Platelets Promote Ang II (Angiotensin II)-Induced Atrial Fibrillation by Releasing TGF-ß1 (Transforming Growth Factor-ß1) and Interacting With Fibroblasts.

Hypertension is a risk factor of atrial fibrillation (AF), and a certain number of patients with hypertension were found with an enlarged left atrium. Platelet activation is found in patients with hypertension or pressure overload/Ang II (angiotensin II)-induced hypertensive animal models and contribute to ventricular fibrosis. Whether hypertension-induced atrial fibrosis is mediated by platelets remains unknown. Our previous experimental data showed that platelet-derived TGF-ß1 (transforming growth factor-ß1) was reduced in patients with hypertensive AF. The present study is to investigate whether platelet-derived TGF-ß1 promotes Ang II-induced atrial fibrosis and AF. Platelet activation and atrial platelet accumulation were measured in sinus rhythm controls, normotensive AF, and patients with hypertensive AF. Ang II (1500 ng/kg per minute, 3 weeks) infused mice with pharmacological (clopidogrel) and genetic platelet inhibition (TGF-ß1 deletion in platelets) were used. Platelet activation, atrial structural remodeling, atrial electrical transmission, AF inducibility, inflammation, and fibrosis were measured in mice. We found that circulating platelets were activated in patients with hypertensive AF. A large amount of platelet was accumulated in the atriums of patients with hypertensive AF. Both clopidogrel treatment and platelet-specific deletion of TGF-ß1 attenuated Ang II-induced structural remodeling, atrial electrical transmission, AF inducibility, as well as atrial inflammation and fibrosis than mice without interventions. Furthermore, clopidogrel blocked atrial platelet accumulation and platelet-fibroblast conjugation. Platelets promoted atrial fibroblast differentiation in cell culture. Profibrotic actions of platelets are largely via activation of atrial fibroblasts by releasing TGF-ß1 and inducing platelet-fibroblast conjugation, and platelet inhibition is sufficient to inhibit atrial fibrosis and AF inducibility.

Elevated Circulating Fibrocytes Is a Marker of Left Atrial Fibrosis and Recurrence of Persistent Atrial Fibrillation.

BACKGROUND: In atrial fibrillation (AF), a more extensively fibrotic left atrium (LA) provides a substrate for arrhythmias and increases risk of relapse following ablation. Fibrocytes are bone marrow-derived circulating mesenchymal progenitors that have been identified in the atrium of patients with AF who have valvular diseases. The present study investigates the associations between circulating fibrocytes and LA fibrosis or the prevalence of recurrence after ablation in patients with persistent AF. METHODS AND RESULTS: We measured the proportion, differentiation, and migration of circulating fibrocytes from patients with persistent AF (n=40), those with paroxysmal AF (n=30), and sinus rhythm controls (n=30). LA low-voltage (fibrosis) area was identified by an electroanatomic mapping system, and patients were followed up for 1 year after ablation. The relationship between circulating fibrocyte percentage and LA low-voltage area or recurrence was assessed by multivariate regression analysis. Circulating fibrocyte percentage positively associated with LA low-voltage area in the persistent AF group, and circulating fibrocyte (≥4.05%) was a significant predictor of 1-year recurrence after ablation. Cultured fibrocytes exhibited enhanced potential of differentiation in the persistent AF group (67.58±1.54%) versus the paroxysmal AF group (56.67±1.52%) and sinus rhythm controls (48.43±1.79%). Furthermore, expression of fibroblast activation markers and cell migratory ability were also elevated in differentiated fibrocytes from patients with persistent AF. Transforming growth factor ß1 and stromal cell-derived factor 1 were elevated in the plasma of patients with persistent AF and were shown to promote fibrocyte differentiation and migration, respectively. CONCLUSIONS: In patients with persistent AF, increased circulating fibrocytes served as a marker of LA fibrosis and recurrence.

Activated platelets inhibit hepatocellular carcinoma cell differentiation and promote tumor progression via platelet-tumor cell binding.

Lack of differentiation in hepatocellular carcinoma (HCC) is associated with increased circulating platelet size. We measured platelet activation and plasma adenosine diphosphate (ADP) levels in HCC patients based on differentiation status. Local platelet accumulation and platelet-hepatoma cell binding were measured using immunohistochemistry (IHC) or flow cytometry. Using a xenograft assay in NON/SCID mice, we tested the effects of the anti-platelet drug clopidogrel on platelet activation, platelet infiltration, platelet-tumor cell binding and tumor cell differentiation. HCC patients with poor differentiation status displayed elevated platelet activation and higher ADP levels. Platelets accumulated within poorly differentiated tissues and localized at hepatoma cell membranes. Platelet-tumor cell binding was existed in carcinoma tissues, largely mediated by P-selectin on platelets. NOD/SCID mice with xenograft tumors also exhibited increased platelet activation and platelet-tumor cell binding. Clopidogrel therapy triggered hepatoma cell differentiation by attenuating platelet activation and platelet-tumor cell binding. TCF4 knockdown promoted HepG-2 cell differentiation and inhibited tumor formation, and TCF4 could be the potential downstream target for clopidogrel therapy.

Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro.

OBJECTIVE: Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. METHODS: HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. RESULTS: UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells. CONCLUSIONS: The induction of HCC cell apoptosis by UDCA was dose-dependent and time-dependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity.

Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways.

Lewis Y (LeY) is a carbohydrate tumor-asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen-activated protein kinases (MAPKs) and phosphoinositide-3 kinase (PI3K)/Akt signaling pathways on FUT4-induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4-induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S-phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin-dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways.

[Immunophenotype characteristics in multiple myeloma cells and their significance].

This study was purposed to investigate the immunophenotype characteristics in multiple myeloma (MM) cells and their significance. The expressions of CD138, CD38, CD56, CD117, HLA-DR, CD3, CD7, CD13, CD33, CD19, CD20, CD22, CD34 in myeloma cells from 31 MM patients were detected by using CD45/SSC immunofluorescent flow cytometry and were confirmed with morphologic observation of myeloma cells. The results indicated that the proportion of myeloma cells detected by morphologic examination was 10%-68%, the proportion of myeloma cells detected by CD45/SSC gating was 9.72%-67.77%. The antigen positive expression rate in myeloma cells was as follows: CD138 61.29%, CD38 100%, CD56 46.15%, CD13 70.00%, CD33 29.03%, HLA-DR 74.19%, CD117 33.33%; the other antigen expressions were negative. It is concluded that the use of CD45/SSC gating technique can identify multiple myeloma cells. The proportion of myeloma cells gated was close to the result of morphological examination. The myeloma cells mainly express the antigens CD138, CD88, CD56, while the expressions of CD117, CD13, CD33 were seen in some MM patients. Myeloma cells don't express antigens of B- and T-lymphocytes, which suggest the heterogenicity of multiple antigens expressed by myeloma cells. However, the biological significance of antigen expression in myeloma cells is worthy to be further investigated.

[Relationship between clinical features and prognosis of highly pathogenic avian influenza A/H5N1 infection in humans in mainland China].

OBJECTIVE: To investigate the relationship between clinical features of patients with A/H5N1 infection and their prognosis in mainland China. METHODS: This study included 28 human cases with A/H5N1 infection in mainland China from October 2005 to May 2008. Data were collected and reviewed from hospital medical records and publishied papers. A database was built by EPIDATA 3.02 and statistical analyses were performed with SPSS 13.0. RESULTS: The median age of the 28 cases was 29 years (range 6-62), and 15 were females. Ten patients survived, and 18 died. The typically clinical manifestations of human influenza A/H5N1 infection included fever and lower respiratory infection. The numbers of peripheral white blood cells, lymphocytes and platelets in the survival and non-survival groups were (4.01 +/- 1.86) x 10(9)/L vs (5.1 +/- 2.9) x 10(9)/L, (1.09 +/- 0.49) x 10(9)/L vs (0.98 +/- 0.44) x 10(9)/L, and (116 +/- 39) x 10(9)/L vs (101 +/- 40) x 10(9)/L, respectively; the differences were not statistically significant between the 2 groups (P>0.05). There was also no statistically significant difference in the increased serum enzymes, such as aspartate aminotransferase [(173 +/- 246) U/L vs (272 +/- 263) U/L], lactate dehydrogenase [(1016 +/- 568) U/L vs (1512 +/- 1052) U/L], creatine kinase [(1099 +/- 1590) U/L vs (2534 +/- 4281) U/L] and MB isoenzyme of creatine kinase [(28 +/- 30) U/L vs (125 +/- 197) U/L] (P>0.05) between the survival and the non-survival groups. However, there was a statistically significant difference in the number of patients with an initial LDH level more than 8 fold of the normal value between the survival and the non-survival groups (none vs 6, P<0.05). All of the 28 cases developed bilateral multiple infiltrates and consolidation in chest radiographs. Acute respiratory distress syndrome occurred in 22 cases, 17 of them died. All the 9 patients with acute kidney injury died. Ten patients received antiviral treatment with oseltamivir, and 6 of them survived. There was a statistical difference in the time of initiating oseltamivir treatment between the survival and the non-survival cases [(6.5 +/- 3.0) d vs (11.8 +/- 3.3) d, Z = 3.70, P<0.05]. Broad spectrum antibiotics and corticosteroids were administered in all of the 28 cases. There was no statistical difference between the survival and the non-survival groups regarding to the corticosteroid treatment (P>0.05). CONCLUSIONS: Initial LDH level reaching more than 8 fold of the normal value suggests a poor prognosis for human H5N1 infection. Patients complicated with either ARDS or acute kidney injury had a higher risk of death. Early administration of effective antiviral agents might improve the prognosis and decrease case fatality.

[Analysis of immunophenotypes of acute lymphoblastic leukemia by three color flow cytometry].

This study was purposed to investigate the immunophenotype characteristics and their significance in subtypes of acute lymphoblastic leukemia (ALL). The immunophenotypes in 40 cases of ALL were analyzed with three color flow cytometry using CD45/SSC two-parametric gating. The results showed that the three color flow cytometry assay using CD45/SSC two-parametric gating could accurately distinguish each other from lymphocytes, monocytes, granulocytes, erythroblasts and primitive cells in bone marrow and/or peripheral blood. Among 40 cases of ALL, B-ALL was 26 cases, T-ALL was 11 cases, HAL was 3 cases. All of the 26 cases of B-ALL expressed CD19 with positive rate of 100%, meanwhile 11 cases of T-ALL most highly expressed CD17 with positive rate of 100%. 12 cases of ALL with myeloid antigen expression (My-ALL) were involved in ALL, the incidence of these cases was almost 30% (12/40). The CD13 was expressed most highly in myeloid antigens. All 3 cases of HAL coexpressed myeloid and B-lineage antigens, among them CD34 was expressed in 2 cases with positive rate of 66.67%. It is concluded that three color flow cytometry assay using CD45/SSC two-parametric gating can exclude the interference of normal cells, thereby the results are more reliable and more accurate.