The Effects Of High Mobility Group Box-1 Protein On Cardiac Fibroblasts And Its Potential Mechanism

PREFACECardiac remodeling is a comment process of a variety of heart diseases, such as ischemic heart disease and hypertension, and is a vital process of development to heart failure. Cardiac remodeling includes cardiomyocyte hypertrophy, interstitial fibrosis and electrical remodeling.Cardiac fibroblasts, which account for 60-70% of the cells in the heart, are a key source of components of the extracellular matrix (ECM) that maintains structural and functional integrity of the heart [1]. Cardiac fibroblasts responded to injurious stimuli in multiple ways, including proliferation, migration, transformation to myofibroblast, secretion of cytokines, and dysregulation of MMP and TIMP [2], which initially was favor for myocardium recover but could lead to pathological fibrosis [3].MMPs is a family of zinc-dependent proteases that degrade various ECM proteins, including collagens, gelatins, fibronectin, and laminins [4]. MMP-9 (gelatinase B) expression by cardiac fibroblasts is very low under normal conditions, but is markedly increased in response to proinflammatory cytokines, and it mainly degrades interstitial collagens IV in the myocardium [2]. Inflammatory cytokines initially induce fibroblasts accumulation to repair damages. However, as cytokines persist, they stimulate fibrosis following repair, leading to cardiac remodeling [5].Various cytokines have been shown to regulate MMP-9 expression such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β induced dysregulation of MMPs and TIMPs, leading to disturbance of ECM [6,7]. The role of inflammatory factors in cardiovascular disease has attracted more and more attention. It has been found, a variety of inflammatory cytokines involved in myocardial remodeling process, but they played different roles in cardiac remodeling.High mobility group box 1 (HMGB1), a recently discovered proinflammatory cytokine, expressed in the nucleus of eukaryotic cells, is identified as a DNA-binding protein that functions as a structural co-factor critical for proper transcriptional regulation in eukaryotic cells. HMGB1 is initiatively secreted by activated immune cells or passively released by necrotic cells [8]. Extracellular HMGB1 has been reported involved in various models such as inflammation, atherosclerosis, myocardial ischemia/reperfusion injury and heart failure [9].Andrassy et al [10] demonstrated that treatment of mice with recombinant HMGB1 worsened ischemia-reperfusion (I/R) injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. Furthermore, it had been reported that HMGB1 induced cardiomyocyte hypertrophy and down-regulated cardiac transient outward potassium current (Ito), which participated in cardiac electrical remodeling [11,12].Furthermore, in contrast to its proinflammatory functions, there is evidence that HMGB1 also has restorative effects leading to tissue repair and regeneration [8].However, the role of HMGBlin cardiac remodeling has not been thoroughly described. Since cardiac fibroblasts play a critical role in cardiac remodeling. The aims of this study are to determine the role of HMGB1 on cardiac fibroblasts and to explore the underlying mechanisms, which may be a potential therapeutic target for heart diseases.Part One Effects of High mobility group box 1 protein on cardiac fibroblasts proliferation, migration and differentiation to myofibroblastsObjectives The aims of this study were to determine the role of high mobility group boxl protein (HMGB1) in cardiac fibroblasts proliferation, migration and differentiation to myofibroblasts.Methods Primary cardiac fibroblasts were obtained from the ventricles of 1-2-day-old neonatal Sprague-Dawley rats. Cardiac fibroblasts were incubated for 24 hours with different concentration of recombinant HMGB1 (0,10,100, 1000ng/ml). Cell vitality was measured by cell counting kit (CCK-8/WST-8). Cell proliferation was measured by Cell-IQ. Cell migration was measured both by cell scratch assay and transwell chambers. Western blot analysis performed to demine the expression of receptor for alpha-smooth muscle actin (a-SMA), which was the marker of cardiac fibroblasts differentiation to myofibroblasts.Results CCK-8 assay revealed HMGB1 did not cause any cytotoxic effects at any concentration (0,10,100, 1000ng/ml). Cell-IQ revealed that HMGB1 (0,10,100, 1000ng/ml) treatment for 24 hours had no effect on cardiac fibroblasts proliferation. Both cell scratch assay and transwell assay demonstrated that 10ng/ml HMGB1 significantly stimulated fibroblasts migration (P<0.05) but the higher dose of 100ng/ml and 1000ng/ml failed to enhance cell migration. Western blot analysis revealed no differences in a-SMA expression between control and HMGB1-treated cardiac fibroblasts, which indicated that HMGB1 had no effect on cardiac fibroblasts differentiation to myofibroblasts.Conclusions Different concentration of HMGB1 (0,10,100, 1000ng/ml) treatment for 24 hours had no effect on cardiac fibroblasts proliferation, differentiation to myofibroblasts, and it did not cause any cytotoxic effects. However, 10ng/ml HMGB1 significantly stimulated fibroblasts migration, but the higher dose of 100ng/ml and 1000ng/ml failed to enhance cell migration.Methods Primary cardiac fibroblasts were obtained from the ventricles of 1-2-day-old neonatal Sprague-Dawley rats. Cardiac fibroblasts were incubated for 24 hours with different concentration of recombinant HMGB1 (0,10,100, 1000ng/ml). Cell vitality was measured by cell counting kit (CCK-8/WST-8). Cell proliferation was measured by Cell-IQ. Cell migration was measured both by cell scratch assay and transwell chambers. Western blot analysis performed to demine the expression of receptor for alpha-smooth muscle actin (a-SMA), which was the marker of cardiac fibroblasts differentiation to myofibroblasts.Results CCK-8 assay revealed HMGB1 did not cause any cytotoxic effects at any concentration (0,10,100, 1000ng/ml). Cell-IQ revealed that HMGB1 (0,10,100, 1000ng/ml) treatment for 24 hours had no effect on cardiac fibroblasts proliferation. Both cell scratch assay and transwell assay demonstrated that 10ng/ml HMGB1 significantly stimulated fibroblasts migration (P<0.05) but the higher dose of 100ng/ml and 1000ng/ml failed to enhance cell migration. Western blot analysis revealed no differences in a-SMA expression between control and HMGB1-treated cardiac fibroblasts, which indicated that HMGB1 had no effect on cardiac fibroblasts differentiation to myofibroblasts.Conclusions Different concentration of HMGB1 (0,10,100, 1000ng/ml) treatment for 24 hours had no effect on cardiac fibroblasts proliferation, differentiation to myofibroblasts, and it did not cause any cytotoxic effects. However, 10ng/ml HMGB1 significantly stimulated fibroblasts migration, but the higher dose of 100ng/ml and 1000ng/ml failed to enhance cell migration.Part Two Effects of High mobility group box 1 protein on MMP-9 and TIMP-1 expression in cardiac fibroblastsObjectives The aims of this study were to determine the role of high mobility group boxl protein (HMGB1) in cardiac fibroblasts expression of matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1).Methods Primary cardiac fibroblasts were obtained from the ventricles of 1-2-day-old neonatal Sprague-Dawley rats. Cardiac fibroblasts were incubated for 24 hours with different concentration of recombinant HMGB1 (0,10,100, 1000ng/ml). Isolating protein in the cell, western blot analysis performed to demine the level of intracellular matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1). Isolating protein in the medium by Millipore filter devices, western blot analysis performed to demine the level of matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1) in supernatant.Results Cardiac fibroblasts were incubated with various concentration (0,10,100, 1000ng/ml) of HMGB1 for 24 hours. 100ng/ml HMGB1 could significantly raised the level of MMP-9 in the supernatant (P<0.05), while the higher dose of 1000ng/ml failed to enhance MMP-9 expression. Both 10 and 100ng/ml HMGB1 increased the level of TIMP-1 in the supernatant (P<0.05). However, there were no differences both in MMP-9 and TIMP-1 between untreated and HMGB1-treated cardiac fibroblasts.Conclusions These results demonstrated that HMGB1 could not modulate intracellular MMP-9 and TIMP-1, but up-regulate extracellular MMP-9 and TIMP-1 to induce the dysregulation of MMPs and TIMPs. Furthermore, in all subsequent treatment experiments, HMGB1 was used at the concentration of 100ng/ml. hours with different concentration of recombinant HMGB1 (0,10,100, 1000ng/ml). Isolating protein in the cell, western blot analysis performed to demine the level of intracellular matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1). Isolating protein in the medium by Millipore filter devices, western blot analysis performed to demine the level of matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1) in supernatant.Results Cardiac fibroblasts were incubated with various concentration (0,10,100, 1000ng/ml) of HMGB1 for 24 hours. 100ng/ml HMGB1 could significantly raised the level of MMP-9 in the supernatant (P<0.05), while the higher dose of 1000ng/ml failed to enhance MMP-9 expression. Both 10 and 100ng/ml HMGB1 increased the level of TIMP-1 in the supernatant (P<0.05). However, there were no differences both in MMP-9 and TIMP-1 between untreated and HMGB1-treated cardiac fibroblasts.Conclusions These results demonstrated that HMGB1 could not modulate intracellular MMP-9 and TIMP-1, but up-regulate extracellular MMP-9 and TIMP-1 to induce the dysregulation of MMPs and TIMPs. Furthermore, in all subsequent treatment experiments, HMGB1 was used at the concentration of 100ng/ml.Part Three Effects of high mobility group box 1 protein on its receptors and MAPKs pathwaysObjectives The aims of this study was to explore the underlying receptor and signaling pathways involved in the effects of high mobility group box 1 protein (HMGB1) on cardiac fibroblasts.Methods Primary cardiac fibroblasts were obtained from the ventricles of 1-2-day-old neonatal Sprague-Dawley rats. Cardiac fibroblasts were incubated for 24 hours with recombinant HMGB1 (100ng/ml). Isolating protein in the cell, western blot analysis performed to demine the expression of receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). Meanwhile, cardiac fibroblasts were incubated with HMGB1 (100ng/ml) for the indicated time (0,5,10,30minutes), western blot analysis performed to demine the level of phosphorylation levels of MAPKs (p-ERKl/2, p-JNK, p-P38) expression.Results Cardiac fibroblasts were incubated with HMGB1 (100ng/ml) for 24 hours, TLR4 expression was significantly higher than control group (P<0.05). However, HMGB1 (100ng/ml) could not increase the RAGE expression. Cardiac fibroblasts were incubated with HMGB1 (100ng/ml) for the indicated time (0,5,10,30minutes). HMGB1 significantly provoked ERK1/2 phosphorylation after 5 minutes (P<0.05). However, HMGB1 failed to significantly induce JNK and P38 activation.Conclusions HMGB1 (100ng/ml) up-regulated the expression of TLR4, and specially activated ERK1/2, indicated that HMGB1 provoke ERK1/2 phosphorylation through TLR4. MAPKs (p-ERKl/2, p-JNK, p-P38) expression.Results Cardiac fibroblasts were incubated with HMGB1 (100ng/ml) for 24 hours, TLR4 expression was significantly higher than control group (P<0.05). However, HMGB1 (100ng/ml) could not increase the RAGE expression. Cardiac fibroblasts were incubated with HMGB1 (100ng/ml) for the indicated time (0,5,10,30minutes). HMGB1 significantly provoked ERK1/2 phosphorylation after 5 minutes (P<0.05). However, HMGB1 failed to significantly induce JNK and P38 activation.Conclusions HMGB1 (100ng/ml) up-regulated the expression of TLR4, and specially activated ERK1/2, indicated that HMGB1 provoke ERK1/2 phosphorylation through TLR4.Part Four The role of ERK1/2 in high mobility group box 1 protein up-regulating MMP-9 and TIMP-1 in cardiac fibroblastsObjectives The aims of this study were to determine the expression of matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1) in cardiac fibroblasts which were pre-treated with PD98059 and then with 100ng/ml HMGB1.Methods Primary cardiac fibroblasts were obtained from the ventricles of 1-2-day-old neonatal Sprague-Dawley rats. Quiescent cardiac fibroblasts were pre-treated with PD98059 (12.5uM) to block ERK1/2 for 2 hours and then with 100ng/ml HMGB1 for 24 hours. Isolating protein in the cell, western blot analysis performed to demine the level of intracellular matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1). Isolating protein in the medium by Millipore filter devices, western blot analysis performed to demine the level of matrix metalloproteases (MMP-9) and tissue inhibitors of metalloproteinase (TIMP-1) in supernatant.Results PD98059 attenuated HMGB1-mediated extracellular MMP-9 and TIMP-1 expression (P<0.05). Furthermore, PD98059 attenuated basal intracellular TIMP-1 expression in cardiac fibroblasts (P<0.05) but had no effect on intracellular MMP-9 expression.Conclusions These results demonstrated that HMGB1 stimulated extracellular MMP-9 and TIMP-1 expression via ERK1/2 activation.