solvent or sGC activator after discontinuing TAA does not alter α-SMA expression, but α-SMA expression is significantly reduced when the 1week of sGC activator is followed by 1 week of solvent (group 5) compared to 2 weeks of solvent after TAA (group 4). * p<0.001. Scale bar, 30 μm. (D) Representative Sirius red stain shows early cirrhosis in 3-week TAA treated rats (top middle). Cirrhosis persists after 1 week of either solvent (top right) or sGC activator (bottom left) after discontinuation of TAA, but only bridging fibrosis is observed when the 1 week of sGC activator is followed by 1 week of solvent (bottom right), compared to persistent cirrhosis in rats that received 2 weeks of solvent after TAA (bottom middle). Scale bar, 1.2 mm. (E) Quantification of fibrosis by scanning morphometry. * p<0.05.

Figure 6

Figure 6. In vivo sGC activation prevents progression and accelerates regression of cirrhosis by restoration of LSEC differentiation

(A) Protocol for 5 treatment groups (n = 5). (B) Representative SEM (scale bar, 2μm) and porosity measurements (n=4) show that sGC activator treatment complete reverses capillarization despite ongoing TAA (group III) compared to solvent control (group II). * P<0. 001. (C) Immunoblot (n = 5) and (D) immunohistochemistry show that co-treatment of sGC with TAA from week 4 to 6 (group III) reduced α-SMA expression compared to 6-week TAA treated rats (group II). 4 weeks after discontinuation of treatment, α-SMA expression in group V was further decreased compared to 4 weeks earlier (group III) or compared to the TAA solvent control group (group IV). * P<0.05. Scale bar, 30 μm. (E) Representative Sirius red staining and (F) scanning morphometry thereof (n=5) show more fibrosis after 6 weeks TAA (group II) compared to 3 weeks TAA (group I). Extent of fibrosis is unchanged after 6 weeks TAA plus sGC activator from week 4 to 6 (group III) compared to 3 weeks TAA (group I). 4 weeks after discontinuation of treatment, regression of fibrosis is greater after sGC activator (group V) compared to the solvent group (group IV). Scale bar, 1.2 mm. * P<0.001.

Figure 7

Figure 7. Complete reversal of HSC activation by the sGC activator requires the presence of LSEC and is independent of NO

(A) Upper panel: percentage α-SMA-positive HSC determined by confocal microscopy is lower in HSC cultured with LSEC plus BAY60-2770 compared to BAY60-2770 alone (p<0.0001, n=4); lower panel: representative photomicrographs of HSC. HSC day 6: HSC cultured alone for 6 days; HSC day 0_–_6, LSEC day 3_–_6: HSC cultured alone from day 0–3, followed by co-culture from day 3–6 with LSEC isolated on day 3 (note: LSEC cultured with activated HSC for 3 days are capillarized); HSC day 0_–_6, sGC activator day 3_–_6: HSC cultured alone from day 0–6, with BAY 60-2770 from day 3–6; HSC day 0_–_6, sGC activator + LSEC day 3_–_6: HSC cultured alone for 3 days, followed by co-culture from day 3–6 with LSEC isolated on day 3 plus BAY 60-2770 (note: LSEC cultured for 3 days with BAY60-2770 remain differentiated) (Scale bar: 40μm. n = 4–6 for each group). (B) NO does not promote HSC quiescence. The percentage α-SMA-positive HSC is not significantly different in HSC cultured with or without DETA-NONOate or in HSC co-cultured with LSEC and BAY 60-2770 with or without L-NAME. HSC day 6: HSC cultured alone for 6 days; HSC day 0_–_6, DETA-NONOate day 3_–_6: HSC cultured alone from day 0–6,with DETA-NONOate added on day 3; HSC day 0_–_6, BAY + LSEC day 3_–_6: HSC cultured alone for 3 days, followed by co-culture from day 3–6 with LSEC isolated on day 3 plus BAY 60-2770; HSC day 0_–_6, sGC activator + LSEC + L-NAME day 3_–_6: HSC cultured alone for 3 days, followed by co-culture from day 3–6 with LSEC isolated on day 3 plus BAY 60-2770 with L-NAME. n = 3–6 for each group.

Comment in

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