Effects Of Pressure On Bioactivity Of Hepatic Stellate Cells

Background & objectivePortal hypertension is a common syndrome in hepatic cirrhosis, characterized by hemodynamic disorders in portal vein system with the main clinical complication of lethal massive hemorrhage of gastrointestinal tract. All chronic liver diseases shall evolve to reversible hepatic fibrosis and then cirrhosis. Hepatic stellate cell (HSC) plays an essential role in the pathogenesis of hepatic fibrosis because of its transdifferentiation of quiescent HSC to myofibroblast. Thus it is important to elucidate the mechanisms involved in the activation and proliferation of HSC. However, so far there are few reports about the roles of mechanical factors in the proliferation and activation of HSC.Hepatic stellate cells (HSCs) are residents of perisinusoidal space of Disse with extended long cytoplasmic processes encircling the endothelial barrier. And Disse is connected with sinusoid by the fenestra on sinusoidal endothelial cells. In hepatic fibrosis, sinusoidal pressure can reach ten times to the normal. In view of anatomical location and function, HSC could be subjected to the elevated sinusoidal pressure in the progression of hepatic fibrosis. Then the elevated pressure could contribute to the proliferation, activation and migration of HSC. Exploring the role of pressure in the regulation of HSC behaviors is helpful to further clarify the mechanisms of hepatic fibrosis and to provide an early intervention time in the treatments of portal hypertension.In the present study, by using a pressure-loading apparatus we simulated the pressure on HSC in the pathogenesis of portal hypertension. And the effects of pressure on proliferation, activation and migration of HSC were examined as well. The current study consists of three sections:⑴to isolate, identify and culture HSC;⑵to elucidate the effects of pressure on proliferation, activation and migration of HSC;⑶to study the underlying mechanisms in the pressure-induced proliferation, activation and migration of HSC. Methods一,Isolation, identification and culture of HSCHSCs were purified from normal male Sprague-Dawley rats (400 g-600 g) by sequential digestion of the liver with pronase and collagenase, followed by density gradient centrifugation over 12% Nycodenz. Isolated HSCs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), standard antibiotics, and 2 mM L-glutamine in a 95% air, 5% CO2-humidified atmosphere at 37°C. Growth medium was exchanged every other day. The survival rate of HSC was evaluated by Trypan blue staining. The purity of quiescent and culture-activated HSC was assessed by the blue-green autofluorescence under ultraviolet light (328 nm) and by the immunostaining cells using desmin andα-smooth muscle actin.二,Regulations of pressures in proliferation, activation and migration of HSC㈠Pressure-loading modelThe pressure-loading apparatus consisted of a resealable steel chamber with inlet and outlet ports. Pressure was induced by a release of compressed helium into the chamber through the inlet port, and a sphygmomanometer was connected to the exit port through a tube for the pressure confirmation. The plates and the flasks used for experiments were placed on a warm plate (37°C) inside the chamber.㈡Proliferation and activation evaluationQuiescent and activated HSCs were seeded on 6 cm dish at a density of 1×106 cells/ml, followed by the synchronization in DMEM free from FBS. After a culture under diverse pressures (0mmHg, 5mmHg, 10mmHg, 20mmHg, 40mmHg, 80mmHg) for predetermined times (1h, 12h, 24h) followed by an additional incubation in the incubator, proliferation rate of HSC was examined by cell counting kit-8 (CCK-8). Base on these results, HSC and optima intervention times used to further experiments were confirmed. In this study, after one-hour pressurization, total RNA was drawn from HSC, and used to examine the mRNA expressions of typeⅠcollagen andα-SMA by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, after 24-hour incubation in the incubator, total protein was extracted from HSC and used to detect the protein expressions of typeⅠc ollagen,α-SMA and PCNA by Western blot analysis.㈢Cell cycle studyHSCs were seeded on 6 cm dish at a density of 1×106 cells/ml, followed by the synchronization in DMEM free from FBS. After a culture under diverse pressures for one hour followed by 24-hour incubation in the incubator, cells were collected by trypsinization followed by PI staining. Cell cycle was determined by using flow cytometry.㈣Migration analysisHSCs were seed on 6 cm dish at a density of 1×106 cells/ml. After the synchronization in DMEM free from FBS for 24 hours, a wound was created by the tip of pipette. After a culture under diverse pressures for one hour followed by 24-hour incubation in the incubator, the migration rate was calculated by the formula: (vertical dimension of wound before pressurization-vertical dimension of wound after pressurization)/ vertical dimension of wound before pressurization×100%.For migration assays on transwell inserts, HSCs were cultured initially in DMEM with 10 % FBS. The seeding density was 5,000 cells/ insert, with 200μl of medium in the insert and 500μl in the well of 24-well plate. After the synchronization in DMEM free from FBS for 24 hours, HSCs were cultured under diverse pressures for one hour. Then DMEM containing 20% FBS was used as chemotactic factor, which was taken as the zero time point of migration. After a migration period of 24 h, inserts were fixed and stained. Ten fields (×200) with individual cells only (clusters were ignored) were randomly picked per insert. The migration index was defined as the percentage of cells in a field on the bottom side of the filter: (number of cells on the bottom)/ (number of cells on the top and bottom)×100.三,The mechanisms in pressure-induced proliferation, activation and migration of HSCHSC underwent one-hour pressurization at 10 mmHg. Specific inhibitors of Herbimycin A (HA, 900nM), PD98059 (25μM) and LY294002 (25μM) were used to inhibit the phosphorylation of Src (Tyr418), Erk1/2(Thr185/Tyr187) and Akt (Ser473) respectively. The expressions at mRNA level ofβ3-integrin,FAK,ILK,ETA,PDGF-B receptor,TGFβ1 and TGFβ1 receptor were examined by RT-PCR. Western blot analysis was used to detect the phosphorylation of Src(Tyr418), FAK(Tyr397), FAK(Tyr576/577), Akt (Ser473), Erk1/2 (Thr185/ Tyr187), p70S6k(Thr421/Ser424) and protein expressions of FAK , p70S6k, TypeⅠcollagen,α-SMA and PCNA. Migration of HSC was assessed by transwell insert analysis.四,Statistical analysisAll data were expressed as the means±S.E of at least three independent experiments. The significance of changes was evaluated by One-Way ANOVA. When the variance is equal, LSD will be used. Or not, Dunnett's will be used. A P value of≤0.05 was considered statistically significant.Results一,Identification of HSCMore than about 95% fresh HSC survived evaluated by trypan blue staining. By observing autofluorescence and using immunofluorescence analysis of Desmin andα-SMA, the purity of HSC was confirmed nearly above 95%.二,Effects of pressure on the proliferation of HSCPressurization at 10 mmHg for one hour notably promoted the proliferation of quiescent (1 day) and activated (14 days) HSC (P<0.01). A pressure of 20 mmHg for one hour promoted the proliferation of activated HSC (P<0.05). One- or twelve-hour pressurization at 40 mmHg suppressed the proliferation of activated HSC (P<0.05). A pressure of 80 mmHg for 12 hours markedly inhibited the proliferation of quiescent HSC (P<0.01). Twenty-four pressurization at 40 or 80 mmHg significantly inhibited the proliferation of quiescent and activated HSC (P<0.01).Base on these results, one-hour pressurization was used in following experiments on activated HSC.三,Effects of pressure on cell cycle of HSC One-hour pressurization at 10 mmHg and 20 mmHg significantly increased HSC in S (P<0.01). However, 80 mmHg markedly reduced cells in S (P<0.01). Pressures had little effects on cells in G0-G1 and G2-M.四,Effects of pressure on the activation of HSCPressurization at 10 mmHg for one hour significantly promoted mRNA and protein expressions of a-SMA and TypeⅠcollagen (P<0.01). One-hour pressurization at 40 and 80 mmHg markedly declined the protein expressions ofα-SMA, TypeⅠcollagen and PCNA (P<0.01).五,Effects of pressure on migration of HSCBy using injury induction analysis, HSC migrating to the wound area increased compared with 0 mmHg after one-hour pressurization at 10, 20 and 40 mmHg. Meanwhile the vertical dimension of wound was markedly shortened (P<0.01). Compared with other pressures, the decrease in vertical dimension of wound in 10 mmHg was most notable (P<0.01). However, there was little effect of 5 mmHg and 80 mmHg on the vertical dimension of wound.By transwell insert analysis, the migration index after one-hour pressurization at 10 mmHg was the highest than that in other pressures (P<0.01). And under 80 mmHg for one hour, HSC on the bottom of insert was least (P<0.05).六,Effects of pressure on mRNA expression of molecules involved in mechanotransductionPressurization at 10 mmHg for one hour markedly increased mRNA expressions ofβ3-integrin, FAK and TGFβ1 (P<0.01, each). Pressures at 40 mmHg and 80 mmHg significantly decreasedβ3-integrin mRNA expression (P<0.01, each). There was little effect of pressurization for one hour on mRNA expressions of ETA, ILK, PDGF-B receptor and TGFβ1 receptor.七,Effects of pressure on time intervals of the phosphorylation of Src and FAK in HSCAfter one-hour pressurization at 10 mmHg, the phosphorylation of Src(Tyr418) and FAK(Tyr397) detected at 0 min, 2 min, 5 min, 10 min, 20 min, 30 min, 60 min. The chronological changes in phosphorylation of Src(Tyr418) and FAK(Tyr397) were synchronous: increased at 2 min (P<0.01), peaked at 10 min (P<0.01), lasted at 20 min (P<0.01), decreased gradually from 30 min to 60 min. Base on these results, the following detection in phopho-proteins at 10 min after the end of interventions.八,Effects of pressure on protein expressions involved in pressure- induced signaling pathway in HSCIn one-hour pressurization experiment: 5 mmHg had little effects on the expressions of phospho-proteins; both 10 mmHg and 20 mmHg increased phospho-protein expressions of Src (Tyr418), FAK (Tyr397), Erk1/2 (Thr185/Tyr187), Akt (Ser473) and p70S6k (Thr421/ Ser424) (P<0.01, each), while 40 mmHg and 80 mmHg inhibited these expressions; 40 mmHg enhanced the protein expression of FAK (Try576/577) (P<0.01). In 24-hour pressurization experiment: the phosphorylation of Src (Tyr418), FAK(Tyr397), Akt(Ser473), p70S6k(Thr421/Ser424) and FAK (Try576/577) could not be detected but Erk1/2(Thr185/Tyr187); not 10 mmHg but 20, 40, 80 mmHg reinforced the phosphorylation of Erk1/2(Thr185/Tyr187) (P<0.05 or 0.01), which peaked at 80 mmHg (P<0.01).九,Effects of inhibitors on the protein expressions involved in pressure-induced signaling pathway in HSCPressurization at 10 mmHg for one hour significantly elevated the protein expressions of FAK (Tyr397), Akt (Ser473), Erk1/2(Thr185/Tyr187) and p70S6k (Thr421/Ser424) (P<0.01, each). Herbimycin A attenuated phospholation of FAK (Tyr397), Akt (Ser473), Erk1/2(Thr185/Tyr187) and p70S6k (Thr421/Ser424) (P<0.01, each). LY294002 suppressed phospholation of Akt (Ser473) and p70S6k (Thr421/Ser424) not Erk1/2(Thr185/Tyr187) (P<0.01, each). PD98059 inhibited phospholation of Erk1/2(Thr185/Tyr187) not FAK (Tyr397), Akt(Ser473) and p70S6k(Thr421/Ser424) (P<0.01, each).十,Effects of inhibitors on the protein expressions ofα-SMA, TypeⅠcollagen and PCNA in HSCOne-hour pressurization at 10 mmHg markedly increased the protein expressions ofα-SMA, TypeⅠcollagen and PCNA (P<0.01, each). PD98059 had little effects on the elevated protein expressions ofα-SMA, TypeⅠcollagen and PCNA induced by 10 mmHg. Both LY294002 and Herbimycin A notably inhibited the increased protein expressions ofα-SMA, Type I collagen and PCNA induced by 10 mmHg (P<0.01, each). In addition, the inhibitory effect of LY294002 on TypeⅠcollagen protein expression was more notable than that in Herbimycin A (P<0.01).十一,Effects of inhibitors on migration of HSCOne-hour pressurization at 10 mmHg markedly promoted migration of HSC, which was little affected by PD98059. Both LY294002 and Herbimycin A notably inhibited the migration reinforced by 10 mmHg (P<0.01).Conclusions一,The response of HSC to extracellular pressure is dependent on the level and time of the pressurization. One-hour pressurization at 10 mmHg significantly promotes the proliferation, increases the mRNA and protein expressions ofα-SMA, TypeⅠcollagen and PCNA, and elevates the migration of activated HSC, which indicates pathologically elevated sinusoidal pressure contributes to the development of hepatic fibrosis and portal hypertension.二,Integrin-dependent autophosphorylation of FAK is involved into the stimulative effects of one-hour pressurization at 10 mmHg on HSC proliferation, activation and migration, whose upstream mediator is Src and downstream mediators are Akt-p70S6k not Erk1/2 MAPK.