Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Separation and purification of highly infectious enterovirus A71 particles using a strong anion-exchange column.

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK.

Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm(2) induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm(2), oscillatory shear stress (OSS) at 0.5±4dyn/cm(2) cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V-FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.

Activation of PPAR-α induces cell cycle arrest and inhibits transforming growth factor-ß1 induction of smooth muscle cell phenotype in 10T1/2 mesenchymal cells.

Transforming growth factor-ß1 (TGF-ß1) regulates the cell cycle and the differentiation of mesenchymal cells into smooth muscle cells (SMCs). However, the precise intracellular signaling pathways involved in these processes have not been fully clarified. It has also been shown that there is an increase in TGF-ß1 expression in human atherosclerotic plaques. Furthermore, peroxisome proliferator-activated receptors (PPARs) and their agonists have recently gained more attention in the study of the pathogenesis of atherosclerosis. In this study, we examined the role of PPARs in the TGF-ß1-mediated cell cycle control and SMC phenotypic modulation of C3H10T1/2 (10T1/2) mesenchymal cells. The results showed the following: (1) the PI3K/Akt/p70S6K signaling cascade is involved in TGF-ß1-induced differentiation of 10T1/2 cells into cells with a SMC phenotype. (2) PPAR-α agonists (i.e., WY14,643 and clofibrate), but not a PPAR-δ/ß agonist (GW501516) or PPAR-γ agonist (troglitazone), inhibit TGF-ß1-induced SMC markers and the DNA binding activity of serum response factor (SRF) in 10T1/2 cells. (3) WY14,643 and clofibrate inhibit the TGF-ß1 activation of the Smad3/Akt/P70S6K signaling cascade. (4) TGF-ß1-induced cell cycle arrest at the G0/G1 phases is mediated by Smad3 in 10T1/2 cells. (5) The PPAR-α-mediated 10T1/2 cell cycle arrest at the G0/G1 phases is TGF-ß receptor independent. These results suggest that PPAR-α mediates cell cycle control and TGF-ß1-induced SMC phenotypic changes in 10T1/2 cells.

Roles of microRNAs in atherosclerosis and restenosis.

Atherosclerosis is commonly appreciated to represent a chronic inflammatory response of the vascular wall, and its complications cause high mortality in patients. Angioplasty with stent replacement is commonly performed in patients with atherosclerotic disease. However, the restenosis usually has a high incidence rate in angioplasty patients. Although the pathophysiological mechanisms underlying atherosclerosis and restenosis have been well established, new signaling molecules that control the progress of these pathologies have continuously been discovered. MicroRNAs (miRs) have recently emerged as a novel class of gene regulators that work via transcriptional degradation and translational inhibition or activation. Over 30% of genes in the cell can be directly regulated by miRs. Thus, miRs are recognized as crucial regulators in normal development, physiology and pathogenesis. Alterations of miR expression profiles have been revealed in diverse vascular diseases. A variety of functions of vascular cells, such as cell differentiation, contraction, migration, proliferation and inflammation that are involved in angiogenesis, neointimal formation and lipid metabolism underlying various vascular diseases, have been found to be regulated by miRs. This review summarizes current research progress and knowledge on the roles of miRs in regulating vascular cell function in atherosclerosis and restenosis. These discoveries are expected to present opportunities for clinical diagnostic and therapeutic approaches in vascular diseases resulting from atherosclerosis and restenosis.

Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-beta1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) is known to induce phenotypic modulation of mesenchymal cells to SMCs. However, the intracellular signals regulating induction of the SMC phenotype of mesenchymal cells have not been fully clarified. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily and phosphatidylinositol 3-kinase (PI3K)/Akt in the TGF-beta1-mediated phenotypic modulation of 10T1/2 mesenchymal cells to SMCs characterized by the expression of SMC-specific markers, including smooth muscle alpha-actin (SMalpha-actin), myosin heavy chain (SM-MHC), and protein 22-alpha (SM22alpha). The results showed the following: (1) TGF-beta1 induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells in a time-dependent manner. (2) TGF-beta1 induced biphasic increases in extracellular signal-regulated kinase (ERK), p38 MAPK, c-Jun-NH2-terminal kinase (JNK), and Akt phosphorylation. (3) The inhibitor for PI3K/Akt (i.e., LY294002), but not those for MAPKs (i.e., SB203580, PD98059, and SP600125), attenuated the TGF-beta1-induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells; in addition, transfection of 10T1/2 cells with the Akt-specific small interfering RNA (siRNA) significantly reduced their SMalpha-actin and SM-MHC expressions. (4) LY294002 and the Akt-specific siRNA inhibited the TGF-beta1-induced SM22alpha gene expression and promoter activity, suggesting that the TGF-beta1-induced gene expression was mediated by PI3K/Akt at the transcriptional level. (5) LY294002 inhibited the TGF-beta1-induced gene expression and DNA binding activity of serum response factor (SRF). These results indicate that TGF-beta1 is capable of inducing the SMC phenotype of 10T1/2 cells and that this induction is mediated through the PI3K/Akt signaling pathway.

Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.

OBJECTIVE: Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes in ECs. METHODS AND RESULTS: Shear stress (20 dynes/cm2) was applied to ECs prior to and/or following the addition of tumor necrosis factor (TNF)-alpha. Shear stress increased the TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) and E-selectin. Transfection studies using promoter reporter gene constructs of ICAM-1, VCAM-1, and E-selectin demonstrated that these shear stress modulations of gene expression occur at the transcriptional levels. After 24-hour preshearing followed by 1 hour of static incubation, the effect of preshearing on TNF-alpha-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-alpha-induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of >6 hours (complete recovery at 24 hours). Pre- and postshearing caused a reduction in the nuclear factor-kappaB-DNA binding activity induced by TNF-alpha in the EC nucleus. CONCLUSIONS: Our findings suggest that shear stress plays differential roles in modulating the TNF-alpha-induced expressions of ICAM-1 versus VCAM-1 and E-selectin genes in ECs.

Activation of PKC-epsilon and ERK1/2 participates in shear-induced endothelial MCP-1 expression that is repressed by nitric oxide.

Vascular endothelial cells (ECs) continuously experience hemodynamic shear stress generated from blood flow. Previous studies have demonstrated that shear stress modulates monocyte chemotactic protein-1 (MCP-1) expression in ECs. This study explored the roles of protein kinase C (PKC), extracellular signal-regulated protein kinase (ERK1/2), and nitric oxide (NO) in sheared-induced MCP-1 expression in ECs. The activation of PKC-alpha and PKC-epsilon isoforms was observed in ECs exposed to shear stress. The use of an inhibitor (calphostin C) to PKC-alpha and PKC-epsilon decreased ERK1/2 activation and MCP-1 induction by shear, whereas an inhibitor (Go6976) to PKC-alpha did not affect ERK1/2 activation or MCP-1 induction. Inhibition of ERK1/2 activation by PD98059 blocked MCP-1 induction. Transfection of ECs with an antisense to PKC-epsilon abolished the shear inducibility of MCP-1 promoter. These results demonstrate that PKC-epsilon and ERK1/2 participate in shear-induced MCP-1 expression. We also examined the regulatory role of NO in MCP-1 expression. An NO donor (NOC18) suppressed shear-induced activation of PKC-epsilon and ERK1/2, and also repressed MCP-1 induction. Consistently, overexpression of endothelial nitric oxide synthase (eNOS) to enhance the endogenous generation of NO in ECs decreased the activation of PKC-epsilon and ERK1/2, and also inhibited MCP-1 expression. Taken together, these findings suggest that PKC-epsilon and ERK1/2 are critical in the signaling pathway(s) leading to the MCP-1 expression induced by shear stress. Additionally, this study indicates that NO, by repressing PKC-epsilon activity and ERK pathway activation, attenuates shear-induced MCP-1 expression.

Shear stress attenuates tumor necrosis factor-alpha-induced monocyte chemotactic protein-1 expressions in endothelial cells.

The interplay between shear stress and cytokines in regulating vascular endothelial function remains largely unexplored. In the present study, the potential role of shear stress in regulating tumor necrosis factor-alpha (TNF-alpha)-induced gene expression in endothelial cells (ECs) was investigated. The TNF-alpha-induced monocyte chemotactic protein-1 (MCP-1) mRNA expressions were significantly attenuated in ECs subjected to a high level of shear stress (20 dynes/cm2) for 4 or 24 h prior to the addition of TNF-alpha in the presence of flow. Less inhibition of TNF-alpha-induced MCP-1 mRNA expression was found in ECs pre-exposed to a low level of shear stress (1.2 dynes/cm2) for 24 h as compared with the cells presheared (pre-exposed to shear stress) for 4 h. Simultaneous exposure of ECs to TNF-alpha and a high or low level of shear stress down-regulated TNF-alpha-induced MCP-1 gene expressions, suggesting that the post-flow condition modulates endothelial responses to cytokine stimulation. Individually or combined, an endothelial nitric oxide synthase (eNOS) inhibitor and a glutathione (GSH) biosynthesis inhibitor had no effect on this shear stress-mediated inhibition. Moreover, in ECs either presheared or remained in a static condition prior to stimulation by TNF-alpha while under shear flow, the ability of TNF-alpha to induce AP-1-DNA binding activity in the nucleus was reduced. Our findings suggest that shear stress plays a protective role in vascular homeostasis by inhibiting endothelial responses to cytokine stimulation.