Experimental observation on a new chimney-shaped mechanical valve completely implanted above the mitral annulus in animals.

Current commercially available prosthetic valves suffer from limited size, high requirements for implantation technique, subvalvular structural destruction, and valve dysfunction due to proliferation of fibrous endothelial tissue. This study aims to perform the preclinical large animal experiments for surgically implanting a chimney-shaped artificial mechanical heart valve with zero left ventricular occupancy, which fully accommodates the movement of the valve leaflets in the valve frame and realizes completely supra-annular surgical implantation. A total of 7 sheep underwent the replacement of artificial valve, and 5 sheep survived normally until anatomical examination. The mechanical properties of these artificial mitral valves remain functionally normal. There was no obvious thromboembolism around the artificial valve and in the important organs. The tissue layer of suture ring was completely organized and endothelialized, and the thickness of tissue layer was about 0.6-1.0 mm. The follow-up of echocardiography showed that the left ventricular ejection fraction was normal (60-70%) before and 6 months after operation. The results of transvalvular pressure gradient and blood flow velocity of artificial valve were normal. Left ventricular retrograde angiography showed that the artificial valve was completely located in the left atrium with good position and normal opening and closing. There was no obvious perivalvular leakage and other abnormalities. At 3 and 6 months, there were no obvious abnormalities in blood routine test, liver and kidney function, and other indexes. The new chimney-shaped artificial mechanical valve implanted completely above the mitral annulus had good wear resistance, histocompatibility, and antithrombotic and hemodynamic performance.

Transjugular Transcatheter Tricuspid Valve Implantation of LuX-Valve Bioprosthesis in a Preclinical Model.

The purpose of this preclinical study in a sheep model was to confirm the feasibility and safety of the LuX-Valve transjugular tricuspid valve (TV) replacement apparatus and to optimize the implantation procedure before beginning first-in-man study. The LuX-Valve was implanted in a sheep model (n = 8) via transjugular approach. Six of eight sheep underwent successful implantation procedure on beating heart. The first two sheep died during the prostheses deployment. In the remaining 6 sheep that survived, postoperative echocardiography results showed there was no paravalvular leakage (PVL) and central tricuspid regurgitation in 5 animals, whereas 1 animal had mild PVL. The mean transvalvular gradient was 1.1 ± 0.9 mm Hg at the 4-week follow-up. No right ventricular outflow tract (RVOT) obstruction, device malposition, pericardial effusion, coronary artery compression, or arrhythmias were observed. This technology may be a promising alternative for TR patients who are at high risk for open-heart surgery. Transjugular tricuspid valved-stent implantation. a Transjugular tricuspid valve replacement in a sheep model. b and c Valved stent. d, e, and f Schematic depiction of the implantation procedure.

A new role for host annexin A2 in establishing bacterial adhesion to vascular endothelial cells: lines of evidence from atomic force microscopy and an in vivo study.

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen-host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis.

Plasmin-mediated fibrinolysis at the surface of vascular endothelial cells (SVEC) plays a key role in maintaining vascular hemostasis, in which the cAMP pathway participates. After externalization to the SVEC, annexin A2 (ANXA2) serves as a platform for conversion of plasminogen to plasmin. Here we describe a regulatory role of the exchange protein directly activated by cAMP (EPAC) in ANXA2 externalization and vascular fibrinolysis. Knockout of EPAC1 in mice results in a decreased ANXA2 expression on the SVEC associated with increased fibrin deposition and fibrinolytic dysfunction. Reduced levels of EPAC1 are also found in endocardial tissues beneath atrial mural thrombi in patients. Notably, administration of recombinant ANXA2 ameliorates fibrinolytic dysfunction in the EPAC1-null mice. Mechanistically, EPAC1 regulates the SVEC plasminogen conversion depended on ANXA2. EPAC1 promotes tyrosine-23 phosphorylation of ANXA2, a prerequisite for its recruitment to the SVEC. Our data thus reveal a novel regulatory role for EPAC1 in vascular fibrinolysis.

Evidence of Osteogenic Regulation in Calcific Porcine Aortic Valves.

BACKGROUND: Chemically cross-linked animal tissues, such as porcine aortic valves (PAVs) have many documented advantages over mechanical valves. However, calcification is the major underlying pathologic process that results in bioprosthetic valve failure. Recently, several reports described the expression of noncollagenous bone matrix proteins in bioprosthetic valves and suggested an actively regulated process of tissue repair. METHODS: Thirty-one explanted PAVs with evidence of calcification were collected and examined for the protein expression implicated in myofibroblast activation, osteoblast differentiation, and bone matrix deposition by using immunohistochemistry. RESULTS: The mean duration that PAVs were implanted was 11.5 ± 5.6 years, ranging from 12 months to 28 years. Pearson correlation analysis showed a significant relationship between the duration and valvular calcification (r = 0.3818, P = .034). The number of vimentin-positive mesenchymal cells in explanted PAVs was significantly lower than that of unused PAVs (P < .01). However, increased expression of α-smooth muscle actin (α-SMA) (P < .01), proliferating cell nuclear antigen (PCNA, P < .01), Cbfa1/Runx2 (P < .01), osterix (P = .0126), bone sialoprotein (BSP, P < .01), osteocalcin (P < .01), and osteopontin (P < .01) was found in explanted PAVs. Immunohistochemical staining of alkaline phosphatase (ALP) and osteocalcin was negative in the unused PAVs. In explanted PAVs, the expression level of these 2 proteins was also significantly increased. CONCLUSIONS: Our results support the view that PAV calcification is an actively regulated process with osteogenic signaling activation.

Prognostic value of association of OCT4 with LEF1 expression in esophageal squamous cell carcinoma and their impact on epithelial-mesenchymal transition, invasion, and migration.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease with poor prognosis. Because of early metastasis prior to diagnosis and therapeutic resistance, ESCC has become one of the leading causes of cancer-related death. Here, we investigated the clinicopathological significance of the association of octamer-binding transcription factor 4 (OCT4) with lymphoid enhancer-binding factor 1 (LEF1) expression and the potential molecular mechanism in the epithelial-mesenchymal transition (EMT), invasion, and migration of ESCC. The expression of OCT4 and LEF1 was detected via immunohistochemistry analysis. High levels of LEF1 expression were observed in 95 ESCC specimens and were obviously associated with aberrant clinicopathological features and poor patient prognosis. Our previous study showed that OCT4 expression level is elevated in ESCC, and statistical analysis showed that the elevated expression of OCT4 and LEF1 in ESCC was significantly associated with histologic grade, lymph node metastasis, TNM stage, and poor patient prognosis. The specific inhibition of OCT4 expression via a lentivirus encoding OCT4-shRNA (LV-shOCT4) in Eca109 cells led to decreased levels of OCT4 and LEF1 in vitro. Additionally, we applied a rescue strategy by infecting LV-shOCT4 Eca109 cells with a LEF1 overexpression plasmid (p-LEF1) and detected changes in EMT, migration, and invasion. Unsurprisingly, the p-LEF1 group exhibited greater EMT, invasion, and migration than did the LV-shOCT4 and negative control groups. This study demonstrates for the first time the relationship between OCT4 and LEF1 expression. The combination of high expression of OCT4 and LEF1 was associated with clinicopathological features of atypical patients, and this combination might be an ideal prognostic factor in ESCC. OCT4 positively regulated LEF1 expression, and LEF1 mediated the effects of OCT4 in cancer cell EMT, invasion, and migration. The data presented here suggest that the inhibition of OCT4-LEF1 signaling may be a new therapeutic target for the treatment of ESCC.

Comparison of detergent-based decellularization protocols for the removal of antigenic cellular components in porcine aortic valve.

BACKGROUND: Various detergent-based protocols are used to remove cells and cellular debris in porcine aortic valve (PAV). However, the removal of antigenic cellular components has not been thoroughly elucidated to date. In this study, we used 4 detergent-based protocols to decellularize PAVs and aimed to evaluate their effects on removing antigenic cellular components. METHODS: Porcine aortic valves were decellularized using sodium dodecyl sulfate (SDS), SDS in combination with sodium deoxycholate (SDS/SD), Triton X-100, and Triton X-100 in combination with SD (Triton X-100/SD), respectively. Untreated PAVs were used as controls. Immunohistochemical and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed to determine the removal of antigenic protein components. Histological, biochemical, and biomechanical analyses were performed to determine the preservation and mechanical properties of the extracellular matrix. PAV tissues were implanted subcutaneously in Sprague Dawley rats to evaluate the host immune response. Implanted PAVs were taken out at the indicated time points for histological and immunohistochemical examinations. RESULTS: All 4 protocols effectively removed the membrane antigenic proteins major histocompatibility complex I molecule and galactose-α-1,3 galactose. The SDS/SD protocol was the most effective method to remove the cytoplasmic cytoskeletal proteins, vimentin and α-SMA, and SDS alone partly removed vimentin protein. The SDS/SD protocol was the most effective method to remove nuclear DNA with residual DNA below 50 ng/mg, followed by the SDS protocol with residual DNA of 74.9 ng/mg. The SDS protocol was the most effective method to remove proteins ranged from 10 to 55 kDa, followed by the SDS/SD protocol. SDS and SDS/SD PAV implants attracted fewer neutrophils in vivo on postoperative day 3. The infiltration of macrophages and T lymphocytes was significantly lower in SDS and SDS/SD implants on days 14 and day 28. All of the decellularized protocols that were examined greatly reduced the contents of collagen, elastin, and glycosaminoglycan compared to controls. Biomechanical analysis revealed significant differences in ultimate tensile strength and Young's modulus between control PAVs and decellularized PAVs generated using SDS, SDS/SD, Triton X-100, or Triton X-100/SD. CONCLUSIONS: These results indicated that SDS-based protocols more effectively removed antigenic cellular components compared to Triton X-100-based protocols. These results are clinically significant because complete removal of antigenic determinants is critical to decrease adverse immune-mediated and inflammatory responses to a PAV when used in xenogeneic application.

Efficient decellularization for bovine pericardium with extracellular matrix preservation and good biocompatibility.

OBJECTIVES: In this study, we sought to explore an efficient decellularization protocol for bovine pericardia with better extracellular matrix preservation and good biocompatibility. METHODS: Bovine pericardia were decellularized by sodium dodecyl sulphate (SDS), SDS + sodium deoxycholate (SD), Triton X-100 (TX), TX + SD (TS), freeze-thaw cycles + SDS + SD (FSS) and freeze-thaw cycles + TX + SD (FTS), respectively. Untreated pericardia were used as native control. Histological examination, residual cellular content analysis, biochemical and biomechanical evaluations and cytotoxicity assay were performed to investigate decellularization efficiency, xenoantigens removal, extracellular matrix preservation and biocompatibility. In vivo biocompatibility was evaluated using a subcutaneous implantation method in rats. RESULTS: Among these protocols, FSS and FTS protocols were the most effective methods to remove both the DNA material and the galactose-α-1,3-galactose antigen. TX, TS and FTS bovine pericardia maintained the collagen content and had no cytotoxicity to human umbilical vein endothelial cells. The contents of elastin and glycosaminoglycan were lost to different degrees after decellularization, with the highest content of preservation with TX, followed by TS and FTS. Consistently, no significant difference was found between native bovine pericardia and TX, TS or FTS bovine pericardia. In vivo, FTS implants had minimal infiltration of macrophages and T-lymphocytes, with no histological evidence of peri-implant necrosis and calcification. CONCLUSIONS: These results suggested that the FTS protocol showed optimal decellularization results with better extracellular matrix preservation and good biocompatibility. It may be a suitable protocol for producing a suitable scaffold for heart tissue engineering.

Pericardial interstitial cell senescence responsible for pericardial structural remodeling in idiopathic and postsurgical constrictive pericarditis.

OBJECTIVE: Idiopathic and postsurgical constrictive pericarditis is characterized by pericardial structural remodeling that involves fibrosis, calcification, and inflammation. This study aimed to determine whether cell senescence was responsible for pericardial structural remodeling. METHODS: Pericardial interstitial cells derived from patients with idiopathic or postsurgical pericarditis (pericarditis cells) were harvested. Timing of senescence and differences in telomere length were compared between age- and sex-matched controls (nonpericarditis cells). Pericardial interstitial cells derived from normal pericardia were serially passaged until senescence (senescent cells). Apoptosis, collagen matrix, calcium deposition, chemoattractant properties, gene expression profiles, and paracrine effects of senescent cells were compared with nonsenescent cells of passage 2 (nonsenescent cells). RESULTS: Pericarditis cells displayed senescent changes, including short telomere length, large flattened cell sizes, positive staining for senescence-associated ß-galactosidase, and limited growth capacity. These senescent cells were resistant to apoptosis, produced more collagen matrix, deposited more calcium, and attracted more monocytes/lymphocytes than the nonsenescent cells. A cluster of genes involved in extracellular matrix deposition (connective tissue growth factor, fibronectin, collagen type I, collagen type III, and tissue inhibitors of metalloproteinase-1), calcium deposition (osteopontin, bone sialoprotein, osteonectin, and matrix Gla protein), and inflammatory cell recruitment (interleukin-6, chemoattractant protein-1, and tumor necrosis factor-α) were upregulated in senescent cells, whereas extracellular matrix-degrading enzyme (metalloproteinase-1 and metalloproteinase-3) was downregulated. Furthermore, senescent cells had the ability to promote the proliferation, differentiation, and senescence of neighboring cells. CONCLUSIONS: These findings suggest that senescent cells have characteristics promoting pericardial structural remodeling, but further work is needed to establish causation.

The size-effect of gold nanoparticles and nanoclusters in the inhibition of amyloid-ß fibrillation.

A significant pathological signature of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) plaques in the brain and the synaptic dysfunction and neurodegeneration associated with it. Compounds or drugs that inhibit Aß fibrillation are thus desirable to develop novel therapeutic strategies against AD. Conventional strategies usually require an elaborate design of their molecular structures. Here we report the size-effect of gold nanoparticles (AuNPs) and nanoclusters (AuNCs) in the inhibition of protein amyloidosis. Using l-glutathione stabilized AuNPs with different sizes and AuNCs as examples, we show that large AuNPs accelerate Aß fibrillation, whereas small AuNPs significantly suppress this process. More interestingly, AuNCs with smaller sizes can completely inhibit amyloidosis. Dynamic light scattering (DLS) experiments show that AuNCs can efficiently prevent Aß peptides from aggregation to larger oligomers (e.g. micelles) and thus avoid nucleation to form fibrils. This is crucially important for developing novel AD therapies because oligomers are the main source of Aß toxicity. This work presents a novel strategy to design anti-amyloidosis drugs, which also provides interesting insights to understand how biological nanostructures participate in vivo in Aß fibrillation from a new perspective.

Twist-related protein 1 negatively regulated osteoblastic transdifferentiation of human aortic valve interstitial cells by directly inhibiting runt-related transcription factor 2.

OBJECTIVE: Valve calcification involves transdifferentiation of valve interstitial cells (VICs) into osteoblasts. Twist-related protein 1 (TWIST1) has been established as a negative regulator of osteoblast differentiation in both mouse and human mesenchymal stem cells, but its function in human aortic VICs is unknown. In our study, we determined the mechanism of TWIST1 action in regulating osteoblastic transdifferentiation of human aortic VICs. METHODS: Human calcified and noncalcified aortic valves were examined for TWIST1 expression. Human aortic VICs were isolated and cultured. RESULTS: The data showed that calcified aortic valves express lower levels of TWIST1. In vitro experiments showed that TWIST1 overexpression inhibited the transdifferentiation of VICs into osteoblasts by decreasing the expression of runt-related transcription factor 2 (RUNX2) and its downstream osteoblastic markers. Through chromatin immunoprecipitation and dual luciferase assays, we found that TWIST1 repressed the expression of RUNX2 by directly binding to an E-box located at -820 bp of the RUNX2 P2 promoter region and inhibiting its activity. CONCLUSIONS: Our study results suggest that TWIST1 could play an important role in preventing human aortic valve calcification by negatively regulating osteoblastic transdifferentiation of human aortic VICs through direct inhibition of RUNX2.

Tumor cells positive and negative for the common cancer stem cell markers are capable of initiating tumor growth and generating both progenies.

The cancer stem cell (CSC) model depicts that tumors are hierarchically organized and maintained by CSCs lying at the apex. CSCs have been "identified" in a variety of tumors through the tumor-forming assay, in which tumor cells distinguished by a certain cell surface marker (known as a CSC marker) were separately transplanted into immunodeficient mice. In such assays, tumor cells positive but not negative for the CSC marker (hereby defined as CSC(+) and CSC(-) cells, respectively) have the ability of tumor-forming and generating both progenies. However, here we show that CSC(+) and CSC(-) cells exhibit similar proliferation in the native states. Using a cell tracing method, we demonstrate that CSC(-) cells exhibit similar tumorigenesis and proliferation as CSC(+) cells when they were co-transplanted into immunodeficient mice. Through serial single-cell derived subline construction, we further demonstrated that CSC(+) and CSC(-) cells from CSC marker expressing tumors could invariably generate both progenies, and their characteristics are maintained among different generations irrespective of the origins (CSC(+)-derived or CSC(-)-derived). These findings demonstrate that tumorigenic cells cannot be distinguished by common CSC markers alone and we propose that cautions should be taken when using these markers independently to identify cancer stem cells due to the phenotypic plasticity of tumor cells.

MSCs transfected with hepatocyte growth factor or vascular endothelial growth factor improve cardiac function in the infarcted porcine heart by increasing angiogenesis and reducing fibrosis.

BACKGROUND: Cell transplantation and gene therapy have been demonstrated to have beneficial effects after a myocardial infarction (MI). Here, we used a large animal model of MI to investigate the beneficial effects of mesenchymal stem cells (MSCs) transfected with hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) genes. METHODS: A porcine MI model was created by balloon occlusion of the distal left anterior descending artery for 90 min followed by reperfusion. At 1 week after MI, the pigs were infused via the coronary vein with saline (n=8), MSCs + AdNull(n=8), MSC+VEGF(n=10), or MSC+HGF(n=10). Cardiac function and myocardial perfusion were evaluated by using echocardiography and gated cardiac perfusion imaging before and 4 weeks after transplantation. Morphometric and histological analyses were performed. RESULTS: All cell-implanted groups had better cardiac function than the saline control group. There were further functional improvements in the MSC+HGF group, accompanied by smaller infarct sizes, increased cell survival, and less collagen deposition. Blood vessel densities in the damaged area and cardiac perfusion were significantly greater in the MSC+AdNull group than in the saline control group, and further increased in the MSC+VEGF/HGF groups. Tissue fibrosis was significantly less extensive in the MSC and MSC+VEGF groups than in the saline control group and was most reduced in the MSC+HGF group. CONCLUSION: MSCs (alone or transfected with VEGF/HGF) delivered into the infarcted porcine heart via the coronary vein improved cardiac function and perfusion, probably by increasing angiogenesis and reducing fibrosis. MSC+HGF was superior to MSC+VEGF, possibly owing to its enhanced antifibrotic effect.

Aortic valve replacement with autologous pericardium: long-term follow-up of 15 patients and in vivo histopathological changes of autologous pericardium.

OBJECTIVES: The study aimed to assess the long-term follow-up of patients with an autologous pericardial aortic valve (APAV) replacement and to analyse in vivo histopathological changes in implanted APAVs. METHODS: From 1996 to 1997, 15 patients (mean age, 34 years) underwent aortic valve replacement with the glutaraldehyde-treated autologous pericardium. All patients were followed up after discharge. The excised APAVs were processed for haematoxylin-eosin, Victoria blue-van Gieson and immunohistochemical staining. RESULTS: The mean clinical follow-up was 11.43 ± 4.50 years. APAV-related in-hospital and late mortalities were both 0%. Five (33%) patients required reoperation because of a prolapse of the right coronary cusp (n = 1), infective endocarditis (n = 1) or fibrocalcific degeneration (n = 3). Freedom from endocarditis, fibrocalcific degeneration and reoperation at the end of follow-up was 93, 80 and 67%, respectively. The remaining 10 patients were alive and well with a mean New York Heart Association class of 1.10 ± 0.32 and normally functioning aortic valves (peak pressure gradient: 7.70 ± 3.41 mmHg; mean pressure gradient: 1.79 ± 0.64 mmHg). Histopathology revealed that (i) a thin factor VIII-positive layer (endothelialization) was found on all non-endocarditis APAVs; (ii) pericardial cells in all APAVs were positive for α-smooth muscle actin (myofibroblast phenotype) and some cells in the fibrocalcific APAVs were positive for alkaline phosphatase (osteoblast phenotype) and (iii) an elastic band was found in 3 cases (in vivo >9 years). CONCLUSIONS: APAV replacement is a procedure with a low mortality. APAVs adapt to new environmental demands by producing an elastic band and by endothelialization, whereas myofibroblast/osteoblast transdifferentiation seems to be responsible for the fibrocalcification of APAVs.

Characteristics of pericardial interstitial cells and their implications in pericardial fibrocalcification.

Pericardial fibrocalcification (PF) is a prominent feature of human pericardial pathology, including constrictive pericarditis and, to a lesser extent, degenerated autologous pericardial substitutes. However, the role of pericardial interstitial cells (PICs) in the pathogenesis of PF has yet to be established. Using a combination of histology and immunohistochemistry, we showed that the critical cellular event in PF in situ was the transdifferentiation of PICs into myofibroblasts/osteoblasts and that the percentage of myofibroblasts/osteoblasts correlated positively with the severity of PF. In vitro studies demonstrated that PICs, similar to mesenchymal stem cells, had the potential to differentiate along adipogenic, osteogenic, chondrogenic or myogenic lineages. However, PICs exhibited a more limited self-renewal capacity and a lower expression of Oct4 (POU5F1) and Kruppel-like transcription factor Klf4, underwent earlier senescence and spontaneously transdifferentiated into myofibroblasts/osteoblasts. Quantitative-real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed that the mRNA levels of α-smooth muscle actin (α-SMA), alkaline phosphatase (ALP), core-binding factor α1/runt-related transcription factor2 (Cbfa1/Runx2), transforming growth factor (TGF)-ß1 and bone morphogenetic protein (BMP)-2 were upregulated as the passage number increased. The mRNA level of platelet-derived growth factor (PDGF)-AA was also significantly upregulated with higher levels at passage 3. Ectopic expression of Oct4 and Klf4 enhanced the colony formation of PICs and selectively impaired induction of genes involved in transdifferentiation into myofibroblasts/osteoblasts (α-SMA, ALP, Cbfa1/Runx2, PDGF-AA and BMP-2). These data, while offering new insights into the biology of PICs, reinforce the central role of these cells in cell-mediated PF and may assist in future strategies to treat fibrocalcific pericardial diseases.

MicroRNA-98 and microRNA-214 post-transcriptionally regulate enhancer of zeste homolog 2 and inhibit migration and invasion in human esophageal squamous cell carcinoma.

BACKGROUND: The enhancer of zeste homolog 2 (EZH2) was found to be overexpressed and associated with tumor metastasis in esophageal squamous cell carcinoma (ESCC). On the other hand, it was reported that miR-26a, miR-98, miR-101, miR-124, miR-138 and miR-214 could inhibit the expression of EZH2 in some tumors. However, the role of miRNAs in the regulation of EZH2 expression in human ESCC has not been documented. The aim of this study was to determine the role of these miRNAs in the regulation of tumor metastasis via EZH2 overexpression in human ESCC. METHODS AND RESULTS: The expression of these miRNAs and EZH2 mRNA were examined by qPCR and the expression of EZH2 protein was detected by western blot. The role of these miRNAs in migration and invasion was studied in ESCC cell line (Eca109) transfected with miRNA mimics or cotransfected with miRNA mimics and pcDNA-EZH2 plasmid (without the 3'-UTR of EZH2). Through clinical investigation, we found that miR-98 and miR-214 expression was significantly lower in ESCC tissues than in matched normal tissues, and the expression level of miR-98 and miR-214 was inversely correlated to EZH2 protein expression and the clinical features such as pathological grade, tumor stage and lymph node metastasis in ESCC. In Eca109 cells, overexpression of miR-98 and miR-214 significantly inhibited the migration and invasion of ESCC cells, which was reversed by transfection of EZH2. CONCLUSIONS: These findings suggest that decreased expression of miR-98 and miR-214 might promote metastasis of human ESCC by inducing accumulation of EZH2 protein.

[Establishment and characterization of a human lung adenocarcinoma cell line, Ch-Huang-1].

BACKGROUND AND OBJECTIVE: Lung cancer is a major worldwide health problem. The aim of this study is to establish a novel Chinese human lung adenocarcinoma cell line and examine its biological characteristics. METHODS: Lung adenocarcinoma specimens were freshly resected during surgery. The tissues were incubated in vitro and the cell line was named Ch-Huang-1. The biological characteristics of the cells were investigated by light microscopy, chromosome analysis, and transplantation experiment. RESULTS: Light microscopy revealed that cells from the primary tumor, Ch-Huang-1 cell line, and transplanted tumor possessed the characteristics of a malignant glandular epithelial tumor. The cell growth curve, doubling time, and mitotic index were also observed in vitro. Nuclear chromosome analysis revealed that the tumor was a subtriploid with a mode of 35-44 per cell. Tumor nodes were observed under the skin of nude mice by heterogenic transplantation. CONCLUSION: The characteristics of the established cell line suggest that it is a newly established human adenocarcinoma cell line.

PRAF3 induces apoptosis and inhibits migration and invasion in human esophageal squamous cell carcinoma.

BACKGROUND: Prenylated Rab acceptor 1 domain family member 3 (PRAF3) is involved in the regulation of many cellular processes including apoptosis, migration and invasion. This study was conducted to investigate the effect of PRAF3 on apoptosis, migration and invasion in human esophageal squamous cell carcinoma (ESCC). METHODS: The expression of PRAF3 mRNA and protein in primary ESCC and the matched normal tissues (57cases) was determined by quantitative RT-PCR and Western blot. Immunohistochemical analysis of PRAF3 expression was carried out in paraffin-embedded sections of ESCC and correlated with clinical features. The role of PRAF3 in apoptosis, migration and invasion was studied in ESCC cell lines of Eca109 and TE-1 through the adenovirus mediated PRAF3 gene transfer. The effect of PRAF3 on apoptosis was analyzed by annexin V-FITC assay. The regulation of PRAF3 on migration was determined by transwell and wounding healing assay, while the cellular invasion was analyzed by matrigel-coated transwell assay. RESULTS: We found that the expression of PRAF3 was significantly down-regulated in ESCC tissue compared with the matched normal tissue and was correlated with the clinical features of pathological grade, tumor stage and lymph node metastasis. Moreover, overexpression of PRAF3 induced cell apoptosis through both caspase-8 and caspase-9 dependent pathways, and inhibited cell migration and invasion by suppressing the activity of both MMP-2 and MMP-9 in human ESCC cell lines. CONCLUSIONS: Our data suggest that PRAF3 plays an important role in the regulation of tumor progression and metastasis and serves as a tumor suppressor in human ESCC. We propose that PRAF3 might be used as a potential therapeutic agent for human ESCC.

[The influence of inhibiting angiopoietin-2 on the biological characteristics of bronchogenic adenocarcinoma].

BACKGROUND AND OBJECTIVE: It is well-known that angiopoietin-2 (Ang-2) plays an important role in the formation of the blood vascular system. Angiopoietin is involved in many diseases about angiogenesis such as tumor, so may have great prospects for the treatment of these diseases. The aim of this study is to evaluate the influence of inhibiting Ang-2 via adeno-associated virus induced RNA interference (RNAi) on the biological characteristics of bronchogenic adenocarcinoma. METHODS: AAV-Ang-2shRNA driven by H1 promoter was constructed to transfect A549 cell line. Normal and AAV-Null cell line were utilized in the control groups. The influence of RNAi on Ang-2 expression as well as the growth rate, tumorigenic efficiency, proliferation rate, apoptosis, and microvessel density of A549 cell line were analyzed. RESULTS: In vitro experiment indicated that the Ang-2 expression level (P<0.001) and growth rate (P<0.001) of A549 cell line 48 h transfected with AAV-Ang-2shRNA were significantly lower than those in the normal and AAV-Null cell lines. Cell cycle analysis showed the proliferation index (PI) of normal, AAV-Null, and AAV-Ang-2shRNA transfected A549 cell line were 0.51± 0.43, 0.48 ± 0.29, and 0.26 ± 0.31, respectively, which indicated the PI of AAV-Ang-2shRNA transfected cell line was significantly lower, compared with the normal and AAV-Null cell lines. In vivo experiment exhibited that AAV-Ang-2shRNA transfected cell line possessed a lower mass and volume of tumor relative to two control groups. In addition, the apoptosis index (AI) of AAV-Ang-2shRNA transfected, normal, and AAV-Null cell lines were (5.98 ± 3.11)%, (7.51 ± 4.42)% and (17.06 ± 7.43)% respectively, which manifested that AAV-Ang-2shRNA transfected cell line possessed a higher AI (P=0.005, P=0.007). A lower percentage of PCNA-positive cell was observed in AAV-Ang-2shRNA transfected cell line (92.75 ± 9.7)% as well, compared with the normal (85.8 ± 11.8)% and AAV-Null (69.8 ± 16.5)% cell lines. CONCLUSIONS: AAV-mediated expression of shRNA significantly reduces concentration of Ang-2 in A549 cell line, lowers proliferation and growth rate and induce .apoptosis of A549 cell line.