Protein P37 of mycoplasma hyorhinis induces secretion of TNF-α from human peripheral blood mononuclear cells

High mycoplasmal infection ratio in gastric cancer tissues suggests a possible association between my-coplasma infection and tumorigenesis.Because TNF-α plays an important role in carcinogenesis caused by microbes in-fection and P37 is a major immunogen of mycoplasma hy-orhinis(M.hyor.),investigating whether P37 could induce expression and secretion of TNF-α will be very significant fo elucidate the possible molecular mechanism of gastric car-cinogenesis involved with M.hyor.At the present study,we cloned full gene of p37 by PCR and mutated the 7 codes of TGA into TGG firstly,then expressed the P37 protein suc-cessfully with pGEX-4T-1 vector in E.coli,which was veri-fied with Western bolt.By RT-PCR and sensitive L929 cell toxic assay,we found that P37 protein could induce expres-sion and secretion of TNF-α from human peripheral blood mononuclear cells,and the inducing activity of P37 could be dramatically blocked by McAb PD4.These results suggest that the induction of TNF-α secretion by P37 probably plays an importan role in diseases caused by M.hyor.infection and needs to be further investigated.     

IL-1β regulates the mouse Fas ligand expression in corneal endothelial cells

Constitutively expressed Fas ligand (FasL) in several distinct epithelial cell types appears to protect tissues by inducing apoptosis of Fas immune cells during inflammatory reactions. To study the rela-tionship of FasL and inflammation process in cornea,we examined the effects of inflammatory cytokine IL-1β on the FasL production,expression and cytotoxic function in corneal endothelial cells. In this paper,we demonstrate that IL-1β inhibits the FasL production and expression in corneal endothelial cells. The promoter activities of FasL in these cells are reduced by IL-1β in a dose-dependent manner. Finally,we also find that IL-1β block the cytotoxic effects of FasL derived from corneal endothelial cells to the Fas target cells. These data support the view that FasL derived from corneal endothelial cells modulate inflammation within cornea.     

Expression and function of a new angiogenic factor AA98 target molecule at the maternal-embryonic boundary of rhesus monkey

The target molecule of monoclonal antibody AA98 (AA for short) is a new vascular endothelial cell related factor and plays a role in angiogenesis as indicated by the previous data. To investigate its role in angiogenesis and placentation in primate, we examined its expression in the implantation sites on D17, 19, 28 and 34 of gestation in rhesus monkey by immunohistochemistry and Western immunoblot. Western blot analysis showed that the primary antibody used in this study was specific for its epitope. AA protein was mainly expressed in small blood vessels and in some cytotrophoblast cells. The AA staining was found mainly in the endothelial cells and vascular small muscle. This observation supported the AA‘s role in angiogenesis. AA was spatio-temporarily expressed in cytotrophoblasts: weak in proliferating trophoblast within cell column and endovascular trophoblast, strong in trophoblastic subpopulation within the basal plate and vascular trophoblast; AA staining within the basal plate was down-regulated during early placentation. The shift of AA98 expression in extravillous trophoblasts suggestes a role of this new factor during the course of cytotrophoblast metastasis and spiral artery remodeling. The spatio-temporarily expression indicats that AA98 could be also used as a trophoblast cellular marker to characterize the acquisition of a vascular endothelial and invasive phenotype.     

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

Human cerebral cavernous malformation （CM） is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type I gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like structures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to receptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.     

Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs （miRNAs） participate in the radiation-induced DNA damage response. However, a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thor- oughly assessed. We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation （IR）, quantified the ex- pression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms, Gene Ontol- ogy （GO） analysis, pathway analysis, and gene network construction. The results showed that the altered miRNAs were involved in regulation of various cellular functions, miRNA-gene network analyses revealed that miR- 186, miR- 106b, miR- 15 a/b, CCND 1 and CDK6 played vital role in the cellular radiation response. Using qRT-PCR, we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression. This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.     

Effect of simvastatin on the atherosclerotic plaque stability and the angiogenesis in the atherosclerotic plaque of rabbits

In this study, the effect of simvastatin on the atherosclerotic plaque stability and the angiogenesis in the atherosclerotic plaque of rabbits was observed. Thirty New Zealand rabbits were randomly divided into the normal control group, the model control group and the simvastatin group, 10 in each group. Rabbits in the normal control group were fed with normal forage, while rabbits in the rest two groups were fed with high fat forage. The balloon injury was performed two weeks later to establish an abdominal aortic atherosclerosis model, and then high fat forage was successively fed to them. Meanwhile, simvastatin at the daily dose of 2.5 mg/kg body weight was administered to rabbits in the simvastatin group. After 6 weeks of successive administration, levels of blood lipids were measured after blood sampling, and the serum high sensitivity C-reactive protein （hsCRP） and matrix metalloproteinase-3,-9 （MMP-3,-9） were detected using enzyme-linked immunosorbent assay. The macroscopically pathological indices of the plaque tissue were observed using hematoxylin and eosin （HE） staining of abdominal aorta specimens under a light microscope, and the plaque area （PA）, cross-sectional vascular area （CVA） and correcting plaque area （PA/CVA） were determined quantitatively using imaging software. The protein expressions of the vascular endothelial growth factor （VEGF）, factor VIII related antigen （FVIIIRAg）, MMP-3 and cluster of differentiation antigen 40 ligand （CD40L） in the plaque were detected with the immunohistochemical method. Compared with the model control group, the levels of VEGF, FVIIIRAg, MMP-3, CD40L protein expression and the serum expression levels of hsCRP, MMP-3, MMP-9 in the simvastatin group were significantly reduced （P〈0.05, P〈0.01）. The ratio PA/CVA in the simvastatin group was more significantly reduced when compared with that in the model control group （P〈0.01）. The levels of serum total cholesterol （TC）, triglyceride （TG） and low-density lipoprotein cholesterol （LDL） were significantly reduced in the simvastatin group when compared with those in the model control group （P〈0.05, P〈0.01）. Simvastatin plays a certain role in stabilizing the atherosclerotic plaque, and inhibiting the angiogenesis in the atherosclerotic plaque may be one of possible mechanisms.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro， PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3.1His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

Background： The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein （LDL） in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. Methods： We have studied the effect of fetal calf serum （FCS）, which we considered a source of LDL, on the adhesion of monocytes to endothelial cells （ECs） by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells （EC monoculture） and a co-culture with bovine aortic smooth muscle cells （EC-SMC co-culture）. Results： It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP- 1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. Conclusion： The results suggest that the elevation of the LDL （cholesterol） level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.     

Induction of human β-defensin-2 in airway epithelial cells by Streptococcus pneumoniae： involvement of IP3-dependent intracellular calcium release and NF-κB

Human β-definsin-2 （hBD-2） is mainly induced by bacterial factors and pro-inflammation mediators in epithelial cells. As the major cause of community-acquired pneumonia, whether Streptococcus pneumoniae （S. pneu-moniae） stimulation induces hBD-2 expression in airway epithelial cells is elusive. In this study, we found that S. pneumoniae stimulation induced hBD-2 expression in a time-and concentration-dependent manner in primary human airway epithelial cells. To further reveal the mechanism of S. pneumoniae inducing hBD-2, we found that S. pneumoniae stimulation activated NF-κB signaling pathway. Specific NF-κB inhibitor, PDTC, could reverse the induction of hBD-2 by S. pneumoniae. We also found that cellular inner Ca^2＋ signaling is involved in the S. pneumoniae-induced hBD-2. Taken together, our find-ings indicated that S. pneumoniae can stimulate the expression of hBD-2 in airway epithelial cells and NF-κB and inositol triphosphate-dependent intracellular calcium release is involved in this induction.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group B

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro, PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3. I/His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

D—galactose induces replicative senescence of rat pulmonary microvascular endothelial cells

Pulmonary vascular endothelial cells, especially pulmonary microvascular endothelial cells (PMVECs) have many important functions. They act as a permeabil-ity barrier to plasma, allowing the selective, active trans-fer and metabolism of many substances. These cells are also involved in endocrine, inflammation and respiration. The recent evidence showed that the structural and func-tional integrity of the PMVECs is essential to supplying oxygen, metabolism, acid-base balance and homeostasis…     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

A novel element (NIRS) participates in the regulation ofinterleukin 2 receptorαgene expression

A novel element at -153/- 143 bp in the interleukin 2 receptor α(IL-2Rα) gene has been coined as NRE-inverse repeat sequence (NIRS) due to its inversely repeated to the known negative regulatory element (NRE) further upstream of the gene. In order to explore the role of NIRS in the expression of IL-2Rαgene,luciferase reporter plasmids driven by 4 individually deleted IL-2Rα genes promoter regions were constructed. Transfection of the reporter plasmids into Jurkat cells and HeLa cells respectively, we found that both NIRS and NRE were critical for repressing the constitutive expression of IL-2Rα gene and were also necessary for promoter activity induced by PHA. EMSA results showed that double-stranded NRE- and NIRS-binding proteins existed in both HeLa cells and Jurkat cells. However, single-stranded NIRS- and NRE-binding protein was only found in HeLa cells. Interestingly, the supershift band showed up in EMSA system with Jurkat cells (no matter whether activated or not) adding to the cell lysate of HeLa cells. UV-crosslinking showed a double stranded NRE- and NIRS-binding protein p83 in both Jurkat cells and HeLa cells. Our results suggest that trans-acting factors play a key role in regulating promoter activity of IL-2Rα gene by interacting with double or single stranded NRE and/or NIRS selectively in different cells.     

Human endothelial senescence can be induced by TNF-α

TNF-α is one of the most important proinfiammatory cytokines in mediating multiple physio-pathological functions during immunological responses. Vascular endothelial cells, when stimulated by TNF-α2 can increase the expression of multiple cytokines and cellular adhesion molecules and, in turn, actively promote the inflammatory responses by recruiting and activating of leukocytes to the inflammatory site. In addition to endothelial death induced by TNF-α2 we found for the first time that TNF-α can also induce the human endothelial cells senescence. The induced senescent endothelial cells will display SA-β-Gal staining and they were arrested in G0-G1 phase. We found that Aψm would always be up-regulated in response to TNF-α stimulation at early time but when the cells become senescent, A ψmshows a tendency to decrease. It may reflect the sthenic function of mitochondria at early time in response to TNF-αstimulation and decay when the endothelial cells were induced senescent. ROS fluctuates at early time and also decreases when the endothelial cells become senescent. Our results show that the change of mitochondrial function may be related to the senescent process.``     

Effects of hypoxic exercise training on microRNA expression and lipid metabolism in obese rat livers

To investigate the effects of hypoxic exercise training on microRNA （miRNA） expression and the role of miRNA expression in regulating lipid metabolism, 20 dietary-induced obese SD rats were divided into a normoxic sedentary group （N, n=10） and a hypoxic exercise training group （H, n=10）. After four weeks, measurements were taken of body weight, body length, fat mass, serum lipid concentration, miRNAs differentially expressed in rat liver, and gene and protein expression levels of perexisome proliferator activated receptor a （PPARα）, fatty acid synthetase （FAS）, and carnitine palmitoyl transferase 1A （CPTIA） in rat liver. Body weight, Lee＇s index, fat mass, fat/weight ratio, and serum levels of total cholesterol （TC） and high density lipoprotein cholesterol （HDL-C） were all significantly lower in the H group than in the N group （P〈0.01）. Six miRNAs expressed significantly differently in the liver （P〈0.05）. Specifically, expression levels of miR-378b were significantly lower in the H group than in the N group （P〈0.05）. Compared with the normoxic sedentary group, hypoxic exercise training resulted in a lower ratio of FAS mRNA to CPTIA mRNA （P〈0.05）, as well as lower CPT1A protein levels （P〈0.01）, while a higher ratio of FAS to CPT1A protein levels （P〈0.01） was observed. In conclusion, hypoxic training may elevate the resistance of high fat diet induced obesity in rats by reducing the expression of miR-378b, and decrease the fatty acid mitochondrial oxidation in obese rat livers by decreasing the protein expression of CPTIA and increasing the protein expression ratio of FAS/CPTIA.