The Role Of High Mobility Group Box-1 Protein And Its Signaling Pathway In Pressure Overload-induced Cardiac Hypertrophy

Cardiac remodelling is associated with many different kinds of heart diseases, such as ischemic cardiomyopathy and hypertensive heart disease. More and more eyes are now focus on inflammatory cells and inflammatory factors, concerning on the mechanisms of cardiovascular diseases. It is well known that inflammatory factors are involved in cardiac remodelling, but the roles they play may not exactly the same in different causes induced cardiac remodelling.Mechanical stress and the influence of neurohumoral factors are of crucial importance in hypertension induced cardiac hypertrophy. Meanwhile, studies have showed that hypertension is also associated with increased mast cells in myocardium' and the activation of monocytes 2,3. Moreover, various inflammatory factors (e.g., IL-1, IL-6, IL-8, TNF-a, CRP), which may involved in hypertension induced hypertrophy 7-10, are highly increased in hypertension 4-6. For instance, pressure-overload may induce the expression of TNF-a in cardiac myocytes, while pressure-overload induced inflammation and cardiac hypertrophy may be inhibited by blocking TNF-?11,12.High mobility group box 1 protein (HMGB1) is an important inflammator factor, which exists in various kinds of organs and cells. It would automaticly or passively be released by cells under stress, and then promote the secrete of TNF-?, IL-6 and IL-8 by monocytes and endothelial cells. In addition, these inflammatory factors would in turn activate HMGB1 13-15, which is highly important in the amplication and maintaining of inflammation reaction 16. Thus, it is plausible that HMGB1 may also be involved in hypertension induced cardiac hypertrophy.The aims of this study are to discuss the relationship between inflammatory markers and cardiac function, and to explore the underlying mechanisms, which may give great impetus to the comprehensive understanding of the disease. Part OneThe relationship among inflammatory markers, cardiac function and structure in patients with chronic heart failureObjectives This meta-analysis was conducted to determine the role of statin therapy in both inflammation markers and cardiac function and structure in patients with chronic heart failure (CHF).Methods Pubmed, MEDLINE, EMBASE, and EBM Reviews databases were searched for randomized controlled trials comparing statin treatment with non-statin treatment in patients with CHF. Two reviews independently assessed studies and extracted data. Weighted mean differences (WMD) with 95% confidence intervals (CI) or standardized mean differences (SMD) with 95% CI were calculated using random effects models.Results Of the initial 4,209 hits, fourteen randomized controlled trials (RCTs) trials with 6,265 patients were included. Pooled analysis showed that statin therapy was associated with significant increase in left ventricular ejection fraction (LVEF, WMD=3.35%,95% CI 0.80% to 5.91%, P=0.01). The beneficial effects of statin treatment were also demenstrated by the reduction of left ventricular end-diastolic diameter (LVEDD, WMD=-3.77 mm,95% CI -6.24 to -1.31 mm, P=0.003), left ventricular end-systolic diameter (LVESD, WMD=-3.57 mm,95% CI -6.37 to -0.76 mm, P= 0.01), B-type natriuretic peptide (BNP, WMD=-83.17 pg/ml,95% CI-121.29 to -45.05 pg/ml, P<0.0001), and NYHA functional class (WMD=-0.30, 95% CI -0.37 to -0.23, P<0.00001). Moreover, statin treatment was associated with significant decrease in high sensitivity C-reactive protein (hsCRP, SMD=-0.74,95% CI -1.16 to -0.32; p=0.0005) and soluble vascular cell adhesion molecule-1 (Svcam-1, SMD=-0.49,95% CI -0.91 to -0.08; p=0.02). Meanwhile, meta-regression analyses and subgroup analyses showed that difference in age, etiology, baseline LVEF, type of statins and follow-up duration might influence the effects of statins on CHF patients. Longer treatment intervals might bring more beneficial effects on the hsCRP, interleukin-6 (IL-6) and LVEF.Conclusions The current cumulative evidence suggests that utilization of statins may not only result in the down-regulation of inflammatory markers, but also in the improvement of cardiac function and clinical symptoms, as well as the amelioration of left ventricular remodeling in patients with CHF. Additionally, more down-regulation of inflammation markers might be associated with more benefits on cardiac function..Part TwoThe expression of high mobility group box 1 protein in myocardium of murine pressure-overload modelObjectives The aims of this study were to build murine pressure-overload model and to detect the expression of high mobility group box 1 protein (HMGB1) in myocardium of the mice.Methods Pressure-overload models were built in male C57BL/6 mice (8-10 weeks old) by transverse aortic constriction (TAC). Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed before the operation and 1 day,3 days,7 days,14 days and 28 days after. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned, stained with hematoxylin and eosin (H-E) and masson's trichrome stain. Cross sectional area (CSA) of cardiomyocytes were measured. Western Blot and immunohistochemistry were performed to detect HMGB1 protein in myocardium. Additionally, the level of HMGB1 protein expression was as well determined in cardiomyocytes, which were stretched in vitro to imitate pressure-overload.Results Both aortic systolic blood pressure and left ventricular end-systolic pressure in mice were highly elevated within 28 days after TAC (P<0.01). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were decreased at 1,3 and 28 days after the operation (P<0.05), while similar changes were observed in left ventricular volume and diameter. Both the anterior wall and the posterior wall of the left ventricle were significantly thicker at 14 days after the operation (P<0.01), and the ratio of heart weight/body weight were up-regulated at 14 and 28 days after TAC (P<0.05), as compared with control. Moreover, HE staining confirmed the results of transthoracic echocardiographic analysis, and CSA of cardiomyocytes at 14 days were significantly larger than any other points of time (P<0.01). Moderate myocardial fibrosis were observed 14 days after TAC, while it deteriorated at 28 days. The expression of HMGB1 protein were increased in myocardium at 3 and 7 days and at the same time, they transferred from nucleus to cytoplasm or even extracelluar region, as indicated by the results of Western Blot and IHC. In addition, the results showed that mechanical stress significantly increased HMGB1 protein expression in cardiomyocytes within 12 hours in vitro (P<0.05).Conclusions Pressure-overload models can be successfully built in mice with TAC. In this model, the function of left ventricle is temporarily impaired within 3 days, followed by cardiac hypertrophy at 2 weeks and cardiac dysfunction at 4 weeks. Cardiomyocytes-derived HMGB1 protein are increased and activated within the first week of pressure-overload.Part ThreeThe role of high mobility group box 1 protein in pressure-overload induced cardiac hypertrophyObjectives The aims of this study were to determine the role of high mobility group box 1 protein (HMGB1) in pressure-overload induced cardiac hypertrophy and to explore the potential mechanisms.Methods Pressure-overload models were built in male C57BL/6 mice (8-10 weeks old) by transverse aortic constriction (TAC), and simultaneously, PBS or HMGB1 recombinant (200ng) were injected into the myocardium. Mice were randomly divided into four groups, accordingly to different intervention:1) Sham group,2) HMGB1 group,3) TAC+PBS group and 4) TAC+HMGB1 group. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed at 2 weeks and 4 weeks after the operation. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned, stained with hematoxylin and eosin (H-E) and masson's trichrome stain. Cross sectional area (CSA) of cardiomyocytes were measured. In addition, western Blot were performed to detect phosphorylated extracellular signal-related kinases (p-ERK1/2) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in myocardium. Results HMGB1 injection alone had no impact on both aortic systolic blood pressure (SBP) and left ventricular end-systolic pressure (LVESP), as compared with sham-operated mice. However, SBP and LVESP were equally elevated in TAC mice (P<0.01). No difference were detected among the 4 groups before the operation. At 2 weeks after the operation, increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), as well as decreased left ventricular volume, were only observed in TAC+HMGB1 group (P<0.05). Moreover, both the anterior wall and the posterior wall of the left ventricle in TAC mice were significantly thicker than sham-operated ones (P<0.01), while the walls in TAC+HMGB1 group were the thickest (P<0.01). At 4 weeks after the operation, both LVEF and LVFS of the mice in HMGB1 group, TAC+PBS group and TAC+HMGB1 group were apparently lower than in Sham group (P<0.05), and specifically, it decreased more in TAC+HMGB1 group, as compared with TAC+PBS group (P<0.05). The thickness of the walls in TAC+PBS and TAC+HMGB1 operated mice at 4 weeks, which were decreased to the same level of sham-operated ones, were significantly thinner than that of 2 weeks (P<0.05). Meanwhile, the walls of the mice in HMGB1 group were thinner than that in sham group at 4 weeks (P<0.05). Furthermore, the ratio of heart weight/body weight in both TAC-operated groups were higher than in sham group at 2 and 4 weeks (P<0.05). HE staining confirmed the results of transthoracic echocardiographic analysis, and CSA of cardiomyocytes at 2 weeks were significantly larger in TAC-operated mice, as compared with sham-operated ones (P<0.01), and furtherly, it was larger in TAC+HMGB1 group than that in TAC+PBS group (P<0.01). The results of masson's trichrome stain suggested that HMGB1 might directly induce myocardial fibrosis as well as aggravate such pathological changes under overloaded pressure. Western Blot showed that TAC significantly up-regulated p-ERK1/2 and p-STAT3 expression in myocardium at 2 weeks, and simultaneous overexpression of HMGB1 could exaggerate this effects.Conclusions Pressure-overload can induce cardiac hypertrophy and cardiac dysfunction in mice. Over-expression of HMGB1 may aggravate pressure-overload induced cardiac hypertrophy, myocardial fibrosis and cardiac dysfunction through the activation of ERK1/2 and STAT3. Part FourSignaling pathways involved in the effects of high mobility group box 1 protein on cardiac myocytes in vitroObjectives The aims of this study was to explore the underlying receptor signaling pathways involved in the effects of high mobility group box 1 protein (HMGB1) on cardiac myocytes in vitro.Methods Primary cultures of rat ventricular myocytes were obtained using one or two-day-old Sprague-Dawley rat pups. Cultured cardiomyocytes were stimulated by mechanical stress and/or HMGB1 for 0 minute,5 minutes,10 minutes,30 minutes,1 hour,2 hours,4 hours,8 hours,12 hours and 24 hours. Western Blot performed to demine the expression of receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR-4), extracellular signal-related kinases (ERK1/2), P38 mitogen-activated protein kinase (P38 MAPK), janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Moreover, the aforementioned kinases were detected again after HMGB1 stimuli for 30 minutes, on the condition of RAGE or TLR-4 blocking.Results Both mechanical stress and HMGB1 stimuli significantly up-regulated phosphorylated extracellular signal-related kinases (p-ERK1/2), phosphorylated P38 mitogen-activated protein kinase (p-P38 MAPK), phosphorylated janus kinase 2 (p-JAK2) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) within 1 hour (all P<0.05). Interestingly, the synergetic effec of mechanical stress and HMGB1 stimuli on the activation of ERK1/2 was observed. Moreover, mechanical stress induced not only RAGE but also TLR-4 expression in cardiac myocytes with 24 hours (P<0.05). The blockage of RAGE significantly inhibited the effect of HMGB1 on the activation of ERK1/2 (P<0.05), whereas blockage of TLR-4 had no impact on such activation.Conclusions Mechanical stress may up-regulate the expression of RAGE and TLR-4 in cardiac myocytes. Similar to mechanical stress, HMGB1 stimuli activate MAPKs and JAK2/STAT3 pathways in cardiac myocytes, which may partially be mediated by RAGE.