Study On The Role And Mechanisms Of Hepatic Stellate Cells Migration And RhoA Signaling Transduction Pathway In Liver Fibrosis

Part One Effect of cytoskeletal reorganization and cell migration in hepatic stellate cells induced by collagens and growth factorsBackground and Aims: Hepatic stellate cells(HSCs) are increasingly being recognized as the key mediators of progressive liver disease. In liver fibrosis, HSCs undergo a process of activation, characterized by increased proliferation, motility, cytokine release and synthesis of extracellular matrix components, which altered the space of Disse microenvironment. The activation of HSCs and cells migration comprise cell transmogrification, fibrogenesis, contractility and cytokine release through extracellular signaling transduction pathways, which all involve actin cytoskeleton reorganization. The aim of this study was to evaluate the effects of collagens and these two cytokine on cytoskeletal components and migration in cultured rat HSCs.Methods: Primary rat hepatic stellate cells were isolated and cultured. A improved Transwell Chamber system was used to observe the changes of serum starved HSCs haptotactic migration (direct stimulation) and chemotactic migration (indirect stimulation) after different concentration of collagen type I , collagen type IV, TGFβ₁, PDGF-BB treatment. Changes in actin cytoskeletal organization were visualized by fluorescence staining and fluorescence images were recorded using confocal microscopy.Results: 1. Both PDGF-BB and TGFβ₁ treatment of hepatic stellate cells resulted in the remarkable enhancement of migration in response to haptotactic and chemotactic stimuli compared with control group (P<0.01). Chemotactic stimuli with collagen type I significantly increased HSCsmigration when its concentration exceed 50μg/ml ( P<0.05 ) , while haptotactic stimuli with 100μg/ml collagen type I marked increased HSCs migration (P<0.05) . Collagen type IV had no effect on HSCs migration.2. Serum-starved, untreated cells had a rounded-up morphology. Stimulation of hepatic stellate cells with PDGF-BB and TGFβ₁ induced a rapid morphological change concomitant with a robust reorganization of actin cytoskeleton. 5 minutes after 5ng/ml TGFβ₁ stimulation, the cells flattened out and the formation of filopodia occurred, after 15-30 minutes, the cells became well spread with fully developed stress fibers and focal adhesion. 5 minutes after 10ng/ml PDGF-BB stimulation, the formation of lamellipodia occurred, 15 minutes later, stress fibers and focal adhesion assembled with little filopodia, after 30minutes, the cells had fully developed stress fibers and focal adhesion.Conclusion: Collagen type I , PDGF-BB and TGF β₁ promote HSCs migration while collagen type IV has no such effect. The stimulation of PDGF-BB and TGFβ₁ induce the actin cytoskeleton reorganization. These effects may play a crucial role in liver fibrosis.Part Two Effects of Rho GTPase expression in hepatic stellate cells in response to PDGF-BB and TGFP,Background and Aims: The Rho family of small GTPases, including the Rho, Rac, and Cdc42 subfamilies, are key regulators of the actin cytoskeleton. During a variety of cellular events, including polarization, shape reorganization, and migration, Rho GTPases serve to transducer signals between extracellular ligands and cytoskeleton reorganization. We have validated that PDGF-BB and TGF β₁ could induce a reorganization of the actin cytoskeletal network and migration in HSCs, therefore aims of the present study were to investigate the expression of Rho GTPases and potential intracellular signal pathways during the processes.Methods: After different concentration of PDGF-BB, TGFβ₁ treatment.mRNA level of RhoA, Rac1, Cdc42 in HSCs was evaluated by Real time-PCR, the protein level of those was by Western blot, and GTP-loaded protein level was by GST-pull down assay. Results:1. Compare with control group, the mRNA level of RhoA is significant up-regulation in HSCs stimulated by TGF β₁ and dose dependent, while the mRNA level of Rac1 and Cdc42 do not change. Different concentrations of PDGF-BB do not affect the mRNA level of RhoA, Racl and Cdc42.2. Each concentration of PDGF-BB, TGF β₁ do not affect the whole protein level of RhoA, Racl and Cdc42 in HSCs.3. Compare with control group, TGF β₁ stimulation do not alter the amount of GTP-bound Rac1. In contrast, Cdc42 and RhoA GTPase activity is significantly augmented. Different concentrations of PDGF-BB increase the GTP-loaded protein level of RhoA, Rac1 and Cdc42 in HSCs and the peak values of these three protein expression are achieved under 10ng/ml concentration.Conclusions: TGF β₁ induced cytoskeletal reorganization in HSC is dependent on Cdc42 and RhoA, not Rac1, while PDGF-BB is dependent on Cdc42, RhoA and Racl, thus suggesting that Rho GTPases pathway may be the cytobiological basis of activation and migration of HSC.Part Three Role of RhoA in HSC migration and cytoskeleton reorganizationAims: To investigate the role of RhoA in hepatic stellate cell migration and actin cytoskeleton changes induced by PDGF-BB and TGF β₁ through observing the effect of constitutively active and dominant negative RhoA.Methods: HSC were transfected with vectors containing wide type (wt)RhoA, constitutively active(Q63L)or dominant negative RhoA(T19N) by cationic lipofectamine 2000. RhoA was detected by western blot. A improvedTranswell Chamber system was used to observe the changes of transfected HSC haptotactic migration and chemotactic migration after collagen type I , TGF β₁ , PDGF-BB treatment. Changes in actin cytoskeletal organization were visualized by fluorescence staining and fluorescence images were recorded using confocal microscopy. Results:1. Compare with HSC, RhoA protein level were significantly increased in wt-RhoA and Q63L-RhoA cells, while remarkable decreased in T19N-RhoA cells.2. Cells transfected with wide-type RhoA formed membrane ruffles and stress fibers at a normal ratio; Cells transfected with constitutively active RhoA stimulated actin cable assembly to form the enhancement of stress fibers and focal adhesion after inducement by PDGF-BB, TGF β₁; In contrast, dominant negative RhoA inhibited PDGF-BB, TGF β₁ induced stress fibers and focal adhesion formation.3. Compare with control group, both constitutively active RhoA and dominant negative RhoA inhibited cells migration induced by collagen type I, PDGF-BB, TGFβ₁.Conclusions: RhoA regulate the formation of actin filament-based structures induced by PDGF-BB, TGF β₁ in HSC. Collagens and growth factors induced HSC migrations occur via RhoA GTPase-dependent pathway.