urvival, migration, and angiogenesis of EPCs. Moreover, NO derived from eNOS has been identified as a critical molecule in mobilizing EPCs from the bone marrow, in reducing their senescence, and in promoting their proliferation. All of this indicates the important role of NO in maintaining EPC function. One may then naturally 
ask why the PP 242 price increase of eNOS phosphorylation and NO production is accompanied with impaired late EPC function, such as proliferation, migration and adhesion, in the present study. To answer this, we propose that the increase of eNOS and NO may be related to the intrinsic abilities of the late EPCs to compensate for the impaired function induced by jasplakinolide. Even with this, however, the increase of eNOS phosphorylation and NO production might still not be sufficient to overcome late EPC dysfunction induced by jasplakinolide. Our data further demonstrate that the proliferation of jasplakinolide-stressed late EPCs is rescued after treatment with the NO donor sodium nitroprusside. Moreover, incubation with endothelial NO synthase inhibitor L-NAME aggravates late EPCs dysfunction induced by jasplakinolide. In conclusion, the results in the present study provide evidence that jasplakinolide exacerbates the apoptosis induced by VEGF deprivation, and impairs the function of late EPCs both in vitro and in vivo. Furthermore, NOrelated mechanisms could be the main contributor to jasplakinolideinduced EPC dysfunction. These findings not only indicate that the actin cytoskeleton plays a pivotal role in regulating late EPC function, but also provide further insights into the complex cellular mechanisms of late EPCs in vascular repair. 9 Jasplakinolide Affects Late EPC Function 10 Jasplakinolide Affects Late EPC Function Materials and Methods Isolation of Bone Marrow Mononuclear Cells and Cell Culture Whole bone marrow was isolated from both the femurs and tibias of Sprague-Dawley rats . The bone marrow mononuclear cells were fractionated by density gradient centrifugation and the leukocyte marker expressions on MNC were evaluated by FACS. MNCs were plated on dishes precoated with fibronectin, and were maintained in complete EGM-2 medium. After 4 days in culture, unattached cells were removed by a single washing step with PBS, after which fresh medium was added. Endothelial colonies subsequently appeared. Highly proliferative endothelial cells grew out from these colonies which then formed a confluent monolayer. Cells under passage third-fifth, namely late EPCs, were used for the experiments. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Weifang medical college. Identification of Late EPCs To identify the late EPCs, the cells were characterized by the uptake of 1,19-dioctadecyl- 3,3,39,39-tetramethylindo-carbo-cyanine-labeled acetylated low density lipoprotein, and by fluorescein isothiocyanate labeled Anti-Ulex Europaeus Lectin 1/UEA1 staining. In short, the adherent cells were first incubated with 2 mg/ml Dil-acLDL for 1 h, after which they were fixed in 2% paraformaldehyde for 10 min, and then counterstained with Jasplakinolide Affects Late EPC Function 10 mg/ml FITC-UEA-1 for 1 h. After the staining, the samples were viewed with inverted fluorescence microscope. Furthermore, the cellular expressions of CD45, VEGFR2,
