Volume 142, Issue 4, Pages 918-927.e6 (April 2012) Role of Differentiation of Liver Sinusoidal Endothelial Cells in Progression and Regression of Hepatic Fibrosis in Rats  Guanhua Xie, Xiangdong Wang, Lei Wang, Lin Wang, Roscoe D. Atkinson, Gary C. Kanel, William A. Gaarde, Laurie D. DeLeve  Gastroenterology  Volume 142, Issue 4, Pages 918-927.e6 (April 2012) DOI: 10.1053/j.gastro.2011.12.017 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 VEGF is required to maintain LSEC phenotype both in vitro and in vivo. (A) Representative scanning electron microscopy of LSECs 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, 5 μm. (B) Hepatic expression of VEGF on immunoblot with densitometry and (C) representative scanning electron microscopy of hepatic sinusoids from rats treated with VEGF anti-sense oligonucleotides (ASO) or control oligonucleotides. *P < .05. Scale bar, 2 μm. All figures represent n ≥ 3. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

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 (protein kinase G inhibitor) completely blocks VEGF-stimulated (A) fenestration and (B) porosity in LSECs cultured for 2 days. Scale bar, 5 μm. *P < .001 vs 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 shows that protein S-nitrosylation is not necessary. Scale bar, 5 μm. (D) Porosity measurement of experiments described in panel C. *P < .001 vs VEGF control; n = 3. NS, not significant. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Both VEGF independent of NO plus VEGF-stimulated NO are required to maintain normal LSEC fenestration. LSECs cultured with VEGF + L-NAME (top left panel) or DETA-NONOate (NO donor; top right panel) for 2 days lack fenestration, whereas LSECs cultured with VEGF + L-NAME + DETA-NONOate (bottom left panel) for 2 days show normal fenestration. Porosity is shown in the bottom right panel. Scale bar, 5 μm. *P < .001 vs VEGF control; n = 3. NS, not significant. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 In vivo sGC activation restores LSEC phenotype in thioacetamide (TAA)-induced capillarization. (A) Representative scanning electron microscopy 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, 2 μm. (B) Porosity from rats treated as in panel A. *P <. 001. (C) Cellular cGMP level in LSECs isolated from rats treated as indicated in panel A. *P < .001. n = 3. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

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 increased α-SMA expression after 3 weeks of TAA treatment. One week of solvent or sGC activator after discontinuing TAA does not alter α-SMA expression, but α-SMA expression is reduced significantly when 1 week of sGC activator is followed by 1 week of solvent (group 5) compared with 2 weeks of solvent after TAA (group 4). *P < .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 1 week of sGC activator is followed by 1 week of solvent (bottom right), compared with 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 < .05. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

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 scanning electron microscopy (scale bar, 2 μm) and porosity measurements (n = 4) show that sGC activator treatment completely reverses capillarization despite ongoing TAA (group III) compared with solvent control (group II). *P < .001. (C) Immunoblot (n = 5) and (D) immunohistochemistry show that co-treatment of sGC with TAA from weeks 4 to 6 (group III) reduced α-SMA expression compared with 6-week TAA-treated rats (group II). Four weeks after discontinuation of treatment, α-SMA expression in group V was decreased further compared with 4 weeks earlier (group III) or compared with the TAA solvent control group (group IV). *P < .05. Scale bar, 30 μm. (E) Representative Sirius red staining and (F) scanning morphometry therefore (n = 5) show more fibrosis after 6 weeks TAA (group II) compared with 3 weeks TAA (group I). Extent of fibrosis is unchanged after 6 weeks TAA plus sGC activator from weeks 4 to 6 (group III) compared with 3 weeks TAA (group I). Four weeks after discontinuation of treatment, regression of fibrosis is greater after sGC activator (group V) compared with the solvent group (group IV). Scale bar, 1.2 mm. *P < .001. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Complete reversal of HSC activation by the sGC activator requires the presence of LSECs and is independent of NO. (A) Upper panel: percentage of α-SMA–positive HSCs determined by confocal microscopy is lower in HSCs cultured with LSECs plus BAY60-2770 compared with BAY60-2770 alone (P < .0001, n = 4). Lower panel: representative photomicrographs of HSC. HSCs day 6: HSCs cultured alone for 6 days; HSCs days 0–6, LSECs days 3–6: HSCs cultured alone from days 0 to 3, followed by co-culture from days 3 to 6 with LSECs isolated on day 3 (note: LSECs cultured with activated HSCs for 3 days are capillarized); HSCs days 0–6, sGC activator days 3–6: HSCs cultured alone from days 0 to 6, with BAY 60-2770 from days 3 to 6; HSCs days 0–6, sGC activator + LSECs days 3–6: HSCs cultured alone for 3 days, followed by co-culture from days 3 to 6 with LSECs isolated on day 3 plus BAY 60-2770 (note: LSECs cultured for 3 days with BAY60-2770 remain differentiated) (scale bar: 40 μm; n = 4–6 for each group). (B) NO does not promote HSC quiescence. The percentage of α-SMA–positive HSCs is not significantly different in HSCs cultured with or without DETA-NONOate or in HSCs co-cultured with LSECs and BAY 60-2770 with or without L-NAME. HSCs day 6: HSCs cultured alone for 6 days; HSCs days 0–6, DETA-NONOate days 3-6: HSCs cultured alone from days 0 to 6, with DETA-NONOate added on day 3; HSCs days 0–6, BAY + LSECs days 3–6: HSCs cultured alone for 3 days, followed by co-culture from days 3 to 6 with LSECs isolated on day 3 plus BAY 60-2770; HSCs days 0–6, sGC activator + LSEC + L-NAME days 3–6: HSCs cultured alone for 3 days, followed by co-culture from days 3 to 6 with LSECs isolated on day 3 plus BAY 60-2770 with L-NAME. n = 3–6 for each group. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Dose-ranging Studies of Inhibitors of the VEGF-stimulated cGMP pathway. (A) ODQ Inhibition of sGC activity. Measurement of intracellular cGMP in LSEC cultured with 40ng/ml VEGF or with VEGF plus 1 μM, 5 μM, or 10 μM ODQ overnight (n = 3). *P < .01, **P <.005. (B) Rp-8-pCPT-PET-cGMPS inhibition of PKG activation and signaling. Immunoblot of VASP and Phospho-VASP (Ser239) from LSEC cultured with VEGF overnight, followed by VEGF (left lane) or VEGF plus 10 μM Rp-8-pCPT-PET-cGMPS (right lane) for 1 hour. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Differentiated LSEC do not promote apoptosis of activated HSC in TAA-induced early cirrhosis. (A) Protocol for 5 treatment groups (same as Figure 5A). (B) Representative immunofluorescence staining of α-SMA and desmin in serially-cut frozen liver sections from different groups described in (A). Although there is a decrease in desmin in group 5, the decrease is substantially less than the decrease in α-SMA expression. Scale bar: 40 μm. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 VEGF pathway in TAA-induced early cirrhosis. (A) Protocol for 5 treatment groups (same as Figure 5A and Supplementary Figure 2). (B) Representative immunofluorescence staining of VEGF, VEGF-R2 and eNOS of frozen liver sections from treatment groups described in (A). Scale bar: 40 μm. (C) Immunoblot and (D) densitometry of VEGF in whole liver lysate. *P < .001. (E) Hepatic vein serum VEGF levels (n = 3). *P < .05. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 VEGF pathway in TAA-induced advanced cirrhosis. (A) Protocol for 5 treatment groups (same as Figure 6A). (B) Representative immunofluorescence staining of VEGF, VEGF-R2 and eNOS of frozen liver sections from different groups described in A. Scale bar: 40 μm. (C) Immunoblot and (D) densitometry of VEGF of the whole liver lysate. Immunoblots for Supplementary Figures 3 and 4 were run at the same time and group I is the same as group 1 described in supplementary figure 3. * P < .05, ** P < 0.001. (E) Hepatic vein serum VEGF level (n = 3). * P<.05, ** P < .01. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 α-SMA and desmin staining of hepatic stellate cells in TAA-induced advanced cirrhosis. (A) Protocol for 5 treatment groups (same as Figure 6A and Supplementary Figure 4). (B) Representative immunofluorescence staining of α-SMA and desmin in serially-cut frozen liver sections from treatment groups described in A. Although there is a decrease in desmin in groups III and V, the decrease is substantially less than the decrease in α-SMA expression. Scale bar: 40 μm. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 LSEC maintain differentiation in the presence of sGC activator and activated HSC. Scanning EM micrograph of LSEC. (A) Freshly isolated LSEC defenestrated when co-cultured from day 3-day 6 with activated HSC (HSC cultured alone from day 0–3). (B) Freshly isolated LSEC remained fenestrated when co-cultured from day 3-day 6 with activated HSC plus 5 μg/ml sGC activator. Scale bars: 10 μm. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 Cardiac Microvascular Endothelial Cells do not Prevent HSC activation in vitro. Confocal imaging of α-SMA expression of HSC cultured alone (top left panel), with LSEC (top right panel), or with rat cardiac microvascular endothelial cell (RCMEC, bottom left panel) for 3 days. Quantification of the percentage α-SMA positive HSC shows that LSEC prevent HSC activation, but RCMEC do not. Scale bar: 50 μm, *P<.01 vs HSC day 3 control. Gastroenterology 2012 142, 918-927.e6DOI: (10.1053/j.gastro.2011.12.017) Copyright © 2012 AGA Institute Terms and Conditions