HMGB1 induces secretion of matrix vesicles which participate in microcalcification of atherosclerotic plaques

AIM: Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1( HMGB1),a cytokine associated with biomineralizing process under physiological and pathological conditions. Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles( MVs) from macrophages. METHODS: HMGB1 was added to the medium of macrophages,the secretion of MVs in the supernatant was tested by flow cytometry analysis. The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining. Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs. Then we subcutaneous injection into mice with MVs to induce regional mineralization. RESULTS: HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis. TNAP activity,considered as a marker of MVs maturation,was higher in HMGB1-induced MVs compared to the control-MVs. HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase 2( n SMase2) that involved the receptor for advanced glycation end products( RAGE) and p38MAPK( upstream of n SMase2). Inhibition of n SMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part,via the RAGE / p38 MAPK /n SMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 may participated in the early calcification of atherosclerotic plaques.

Expression of Interleukin-15 and Its Receptor on the Surface of Stimulated Human Umbilical Vein Endothelial Cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.

Effect of simulated ischemia-reperfusion on I_f in sinoatrial node cells and the intervention of KATP channel opener

Objective: To study the effect of simulated ischemia-reperfusion (I/R) on If of sinoatrial node (SAN) cells and the intervention of KATP channel opener Pinacidil. Methods: The SAN cells of the neonatal rats were detached and purified 2 d before the experiment. The experimental animals were randomly divided into the control group, group of simulated I/R, group intervened with KATP channel opener Pinacidil (P+ I/R) and group intervened with KATP channel blocking agent 5-HD (5-HD + P + I/R & 5-HD + I/R). The If density of each group was measured by technique of routine whole cell patch clamp and multiple-catheter perfusion system and the If activated curve in each group was drawn. Results: ①Under different directive potentials, the If density of the SAN cells in I/R group increased significantly, compared with that in the control group ( P < 0.01); that in P + I/R group decreased significantly, compared with that in I/R group ( P < 0.01); the If density values in 5-HD + P + I/R group and 5-HD + I/R group increased significantly, compared with that in P + I/R group, but showed no significant difference with that in I/R group. ②Compared with that in the control group, the If activated curve of the SAN cells moved rightwards under ultimate activating potential, half of which was from - 108.0 ± 12.4 to - 89.5 ± 7.2 mV ( P <0.01); compared with that in I/R group, If activated curve of the SAN cells moved leftwards under ultimate activating potential, half of which was the range from -99.5± 10.8 mV (P<0.05); KATP channel blocking agent 5-HD could block the effect of Pinacdil on If activated curve. Conclusion: KATP channel opener Pinacidil can antagonize the effect of simulated I/R on the If of SAN cells, which may be beneficial to the maintenance of the relative stability of ion steady state and electrophysiological activities under condition of simulated I/R.

Adenovirus-mediated gene transfer of TIMP-4 reduces neointimal hyperplasia in balloon-injured rat carotid artery

Objective:To determine the effects of a recombinant replication-deficient adenovirus encoding human tissue inhibitor of metalloproteinase-4(Ad.TIMP-4) on vascular smooth muscle cell(VSMC) function in vitro and neointimal development in the injured rat carotid artery.Methods:Western blotting,gelatin zymography and reverse zymography were used to characterize the expression and functional activity of the TIMP-4 secreted by Ad.TIMP-4-infected VSMCs.The migration and proliferation of VSMCs in vitro were separately detected by using Millicell-PCF invasion chambers and [3H]-thymidine incorporation assay.Immunohistochemistry and morphometric analysis were used to determine the local expression of TIMP-4 and its effect on neointima development in a rat carotid artery balloon injury model.Results:VSMCs infected with Ad.TIMP-4 expressed functionally active human TIMP-4 which increased with the duration of infection.TIMP-4 expression inhibited VSMC migration,but not significantly affect VSMC proliferation.In a balloon-injured rat carotid artery model,a significant 62% reduction in neointimal area was found in Ad.TIMP-4-infected vessels at 14 days after injury.Ad.TIMP-4 infection had no effect on medial area.Conclusion:Our results indicated TIMP-4 over expression can significantly inhibit the migration of cultured VSMCs and prevent neointimal formation after vascular injury.Our findings provide additional evidence that TIMP-4 could play an important role in vascular pathophysiology,and may be an important therapeutic target for future drug development.

Effects of Bisoprolol on the ventricular function and hemodynamics in patients with atrial fibrillation and chronic heart failure

Background: Recent data suggest that beta-blockers can be beneficial in patients with chronic heart failure (CHF). Atrial fibrillation (AF) is present in a significant number of patients with CHF and is associateing with significant morbidity and increasing mortality rates. Thus it is necessary to establish therapy to improve the poor prognosis in this high-risk population, but a specific benefit of beta-blockers to the subset with concomitant AF and CHF has been little demonstrated. Objective: To examine the effects of Bisoprolol (6 months treatment) on the ventricular function and hemodynamics in patients with AF and CHF. Methods: 84 patients with stable CHF (NYHA≤Ⅲ class) and AF were assigned to Treated Group( n = 37) or Control group Ⅰ ( n = 22, 24-hour heart mean rate < 70/min) or Control Group Ⅱ ( n = 25, 24-hour heart mean rate ≥ 70/min) . All patients were given the basic therapy for CHF, and Treated Group received Bisopolol. Clinical and echocardiographic variables were measured in 3 groups at baseline and after 6 months, and the results were compared . Results: After 6 months of treatment with Bisoprolol, left ventricular ejection fraction (LVEF) and NYHA class had significandy improved (P < 0.05), and a trend towards a reduction in combined end point of death or CHF hospitalization was also observed (P < 0.20) in Treated Group; The increase of LVEF in Treated Group were associated with a reduction in mitral regurgitation degree and left atrial volume; The heart rate in mean 24-hour and at peak exercise decreased in Treated Group, but were similar to that in Control Group Ⅰ. Conclusion: 6 months of Bisoprolol therapy resulted in an improvement in the NYHA class and LVEF, and also showed a trend towards a reduction in hospitalization or death. The beneficial effects of Bisoprolol on patients with AF and CHF may be partly mediated by improvement of ventricular diastolic function.

Inhibitory effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia

Objective: To investigate the effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia after rat carotid artery balloon injury. Methods :AT2R gene was transferred into rat carotid arteries by recombinant adenovirus pAd-AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested on the 14th day after gene transfer. The efficiency of trans-gene delivery was measured by the expression of adenovirus-encoding green fluorescent protein (GFP) under fluorescent microscope. The expression of AT2R and PCNA (proliferating cell nuclear antigen) was e-valuated by RT-PCR, immunocytochemistry, immunofluorescence staining, confocal microscopy, respectively. The ratio of intimal to medial area (I/M) was quantified with images and determined by an image analysis system. Results: GFP-positive area in adventitia, media and the forming neointima was about 40%. Adenoviral delivery of rat AT2R gene up-regulated AT2R expression in balloon-injured rat carotid and reduced PCNA expression and I/M significantly in neointima(P<0. 01). Double immunofluorescence labeling of AT2R and PCNA also showed that AT2R gene transfer inhibited VSMCs proliferation in neointima. Conclusion: AT2R gene transfer may be a novel promising therapy to limit neointimal hyperplasia.

Protective effect and mechanism of K_(ATp) channel opener on the sinoatrial node cells of neonatal rat cultured in simulated ischemia-reperfusion

Objective: To study the effect of KATp channel opener and its possible mechanism on the sinoatrial node cells of neonatal rats which were cultured under simulated ischemia-reperfusion. Methods: Freshly isolated sinoatrial node (SAN) cells of neonatal rats were purified and cultured for 2 d, and then they were randomly divided into the control, simulated ischemia-reperfusion group (I/R group) , group intervened with KATp channel opener pinacidil (P + I/R group), KATP Channel blocking agent 5-HD (5-HD + I/R group) , and group with the 2 agents at same time (5-HD + P + I/R group) . The survival rate of cells was measured by flow cytometry and the content of intracellular calcium in the cells of each group was detected with laser confocal microscopy. Results: ① The survival rate of SAN cells in I/R group [ (51. 79±6. 28)% ] was remarkably significantly lower than in control [ (95. 08±10. 48)% ] (P < 0.001), and very significantly lower than in P + I/R group [ (63. 77±5. 35) % ] (P<0.01), however, those of 5-HD + P + I/R group [(52. 88±6. 25)% ] and 5-HD+I/R group [ (53. 16±5. 35)% ] was significantly lower compared with that in P + I/R group (P <0. 01) ; ② When the average fluorescence intensity of sinoatrial node cells in the control was regarded as 100% , the relative fluorescence intensities of each group were: ( 374±52) % in I/R group, significantly higher than that of control (P <0. 01) ; ( 162±20)% in P + I/R group, declining significantly than that of I/R group (P<0.01); (385?6)% in 5-HD+ P + I/R group and (379±44)% in 5-HD + I/R group, increasing significantly than that of I/R group (P<0.01). Conclusion: ① Simulated ischemia-reperfusion can significantly reduce the survival rate of SAN cells, and can also lead to the overload of intracellular calcium in them.② KATp channel opener, pinacidil, exerts protective effect on the cells under simulated ischemia-reperfusion, which may be associated with the decrease of intracellular calcium loading in them.

BTEB2 antisense RNA inhibits intimal hyperplasia in a rat carotid balloon injury model

Objective： To investigate the effects of basic transcriptional element binding protein-2 （BTEB2） antisense RNA on vascular smooth muscle cells （VSMCs） proliferation and the neointimal formation after carotid balloon injury in rats. Methods： The cultured VSMCs were transfected with an adenoviral vector containing BTEB2 antisense gene, Ad ASBTEB2. Effects of BTEB2 antisense RNA on the expression of BTEB2 were investigated by Western blot analysis. The cell cycle was analyzed using flow cytometry. Ad ASBTEB2, control adenoviral vector Ad. LacZ or PBS was transduced into the rat carotid artery after balloon injury. The expression of BTEB2 at 7, 14, and 21 d after gene transfer was detected by immunohistochemistry and neointima-to-media （I/M） area ratio at these time points was calculated. Resuits： The cell cycle was arrested in G0/G1 phase and the expression of BETB2 was downregulated after transfection with Ad ASBTEB2. Ad ASBTEB2 treatment reduced I/M area ratios on day 7, 14, and 21 after injury by 45%, 50% and 53% respectively, whereas the Ad LacZ treatment did not significantly alter these ratios compared with control group. Conclusion： BTEB2 antisense RNA mediated by adenoviral vector inhibits proliferation of VSMCs and significantly reduces neointimal hyperplasia in the rat carotid balloon injury model. BTEB2 antisense RNA is a potential therapeutic approach to preventing neointimal formation after balloon injury.

MinK gene G112A polymorphisms and atrial fibrillation:a Meta-analysis

Atrial fibrillation (AF) is the most common arrhythmia with multi-factorial pathogenesis. A number of studies of genetic epidemiology have assessed the association of G112A (G38S) single nucleotide polymorphisms (SNPs) in Mink gene with AF in different populations. However, the results are inconsistent and inconclusive. We performed a Meta-analysis of the association between G112A polymorphisms of MinK gene and AF to estimate the magnitude of the gene effect. Six case-control studies with a combined 854 cases and 1079 controls were summarized. Subgroups in different races were separately analyzed. Heterogeneity and publication bias were also explored. When all groups were pooled, the individuals with G allele had an over 40% higher risk of AF compared with individuals with the A allele. The GG genotype (versus AA genotype) was found to be significant association with increased AF risk. The significant associations were also found in both dominant and recessive genetic model. For subgroup analysis, the results were consistent with above, except that the pooled OR for Chinese population was not significant in a recessive genetic model. In conclusion, G112A polymorphisms in Mink gene may have an important effect on the pathogenesis of AF. This warrants further investigation in large multi-center studies with precise design.

Elevated Levels of Naturally-Occurring Autoantibodies Against the Extracellular Domain of p75NTR Aggravate the Pathology of Alzheimer's Disease

The extracellular domain(p75ECD)of p75 neurotrophin receptor(p75NTR)antagonizes Aβ neurotoxicity and promotes Aβclearance in Alzheimer’s disease(AD).The impaired shedding of p75ECD is a key pathological process in AD,but its regulatory mechanism is largely unknown.This study was designed to investigate the presence and alterations of naturally-occurring autoantibodies against p75ECD(p75ECD-NAbs)in AD patients and their effects on AD pathology.We found that the cerebrospinal fluid(CSF)level of p75ECD-NAbs was increased in AD,and negatively associated with the CSF levels of p75ECD.Transgenic AD mice actively immunized with p75ECD showed a lower level of p75ECD and more severe AD pathology in the brain,as well as worse cognitive functions than the control groups,which were immunized with Re-p75ECD(the reverse sequence of p75ECD)and phosphate-buffered saline,respectively.These findings demonstrate the impact of p75ECD-NAbs on p75NTR/p75ECD imbalance,providing a novel insight into the role of autoimmunity and p75NTR in AD.

Relationship between intravascular ultrasound imaging features of coronary plaques and soluble CD105 level in patients with coronary heart disease

Plaque rupture with subsequent thrombus formation is the common pathophysiological substrate of acute coronary syndrome （ACS）. Moreno et al reported mat neovascularization as manifested by the localized appearance of microvessels is increased in ruptured plaques in the human aorta. Microvessel density is also increased in inflammatory lesions, with intraplaque hemorrhage and in thin-cap fibroatheromas. Microvessels at the base of the plaque are independently correlated with plaque rupture, suggesting a contributory role for neovascularization in the process of plaque rupture. Soluble CD105, a sensitive serum marker of neovascularization, is thought to be associated with cardiovascular disease. The purpose of this study was to assess the relationship between the level of soluble CD105 and the morphological plaques by intravascular ultrasound （IVUS） in patients with stable angina （SA） and those with unstable angina （UA） and whether soluble CD105 may serve as a non-invasive marker of coronary plaque destabilization.

Primary culture and identification of sinoatrial node cells from newborn rat

Objective To establish a reliable approach to primary culture and identification of sinoatrial node (SAN) cells. Methods The SAN cells were cultured from SAN tissue removed from neonatal Wistar rats and purified with differential attachment and 5'-bromodeoxyuridine (BrdU) treatment. The obtained cells were morphologically observed with inverted microscopy and transmission electron microscopy. Its action potential was recorded using electrophysiological methods.Results Three distinctly different cells were observed in the cultured SAN cells: spindle, triangle and irregular. Of these, the spindle cells comprised the greatest proportion, with their shape, structure and electrophysiological characteristics consistent with those of the pacemaker cells of SAN. The triangle cells were similar in features to the similarly shaped myocytes located in the atrial myocardium. Conclusions The culture method of differential attachment combined with BrdU treatment is a reliable approach to growing SAN cells. Of the cells cultured from SAN, the spindle cells appear to function as pacemaker cells.

Recent advances and findings of angiotensin type 2 receptor：a review

Angiotensinogen is a member of the serpin family. It is produced constitutively and released into the circulation mainly by the liver. Angiotensinogen forms angiotensin Ⅰ by action of the circulated renin released from the kidney. Angiotensin Ⅱ （Ang Ⅱ）, an octapeptide hormone with sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe,is converted from angiotensin Ⅰ through removal of two terminal residues by the angiotensin-converting enzyme （ACE） mostly catalyzed in the lung.1 This peptide binds to two subtype receptors, angiotensin type 1 receptor （AT1R） and angiotensin type 2 receptor （AT2R）,members of the superfamily of heptahelical G protein coupled receptors, with different affinities.2 It is well known that AT1R and AT2R crosstalk and lead to counterregulatory functions in many systems, especially the cardiovascular system.3 Accumulating data established the roles of AT1R in the classic actions of Ang Ⅱ including vasoconstriction and cardiovascular hypertrophy, whereas AT2R is suggested to exert direct functions in vasodilation and antigrowth effects.4 Recent publications provide new insights into the roles of AT2R with increasing responsibilities. Recent progresses in AT2R research are reviewed in this article.

Blunt chest impact leading to acute myocardial infarction in a young man： a rare finding of both coronary artery dissection and pseudoaneurysm

To the editor： A 22-year-old man was admitted without significant heart complaint, but with a diagnosis of myocardial infarction at a local hospital. Two months earlier, he suffered blunt chest impact and maxillofacial trauma due to an accident falling from a 3-metero high balcony. He has previously been healthy without any family history of disease. Physical examination on admission revealed mandibular tenderness and a traumatic blindness in the left eye.

Role of circulating long non-coding RNA for the improvement of the predictive ability of the CHA_(2)DS_(2)-VASc score in patients with atrial fibrillation

Background:The CHA_(2)DS_(2)-VASc score was initially applied to stratify stroke risk in patients with atrial fibrillation(AF)and was found to be effective in predicting all-cause mortality outcomes.To date,it is still unclear whether circulating long non-coding RNAs(lncRNAs)as emerging biomarkers,can improve the predictive power of the CHA_(2)DS_(2)-VASc score in stroke and all-cause mortality.Methods:Candidate lncRNAs were screened by searching the literature and analyzing previous RNA sequencing results.After preliminary verification in 29 patients with AF,the final selected lncRNAs were evaluated by Cox proportional hazards regression in 192 patients to determine whether their relative expression levels were associated with stroke and all-cause mortality.The c-statistic,net reclassification improvement(NRI),and integrated discrimination improvement of the patients were calculated to evaluate the discrimination and reclassification power for stroke and all-cause mortality when adding lncRNA expression levels to the CHA_(2)DS_(2)-VASc score model.Results:Five plasma lncRNAs associated with stroke and all-cause mortality in AF patients were selected in our screening process.Patients with elevated H19 levels were found to have a higher risk of stroke(hazard ratio[HR]3.264,95% confidence interval[CI]:1.364–7.813,P=0.008).Adding the H19 expression level to the CHA_(2)DS_(2)-VASc score significantly improved the discrimination and reclassification power of the CHA_(2)DS_(2)-VASc score for stroke in AF patients.In addition,the H19 level showed a marginally significant association with all-cause mortality(HR 2.263,95%CI:0.889–5.760,P=0.087),although it appeared to have no significant improvement for the CHA_(2)DS_(2)-VASc model for predicting all-cause mortality.Conclusions:Plasma expression of H19 was associated with stroke risk in AF patients and improved the discriminatory power of the CHA_(2)DS_(2)-VASc score.Therefore,lncRNA H19 served as an emerging non-invasive biomarker for stroke risk prediction in patients with AF.

A Case of Renal Subcapsular Hematoma Caused by an Accident Injection from Renal Capsular Artery

To the Editor： A 49-year-old woman was admitted with hypertension and diabetes. Electrocardiogram demonstrated inverted Y waves in V1-6. Blood pressure was 180/110 mmHg.

Relationship among soluble CD105, hypersensitive C-reactive protein and coronary plaque morphology： an intravascular ultrasound study

Background Rupture of unstable plaque with subsequent thrombus formation is the common pathophysiological substrate of acute coronary syndrome （ACS）. It is of potential significance to explore the blood indexes predicting plaque characteristics. We investigated the relationship among soluble CD105, hypersensitive C-reactive protein （hs-CRP）, and coronary plaque morphology.Methods A clinical study from April 2004 to December 2006 was conducted in 130 patients who were divided into 3 groups： 56 patients （43.1%） in stable angina （SA） group, 52 patients （40.0%） in unstable angina （UA） group and 22 patients （16.9%） in acute myocardial infarction group. The concentrations of soluble CD105 and hs-CRP were measured in all of the patients by cardioangiography （CAG）. Plasma samples of arterial blood were collected prior to the procedure. The levels of soluble CD105 and hs-CRP were measured by enzyme-linked immunosorbent assay （ELISA）.Results Unstable and ruptured plaque was found more frequently in patients with acute myocardial infarction and UA. External elastic membrane cross-sectional area （EEM CSA）, plaque area, lipid pool area and plaque burden were significantly larger in the ruptured and unstable plaque group. Positive remodeling, thinner fabric-cap, smaller minimal lumen cross-sectional area （MLA）, dissection and thrombus were significantly more frequent in the ruptured and unstable plaque group. Remodeling index （RI） was positively correlated with the levels of soluble CD105 in the UA group （r=0.628, P〈0.01） and the acute myocardial infarction group （r=0.639, P〈0.01）. The levels of soluble CD105 and hs-CRP were higher in the ruptured plaque group. Soluble CD105 〉4.3 ng/ml was used to predict ruptured plaque with a receiver operating characteristic （ROC） curve area of 0.77 （95% confidence interval （CI）, 66.8%-87.2%）, a sensitivity of 72.8%, a specificity of 78.0% and an accuracy of 70.2% （P〈0.01）, similarly for hs-CRP 〉 5.0 mg/ml with a ROC curve area of 0.70 （95% CI, 59.2%-80.2%）, a sensitivity of 70.2%, a specificity of 76.2% and an accuracy of 67.2% （P〈0.01）.Conclusions The plaque characteristics correlate with the clinical presentation. The elevation of soluble CD105 and hs-CRP is related to the plaque instability and rupture.