Apelin attenuates the osteoblastic differentiation of aortic valve interstitial cells via the ERK and PI3-K/Akt pathways.

Aortic valve calcification (AVC), which used to be recognized as a passive and irreversible process, is now widely accepted as an active and regulated process characterized by osteoblastic differentiation of aortic valve interstitial cells (AVICs). Apelin, the endogenous ligand for G-protein-coupled receptor APJ, was found to have protective cardiovascular effects in several studies. However, the effects and mechanisms of apelin on osteoblastic differentiation of AVICs have not been elucidated. Using a pro-calcific medium, we devised a method to produce calcific human AVICs. These cells were used to study the relationship between apelin and the osteoblastic calcification of AVICs and the involved signaling pathways. Alkaline phosphatase (ALP) activity/expression and runt-related transcription factor 2 (Runx2) expression were examined as hallmark proteins in this research. The involved signaling pathways were studied using the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002. The results indicate that apelin attenuates the expression and activity of ALP, the expression of Runx2, and the formation of mineralized nodules. This protective effect was dependent on the dose of apelin, reaching the maximum at 100 pM, and was connected to activity of ERK and Akt (a downstream effector of PI3-K). The activation of ERK and PI3-K initiated the effects of apelin on ALP activity/expression and Runx2, but PD98059 and LY294002 abolished the effect. These results demonstrate that apelin attenuates the osteoblastic differentiation of AVICs via the ERK and PI3-K/Akt pathway.

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway.

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free ß-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing ß-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

[Correlation of serum levels of VEGF and SDF-1 with the number and function of circulating EPCs in children with cyanotic congenital heart disease].

OBJECTIVE: To examine the number and function of circulating endothelial progenitor cells (EPCs) in children with cyanotic congenital heart diseases (CHD) and study their correlation with serum levels of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1). METHODS: Fifteen children with tetralogy of Fallot (cyanotic group) and 15 age-and sex-matched children with ventricular septal defect (control group) were enrolled. Serum levels of VEGF and SDF-1 were measured using ELISA. Mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation and cultured in vitro. EPCs were identified by immunofluorescence and were counted under a microscope. Modified Boyden chamber assay and the MTT assay were used to measure the migration and proliferation capacities of EPCs. EPCs adhesion ability assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. The correlations of serum levels of VEGF and SDF-1 with the number and function of circulating EPCs were assessed by linear regression analysis. RESULTS: Serum levels of VEGF (201.42+/-44.74 ng/L vs 113.56+/-35.62 ng/L; P<0.05) and SDF-1 (3.45+/-1.07 ng/L vs 1.05+/-0.99 ng/L; P<0.05) in the cyanotic group were higher than those in the control group. There was a positive correlation between serum levels of VEGF and SDF-1(r=0.675, P<0.01). The number of EPCs (*200 field) in the cyanotic group significantly increased compared with that of the control group (72.2+/-9.73 vs 51.2+/-3.83; P<0.01). The functional activities of EPCs, including proliferation, migration and adhesion capacities, were augmented in the cyanotic group compared with those in the control group. The increased number and function of EPCs and the increased serum levels of VEGF and SDF-1 were consistent in the cyanotic group, with a correlation coefficient of 0.8395, 0.5491, 0.6376 and 0.7392 respectively. CONCLUSIONS: The number and functional activity of EPCs as well as serum levels of VEGF and SDF-1 increased in children with cyanotic CHD. Serum levels of VEGF and SDF-1 were correlated to the number and functional activity of EPCs. Serum VEGF and SDF-1 together with circulating EPCs may play important roles in the pathology and physiology in these patients.

Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds.

Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional (3D) scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as 3D tumor engineering scaffolds for cancer research. STATEMENT OF SIGNIFICANCE: Natural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor ECMs are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

[Effect of decellular treatment on the framework of extracellular matrix in bovine jugular vein conduit].

OBJECTIVE: To investigate the effect of decellular treatment on the framework constituents of extracellular matrix and tissue stability in bovine jugular vein conduit (BJVC), and to provide an evidence for tissue engineering of vascular prosthesis. METHODS: Bovine jugular veins were obtained fresh from a local slaughterhouse and were stored in chilled PBS. In the laboratory, any fat and loose connective tissue on the outer surface of the vessel was trimmed. BJVCs were decellularized by a 3-step extraction method as detergent Triton X-100 (0.5%), Trypsin (0.025%) EDTA (0.02%), and DNase I(30kU/L) RNaseA(0.3g/L). Histological and transmission electron microscopy (TEM) techniques were used to study the framework constituents of extracellular matrix of treated the examples, and fresh tissues were used as controls. Tissue contents of hydroxyproline(alkaline hydrolysis method) and elastin (Fastin Elastin Assay) were assayed respectively in the fresh and decellularized groups (n=10). The vascular wall heat shrinking temperature and mechanical strength were measured to evaluate the tissue stability (n=10). RESULTS: Histochemical and TEM analysis of BJVCs treated with decellularization proved a complete removal of nuclear and other cell components. Tissue collagen was well kept,but elastin was partly lessened. Tissue content of hydroxyproline increased comparatively [(25.73+/-2.97)mg/g vs. (29.25+/-2.99)mg/g, P<0.05] and the elastin content obviously decreased [(159.71+/-21.06)mg/g vs. (134.91+/-35.40)mg/g, P<0.05] in the decellular treatment group compared with the control group. The heat shrinking temperature and tensile stress of decelluarized tissue were lower than those of the fresh tissue[(72.50+/-0.53) degrees C vs. (69.75+/-0.54)degrees C ,P<0.05], [(5.19+/-0.65)MPa vs. (3.13+/-0.94)MPa, P<0.05]. CONCLUSION: The basic framework of extracellular matrix in the decellularized BJVC is partly damaged and tissue stability is reduced. Decellularized BJVC should be further crosslinked before being used as a tissue engineering scaffold for clinical pulmonary artery graft.

[Bovine jugular venous conduit treated with the polyepoxy compound].

OBJECTIVE: To determine the feasibility whether the bovine jugular venous conduit (BJVC) can be fixed with polyepoxy compound (PC). METHODS: Twenty-four BJVCs were divided into 3 groups and fixed with polyepoxy compound (PC group, n = 8), glutaraldehyde (GA group, n = 8), and unfixed group (Control group, n = 8), respectively. The morphologic and mechanical properties of BJVCs in the 3 groups, including thickness, diameter, moisture content, denaturation temperature, tensile strength, elongation at break, and fixation index were measured. The rat subcutaneous model for the assessment of tissue calcification was used. The calcium content in bovine jugular vein patches and valves was determined by flame atomic absorption spectrophotometer. RESULTS: There was no difference in the wall thickness, diameter, and tissue water content between PC and the control group, but significant difference was found between GA and PC groups. The mechanical properties of PC group and GA group were not significantly different, but they were better than those of the control group. GA-fixed BJVC samples showed clear calcification, while PC fixed BJVC were calcified significantly less. CONCLUSION: PC is an effective and suitable choice for the treatment of BJVC since it can effectively preserve the structure and the anti-reflow function of valves in bovine jugular vein and it has better anti-calcification properties.

[Augmentation of revascularization by implantation of autologous bone marrow mononuclear cells in a rat ischemic hind limb model].

OBJECTIVE: To examine whether the in vivo implantation of autologous bone-marrow mononuclear cells (BM-MNCs) can augment postnatal neovascularization in rat model of hind limb ischemia. METHODS: Rat BM-MNCs were isolated by centrifugation through a Ficoll-paque density gradient. The rat ischemic hind limb model was made by ligation of the right femoral artery and its branches of imbred Wistar rats. BM-MNCs (MNC group) or phosphate saline buffer (PBS group) were injected into 7 points of the ischemic muscles (20 microl/point). To assess angiogenesis, histologic evaluation was performed on tissue slices from adductor muscles with immunohistochemical staining at the 2nd and 4th weeks after the ligation. Severity of ischemic insult was evaluated by the femoral arteriovenous oxgen difference (AVDO2) at the 2nd and 4th weeks after the ligation. RESULTS: The MNC groups had a higher capillary density and higher capillary/muscle fiber ratios than that of the sham and PBS groups (P < 0.01) at the 2nd and 4th weeks after the ligation. AVDO2 of the MNC group significantly decreased compared with that of the PBS groups (P < 0.05). CONCLUSION: The implantation of autologous BM-MNCs induced neovascularization in a rat ischemic hind limb model. The implantation of autologous BM-MNCs augmented the collateral perfusion of ischemic hind limb.

Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering.

Tumor engineering is defined as the construction of three-dimensional (3D) tumors in vitro with tissue engineering approaches. The present 3D scaffolds for tumor engineering have several limitations in terms of structure and function. To get an ideal 3D scaffold for tumor culture, A549 human pulmonary adenocarcinoma cells were implanted into immunodeficient mice to establish xenotransplatation models. Tumors were retrieved at 30-day implantation and sliced into sheets. They were subsequently decellularized by four procedures. Two decellularization methods, Tris-Trypsin-Triton multi-step treatment and sodium dodecyl sulfate (SDS) treatment, achieved complete cellular removal and thus were chosen for evaluation of histological and biochemical properties. Native tumor tissues were used as controls. Human breast cancer MCF-7 cells were cultured onto the two 3D scaffolds for further cell growth and growth factor secretion investigations, with the two-dimensional (2D) culture and cells cultured onto the Matrigel scaffolds used as controls. Results showed that Tris-Trypsin-Triton multi-step treated tumor sheets had well-preserved extracellular matrix structures and components. Their porosity was increased but elastic modulus was decreased compared with the native tumor samples. They supported MCF-7 cell repopulation and proliferation, as well as expression of growth factors. When cultured within the Tris-Trypsin-Triton treated scaffold, A549 cells and human colorectal adenocarcinoma cells (SW-480) had similar behaviors to MCF-7 cells, but human esophageal squamous cell carcinoma cells (KYSE-510) had a relatively slow cell repopulation rate. This study provides evidence that Tris-Trypsin-Triton treated acellular tumor extracellular matrices are promising 3D scaffolds with ideal spatial arrangement, biomechanical properties and biocompatibility for improved modeling of 3D tumor microenvironments.

MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells.

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with ß-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

Calcification resistance for photooxidatively crosslinked acellular bovine jugular vein conduits in right-side heart implantation.

This study aimed to investigate the effect of decellularization plus photooxidative crosslinking and ethanol pretreatment on bioprosthetic tissue calcification. Photooxidatively crosslinked acellular (PCA) bovine jugular vein conduits (BJVCs) and their photooxidized controls (n = 5 each) were sterilized in a graded concentration of ethanol solutions for 4 h, and used to reconstruct dog right ventricular outflow tracts. At 1-year implantation, echocardiography showed similar hemodynamic performance, but obvious calcification for the photooxidized BJVC walls. Further histological examination showed intense calcium deposition colocalized with slightly degraded elastic fibers in the photooxidized BJVC walls, with sparsely distributed punctate calcification in the valves and other areas of walls. But PCA BJVCs had apparent degradation of elastic fibers in the walls, with only sparsely distributed punctate calcification in the walls and valves. Content assay demonstrated comparable calcium content for the two groups at preimplantation, whereas less calcium for the PCA group in the walls and similar calcium in the valvular leaflets compared with the photooxidized group at 1-year retrieval. Elastin content assay presented the conduit walls of PCA group had less elastin content at preimplantation, but similar content at 1-year retrieval compared with the photooxidized group. Phospholipid analysis showed phospholipid extraction by ethanol for the PCA group was more efficacious than the photooxidized group. These results indicate that PCA BJVCs resist calcification in right-side heart implantation owing to decellularization, further photooxidative crosslinking, and subsequent phospholipid extraction by ethanol at preimplantation.

The performance of photooxidatively crosslinked acellular bovine jugular vein conduits in the reconstruction of connections between pulmonary arteries and right ventricles.

In this study, valved photooxidatively crosslinked acellular bovine jugular vein conduits (BJVCs) were implanted in young dogs to reconstruct the connections of pulmonary arteries and right ventricles, with acellular conduits used as controls. All acellular conduits had moderate to severe valvular dysfunction and were explanted at 1-month implantation (n = 5). Histological examination showed inflammatory cell infiltration and intimal hyperplasia in the walls, and severe inflammatory cell infiltration and thrombosis in the valves. The photooxidatively crosslinked acellular conduits were retrieved at 1-month (n = 5) and 6-month (n = 5) implantations respectively. These conduits had excellent valvular function at retrieval. Their walls and valves were still soft and smooth without calcification and hemangioma. Endothelialization in valves and luminal walls was unsatisfied at 1-month retrieval, and was improved at 6-month retrieval. Host cells infiltrated and migrated from outer layer to the middle layer, with tissue remolding and regeneration found in these recellular regions. Histological examination and tissue content assay demonstrated that degeneration and regeneration of collagens and glycosaminoglycans were comparable, but elastic fibers gradually degraded. Photooxidatively crosslinked acellular BJVCs resist calcification and thrombosis and have regeneration patterns, with excellent hemodynamic performance.

Detection of nanobacteria-like material from calcified cardiac valves with rheumatic heart disease.

BACKGROUND: Nanobacterium contributes to pathological calcification in human renal stones and psammoma bodies in ovarian cancer. Pathological calcification is also present in cardiac valves with rheumatic heart disease. The aim of this study was to detect, isolate, culture, and characterize nanobacteria-like material from human calcified cardiac valves with rheumatic heart disease. METHODS: Normal and calcified cardiac valve groups, as well as positive (nanobacteria strain Se90) and negative (serum radiated with 30 kGy of γ-ray) control groups, were included in this study. Part of each valve was immunostained with nanobacterial antibody 8D10, and the remaining parts were homogenized, filtered, and maintained in culture. The cultures were checked with a microscope weekly. Culture medium at different time points was analyzed with a spectrophotometer. The cultures maintained for 3 weeks were further examined with immunofluorescence double staining and transmission electron microscopy. RESULTS: While 26 of 29 calcified valves stained positive for 8D10 antibody, all normal valves stained negative. Mobile tiny particles were observed under a microscope in the calcified valve group and the Se90 group. Optical densities were significantly different among groups (P<.001). Immunofluorescence double staining displayed tiny green fluorescence particles in the calcified valve group, in the Se90 group, and in two samples of the normal valve group. Transmission electron microscopy analysis indicated that cultured particles from calcified valves ranging in size from 88 to 341 nm had an obvious cell membrane structure similar to that of Se90. CONCLUSIONS: The nanobacteria-like material has been isolated and cultured from calcified cardiac valves with rheumatic heart disease, and its characteristics are similar to those of Se90.

[Establishment of patient-derived xenotransplantation models for non-small cell lung cancer in immune deficient mice].

BACKGROUND AND OBJECTIVE: Targeted therapies have become a valuable therapeutic option for cancer. Establishment of different animal tumor models has become necessary. This study was to establish xenotransplantation models for patient-derived non-small cell lung cancer (NSCLC) in immune deficient mice. METHODS: Immune deficient mice, BALB/C-nu, NOD/scid and SCID, 16 in each strain, were used. Sixteen tumor specimens were obtained from patients with NSCLC. Each specimen was subcutaneously transplanted into one mouse from each of the three strains. The tumor formation rate, time to tumor engraftment, tumor volume doubling time were recorded and compared among the three strains of mice. Histology of xenograft tumors was examined. RESULTS: The total tumor formation rate was 75% (12/16). The tumor formation rate was the highest in SCID mice (56.25%). Only four tumors were engrafted in SCID mice, and two in BALB/C-nu mice. The tumor formation rate, time to tumor engraftment, and tumor volume doubling time were not significantly different among the three strains of mice. The incidence of tumor size over 1cm in the upper flanks of the mice (56.25%) was significantly higher than that in the lower flanks (25%) (P=0.037). Haematoxylin Eosin staining revealed a high degree of histological similarity between all xenograft and the parental tumors. CONCLUSIONS: We have established xenotransplantation models for patient-derived NSCLC with a success rate of 75% in BALB/C-nu and SCID mice. The xenograft tumors have the same histological features to those of their parental tumors.