Ginkgo biloba Induces Thrombomodulin Expression and Tissue-Type Plasminogen Activator Secretion via the Activation of Krüppel-Like Factor 2 within Endothelial Cells.

The effects of thrombo-prevention, such as antiplatelet and anticoagulant activity, have been reported with the usage of Ginkgo biloba extract (GbE); however, the detailed mechanism has not yet been fully investigated, especially the role of Krüppel-like factor 2 (KLF2). This study aimed to investigate whether GbE can activate KLF2 and then induce thrombomodulin (TM) and tissue-type plasminogen activator (t-PA) secretion to enhance the effects of thrombo-prevention. Different concentrations of GbE were incubated with human umbilical vein endothelial cells (HUVECs) to evaluate its effect on endothelial cells. We found that KLF2 expression is correlated to the risk of atherosclerosis and venous thromboembolism in clinical practice. In the HUVEC cell model, GbE stimulated the expression of KLF2 in a dose-dependent manner. Moreover, TM and t-PA secretion increased when the cells were cultured with GbE. Both the expressions and activities of TM and t-PA in the GbE-treated cells declined after KLF2 was blocked by shKLF2. In sum, with GbE treatment, KLF2 expression in human endothelial cells was significantly activated, which in turn induced an increase in the protein expression and activity of TM and t-PA. After shRNA inhibited the KLF2 expression, GbE stopped inducing the expression and activity of TM and t-PA. These findings suggest that GbE exerts an antithrombotic effect on endothelial cells by increasing the TM expression and t-PA secretion; further, KLF2 is a key factor in this mechanism.

Activation of Krüppel-Like Factor 2 with Ginkgo Biloba Extract Induces eNOS Expression and Increases NO Production in Cultured Human Umbilical Endothelial Cells.

BACKGROUND: The mechanisms responsible for the effects of Ginkgo biloba extract (GbE) are not fully understood. Krüppel-like factor 2 (KLF2), a zinc transcription factor, has vasculoprotective effects if activated. The present study attempted to explore whether GbE may activate KLF2 and its consequences. METHODS: To determine the effects of GbE on endothelial cells, human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of GbE. KLF2 expression levels were determined by quantitative reverse transcription polymerase chain reaction. Cytoskeleton staining and cell migration assays were performed to determine the effects of KLF2 activation. Moreover, endothelial NO synthase (eNOS) expression levels were detected by PCR and Western blot testing. Nitric oxide (NO) production was also measured with 4,5-diaminofluorescein. A knockdown of KLF2 was performed to identify the role of KLF2 in GbE-induced eNOS expression and NO production. RESULTS: HUVECs that were incubated with GbE increased KLF2 expression. These cells demonstrated an altered cell morphology, cytoskeleton rearrangement, and inhibited migration activity. Moreover, eNOS expression and NO production increased in a dose-dependent manner when cells were treated with GbE. Correspondingly, silencing of KLF2 in HUVECs decreased eNOS expression and NO production in GbE-treated cells. CONCLUSIONS: GbE significantly activated KLF2 expression and KLF2-related endothelial function, including cytoskeleton rearrangement, inhibition of migration, eNOS activation, and NO production. These findings suggest that GbE may induce a vasculoprotective effect in endothelial cells. KEY WORDS: Endothelial cells; eNOS; Ginkgo biloba extract; KLF2; NO.

Differential expression of distinct surface markers in early endothelial progenitor cells and monocyte-derived macrophages.

Bone marrow-derived endothelial progenitor cells (EPCs) play a fundamental role in postnatal angiogenesis. Currently, EPCs are defined as early and late EPCs based on their biological properties and their time of appearance during in vitro culture. Reports have shown that early EPCs share common properties and surface markers with adherent blood cells, especially CD14+ monocytes. Distinguishing early EPCs from circulating monocytes or monocyte-derived macrophages (MDMs) is therefore crucial to obtaining pure endothelial populations before they can be applied as part of clinical therapies. We compared the gene expression profiles of early EPCs, blood cells (including peripheral blood mononuclear cells, monocytes, and MDMs), and various endothelial lineage cells (including mature endothelial cells, late EPCs, and CD133+ stem cells). We found that early EPCs expressed an mRNA profile that showed the greatest similarity to MDMs than any other cell type tested. The functional significance of this molecular profiling data was explored by Gene Ontology database search. Novel plasma membrane genes that might potentially be novel isolation biomarkers were also pinpointed. Specifically, expression of CLEC5A was high in MDMs, whereas early EPCs expressed abundant SIGLEC8 and KCNE1. These detailed mRNA expression profiles and the identified functional modules will help to develop novel cell isolation approaches that will allow EPCs to be purified; these can then be used to target cardiovascular disease, tumor angiogenesis, and various ischemia-related diseases.

Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat.

To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C. By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins.