Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence

Abstract

Capillarization precedes hepatic fibrosis. We hypothesize that capillarization of sinusoidal endothelial cells (SEC) is permissive for hepatic stellate cell (HSC) activation and therefore permissive for fibrosis. We examined whether freshly isolated SECs prevent activation of HSCs and promote reversion to quiescence, and whether this effect was lost in capillarization. HSCs were cultured alone or co-cultured with differentiated or capillarized SECs.

Results: Co-culture with freshly isolated SECs markedly decreased HSC activation after 3 days in culture, but co-culture with capillarized SEC had no effect. Inhibition of nitric oxide (NO) synthesis abolished SEC suppression of HSC activation. Activated HSCs reverted to quiescence when co-cultured with SEC plus vascular endothelial growth factor (VEGF) (that is, with SECs that maintained differentiation), but co-culture with capillarized SECs did not. Reversion of activated HSCs to quiescence in the presence of SECs plus VEGF was abolished by inhibition of NO synthesis. To establish whether there was indeed reversion, activated and quiescent HSCs were counted before and 3 days after adding freshly isolated SECs plus VEGF to activated HSCs, and proliferation was quantified in quiescent HSCs; the stoichiometry demonstrated reversion.

Conclusion: Differentiated SECs prevent HSC activation and promote reversion of activated HSCs to quiescence through VEGF-stimulated NO production. Capillarized SECs do not promote HSC quiescence, because of loss of VEGF-stimulated NO production.

Fig. 1

Alpha-Smooth muscle actin (ASMA) as a marker of HSC activation. This experiment examines whether differentiated or capillarized SECs can prevent stellate cell activation in vitro. ASMA expression by HSCs was examined by confocal microscopy after 3 days. In the left panel, HSCs were cultured alone (open bar) from day 0 (the day of isolation) until day 3, co-cultured from day 0 to day 3 with freshly isolated SECs (black bar), or with SECs allowed to capillarize in vitro over 3 days, which were then added to HSC for 3 days (diagonal hatched bar). In the right panel, from day 0 to day 3, HSCs were cultured alone (open bar) or with capillarized SECs freshly isolated from rats with thioacetamide-induced cirrhosis (vertical hatched bar) (n = 3-8).

Fig. 2

Confocal imaging of ASMA expression of HSCs cultured alone or with SECs for 3 days. The panel on the left shows HSCs cultured alone for 3 days. The panel in the middle shows HSCs cultured with SECs for 3 days. The panel on the right shows a control of HSCs cultured alone for 3 days stained with a nonspecific isotype-controlled primary antibody.

Fig. 3

Scanning electron microscopy of SECs. (A) Sinusoidal endothelial cells cultured for 1 day after isolation from normal liver demonstrate fenestrae in sieve plates. (B) Sinusoidal endothelial cells cultured alone for 3 days lack fenestrae. (C) Sinusoidal endothelial cells cultured for 1 day after isolation from thioacetamide-induced cirrhotic liver lack fenestrae.

Fig. 4

Immunoblot of HSC expression of ASMA and TIMP-1. This experiment examines whether differentiated or capillarized SECs can prevent stellate cell activation in vitro. Three conditions are examined: HSCs cultured alone, HSCs co-cultured with capillarized SECs, and HSCs co-cultured with differentiated SECs. In each condition, equal numbers of HSCs are plated. (A) Immunoblot for ASMA and TIMP-1. Lane 1: HSCs cultured alone from day 0 (day of HSC isolation) to day 3. Lane 2: HSCs cultured from day 0 to day 3 with SECs isolated from thioacetamide-treated cirrhotic rats, that is, SECs that were capillarized. Lane 3: HSCs cultured from day 0 to day 3 with SECs isolated from control liver, that is, differentiated SECs. Lanes 1 and 2 examine whether differentiated or capillarized SECs, respectively, can prevent HSC expression of activation markers on day 3. (B) Densitometry of ASMA corrected for β-actin loading (n = 3). P < 0.0001 by ANOVA, P < 0.001 by least significant difference for HSCs co-cultured with normal SECs compared with HSCs alone (n = 3) (C) Densitometry of TIMP-1, corrected for β-actin loading. P < 0.0001 by ANOVA, P < 0.001 by least significant difference for HSCs co-cultured with normal SECs compared with HSCs alone (n = 3).

Fig. 5

Fluorescence of F-actin stress as a marker of HSC activation. The panel on the left shows fluorescence per cell in HSCs cultured alone or with either capillarized SECs or freshly isolated SECs after 3 days in culture. The two panels on the right are photomicrographs of the F-actin stress fibers in HSCs co-cultured for 3 days with either capillarized or freshly isolated SECs (n = 3).

Fig. 6

Regulation of HSC phenotype by nitric oxide. Left panel: The addition of 3 mM L-NAME blocked the paracrine ability of SECs to suppress HSC activation; from left to right: HSC day 3: HSCs cultured alone for 3 days; HSC/L-NAME: HSCs cultured with L-NAME for 3 days; HSC and SEC day 3: HSCs and SECs co-cultured for 3 days; HSC and SEC day 3/L-NAME: HSCs and SECs co-cultured for 3 days with the addition of L-NAME. Right panel: hepatocytes alone do not prevent HSC activation, but when 100 μM V-PYRRO/NO is added, hepatocytes produce NO and mimic the ability of SEC to prevent HSC activation; from left to right: HSC day 3: HSCs cultured alone for 3 days; HSC and Hep day 3: HSC and hepatocytes co-cultured for 3 days; HSC day 3/V-PYRRO-NO: HSCs cultured alone for 3 days with V-PYRRO-NO; HSC and Hep day 3/V-PYRRO-NO: HSCs and hepatocytes co-cultured for 3 days with V-PYRRO-NO (n = 6).

Fig. 7

Reversal of HSC phenotype. In this experiment, HSCs are allowed to activate over 3 days (days 0-3) by culturing alone on plastic. From day 3 to day 6, HSCs are cultured either: alone, with SEC (which capillarize over the course of the next 3 days), with VEGF, or with VEGF plus SEC (that remain differentiated because of the presence of VEGF. (A) Immunoblot for ASMA and type I collagen. Lane 1 examines HSCs cultured alone for 6 days. Lanes 2-4 examine whether HSCs that are activated by day 3 can revert to quiescence by day 6. Lane 2, HSCs cultured alone from day 0 (the day of HSC isolation) to day 3 followed by co-culture from day 3 to day 6 with SECs isolated on day 3 (note: SECs will capillarize when cultured for 3 days with activated HSCs). Lane 3, HSCs cultured alone from day 0 to day 3, followed by the addition of VEGF from day 3 to day 6. Lane 4, HSCs cultured alone from day 0 to day 3, then from day 3 to day 6 with the addition of VEGF plus co-culture with SECs isolated on day 3 (note: SECs remain differentiated when cultured for 3 days with VEGF). (B) Densitometry of ASMA corrected for β-actin loading. P < 0.0001 by ANOVA, P < 0.001 by least significant difference for HSC co-cultured with SECs plus VEGF compared with HSCs alone (n = 3). (C) Densitometry of type I collagen, corrected for β-actin loading; P < 0.005 for ANOVA, P < 0.005 by least significant difference for HSCs co-cultured with SECs plus VEGF compared with HSCs alone (n = 3).

Fig. 8

SECs with VEGF induces reversal of stellate cell phenotype. The top panel shows the percentage of HSCs that express ASMA determined on confocal microscopy, and the lower panels demonstrate representative photomicrographs of HSCs. From left to right in the upper and lower panels: HSC day 6: HSCs cultured alone for 6 days; HSC day 0-6, SEC day 3-6: HSCs cultured alone from day 0 to day 3, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 (note: SECs cultured with activated HSCs for 3 days will capillarize); HSC day 0-6, VEGF day 3-6: HSCs cultured alone from day 0 to day 6, with the addition of VEGF to the medium from day 3 to day 6; HSC day 0-6, SEC + VEGF day 3-6: HSCs cultured alone for 3 days, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 plus VEGF (note: SECs cultured for 3 days with VEGF remain differentiated) (n = 4-6).

Fig. 9

Reversal of activated HSC to quiescence as demonstrated by loss of F-actin stress fiber fluorescence. Quantitation of F-actin fluorescence per cell demonstrates a marked drop in fluorescence when HSCs are in co-culture with SECs plus VEGF compared with the other conditions. From left to right: HSC day 6: HSCs cultured alone for 6 days; HSC day 0-6, SEC day 3-6: HSCs cultured alone from day 0 to day 3, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 (note: SECs cultured with activated HSCs for 3 days will capillarize); HSC day 0-6, VEGF day 3-6: HSCs cultured alone from day 0 to day 6, with the addition of VEGF to the medium from day 3 to day 6; HSC day 0-6, SEC & VEGF day 3-6: HSCs cultured alone for 3 days, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 plus VEGF (note: SECs cultured for 3 days with VEGF remain differentiated; n = 3).

Fig. 10

Effect of L-NAME on ASMA-positive HSCs in the presence of SECs and VEGF. From left to right: HSC day 6: HSCs cultured alone for 6 days; HSC day 0-6, SEC day 3-6: HSCs cultured alone from day 0 to day 3, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 (note: SECs cultured with activated HSCs for 3 days will capillarize); HSC day 0-6, SEC and VEGF day 3-6: HSCs cultured alone for 3 days, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 plus VEGF (note: SECs cultured for 3 days with VEGF remain differentiated); HSC day 0-6, SEC, VEGF, and L-NAME day 3-6: HSCs cultured alone for 3 days, followed by co-culture from day 3 to day 6 with SECs isolated on day 3 plus VEGF. The addition of L-NAME to the co-culture with SECs and VEGF blocks nitric oxide synthase and prevents the decrease in ASMA-positive HSCs (n = 6).