Volume 125, Issue 1, Pages 117-125 (July 2003) Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II  Ramón Bataller, Pau Sancho-bru, Pere Ginès, José M Lora, Amal Al-garawi, Manel Solé, Jordi Colmenero, Josep M Nicolás, Wladimiro Jiménez, Nadine Weich, José-carlos Gutiérrez-ramos, Vicente Arroyo, Juan Rodés  Gastroenterology  Volume 125, Issue 1, Pages 117-125 (July 2003) DOI: 10.1016/S0016-5085(03)00695-4

Figure 1 Gene expression of the components of the renin-angiotensin system in quiescent, culture-activated, and in vivo activated human hepatic stellate cells (HSCs) (A) and normal primary human hepatocytes (B), as assessed by quantitative PCR (TaqMan; see Materials and Methods). Gene expression for angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin type 1 receptor (AT1), and angiotensin type 2 receptor (AT2) was studied. ∗P < 0.01 with respect to quiescent HSCs. (C) Western blot analysis of AT1 and AT2 receptors in culture-activated HSCs. Human kidney was used as a positive control. Figures are representative of 3 independent experiments. Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)

Figure 2 Demonstration of immunoreactivity for mature ANGII in human hepatic stellate cells (HSCs) by immunofluorescence (see Materials and Methods). A positive green staining is observed in both in vivo activated and culture-activated HSCs. Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)

Figure 3 (A) Secretion of angiotensin II (ANGII) by human activated hepatic stellate cells (HSCs). Cells were cultured in the absence (■) or presence (□) of angiotensinogen (10−8 mol/L), and ANGII was measured in the culture media by radioimmunoassay. Cells incubated in the absence of angiotensinogen secreted moderate amounts of ANGII to the culture media. In contrast, incubation with angiotensinogen resulted in a dramatic increase in ANGII secretion. ∗P < 0.05 with respect to 0 minutes; ∗∗P < 0.001 with respect to cells incubated in the absence of angiotensinogen at the same time point (B). Demonstration of renin and angiotensin-converting enzyme (ACE) enzymatic activities in human activated HSCs. Cells were preincubated with buffer, 10−8 mol/L of CG38560 (a specific renin inhibitor), or 10−7 mol/L of lisinopril (an ACE inhibitor) and then supplemented with angiotensinogen (10−8 mol/L) for 6 hours. Media were collected, and immunoreactive ANGII was measured. Both renin and ACE inhibitors prevented the increase of ANGII synthesis induced by angiotensinogen. ∗P < 0.001 with respect to control; ∗∗P < 0.001 with respect to cells incubated with buffer. (C) Demonstration of ANGII degradation by culture-activated HSCs. Exogenous ANGII (10−9 mol/L) was incubated in 6-well plates in the presence or absence of HSCs, and immunoreactive ANGII was measured in the medium. In the presence of HSCs, ANGII levels markedly decreased after 12 hours in culture. Incubation of HSCs with the protease inhibitor bestatin (10−4 mol/L) decreased the rate of ANGII degradation. The figure is representative of 3 independent experiments. Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)

Figure 4 Effect of different growth factors and proinflammatory cytokines on angiotensin II (ANGII) secretion by activated human hepatic stellate cells (HSCs). Cells were stimulated with buffer, platelet-derived growth factor (PDGF; 5 ng/mL), epidermal growth factor (EGF; 5 ng/mL), transforming growth factor-β (TGF-β; 10 ng/mL), tumor necrosis factor-α (TNF-α; 100 ng/mL), and interleukin-1β (IL-1β; 5 U/mL), and immunoreactive ANGII was measured in the collected media. Only PDGF and EGF increased ANGII synthesis, whereas the rest of substances did not induce a significant change in ANGII secretion. ∗P < 0.001 with respect to buffer. Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)

Figure 5 Effect of platelet-derived growth factor (PDGF) on angiotensin II (ANGII) secretion by activated human hepatic stellate cells (HSCs). (A) Cells were cultured in the absence (■) or presence (□) of PDGF (10 ng/mL), and ANGII was measured in the culture media by radioimmunoassay. Incubation of cells with PDGF resulted in a rapid and dramatic increase in ANGII secretion. ∗P < 0.001 with respect to cells incubated in the absence of PDGF at the same time point; ∗∗P < 0.001 with respect to 0 minutes. (B) Cells were incubated in the absence of angiotensinogen with increasing amounts of PDGF. A dose-dependent effect was observed. ∗χ2 = 15.6; P < 0.001. (C) Effect of angiotensin-converting enzyme (ACE) inhibition on PDGF-induced ANGII secretion by HSCs. Cells were stimulated for 6 hours with PDGF (10 ng/mL) in the presence of either buffer or lisinopril (10−7 mol/L), an ACE inhibitor. Lisinopril partially prevented the increase of ANGII induced by HSCs. ∗P < 0.001 with respect to buffer. Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)

Figure 6 (A) Effect of different vasoactive substances on angiotensin II (ANGII) secretion by activated human hepatic stellate cells (HSCs). Cells were stimulated with buffer, thrombin (5 U/mL), arginine-vasopressin (10−8 mol/L), and endothelin-1 (10−8 mol/L) for 6 hours, and immunoreactive ANGII was measured in the collected media. Thrombin and endothelin-1 increased ANGII synthesis (∗P < 0.001). (B) To test whether vasodilators prevent the effect induced by endothelin-1, cells were incubated with a nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP; 0.05 mmol/L), prostaglandin E2 (PGE2; 10−6 mol/L), and adrenomedullin (10−7 mol/L) and then challenged with endothelin-1 (10−8 mol/L). All vasodilators markedly attenuated the effect of endothelin-1 on ANGII synthesis. This effect was mimicked by the cGMP permeant 8-Br-cGMP (10−4 mol/L). Moreover, chelation of intracellular calcium with 2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (5.10−6 mol/L) completely blocked endothelin-induced ANGII secretion (∗∗P < 0.001 with respect to endothelin-1 alone). Gastroenterology 2003 125, 117-125DOI: (10.1016/S0016-5085(03)00695-4)