Actin Stabilization by Jasplakinolide Affects the Function of Bone Marrow-Derived Late Endothelial Progenitor Cells

<div><h3>Background</h3><p>Bone marrow-derived endothelial progenitor cells (EPCs), especially late EPCs, play a critical role in endothelial maintenance and repair, and postnatal vasculogenesis. Although the actin cytoskeleton has been considered as a modulator that controls the function and modulation of stem cells, its role in the function of EPCs, and in particular late EPCs, remains poorly understood.</p> <h3>Methodology/Principal Finding</h3><p>Bone marrow-derived late EPCs were treated with jasplakinolide, a compound that stabilizes actin filaments. Cell apoptosis, proliferation, adhesion, migration, tube formation, nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation were subsequently assayed in vitro. Moreover, EPCs were locally infused into freshly balloon-injured carotid arteries, and the reendothelialization capacity was evaluated after 14 days. Jasplakinolide affected the actin distribution of late EPCs in a concentration and time dependent manner, and a moderate concentration of (100 nmol/l) jasplakinolide directly stabilized the actin filament of late EPCs. Actin stabilization by jasplakinolide enhanced the late EPC apoptosis induced by VEGF deprivation, and significantly impaired late EPC proliferation, adhesion, migration and tube formation. Furthermore, jasplakinolide attenuated the reendothelialization capacity of transplanted EPCs in the injured arterial segment in vivo. However, eNOS phosphorylation and NO production were increased in late EPCs treated with jasplakinolide. NO donor sodium nitroprusside (SNP) rescued the functional activities of jasplakinolide-stressed late EPCs while the endothelial NO synthase inhibitor L-NAME led to a further dysfunction induced by jasplakinolide in late EPCs.</p> <h3>Conclusions/Significance</h3><p>A moderate concentration of jasplakinolide results in an accumulation of actin filaments, enhancing the apoptosis induced by cytokine deprivation, and impairing the proliferation and function of late EPCs both in vitro and in vivo. NO donor reverses these impairments, suggesting the role of NO-related mechanisms in jasplakinolide-induced EPC downregulation. Actin cytoskeleton may thus play a pivotal role in regulating late EPC function.</p> </div

Jasplakinolide inhibited the adhesion of late EPCs.

<p>(A) Late EPCs were pretreated with either jasplakinolide or DMSO for 1 h and then seeded on either plastic or culture surfaces coated with different ECM proteins, such as fibronectin, collagen I or laminin, and incubated for 1 h at 37°C. After nonadherent cells were removed by washing, adherent cells were counted and analyzed. (B) The cell surface expressions of integrin β1 and β3 were assessed by FACS. Representative FACS profiles of four independent experiments are shown. The relative fluorescence intensity that is normalized with the mean fluorescence intensity of isotype control. Data represent the mean±SE of four different experiments. **P<0.01.</p

Dose- and time-dependent effects of jasplakinolide on actin distribution of late EPCs.

<p>The actin filaments of late EPCs were stained with FITC-phalloidin. (A) Control cells. (B) Cells treated with DMSO for 24 h. (C-N) Effects of jasplakinolide on late EPCs for: 50 nmol/L for 15 min, 30 min, 1 h and 24 h respectively (C-F); 100 nmol/L for 15 min, 30 min, 1 h and 24 h respectively (G-J) and 200 nmol/L for 15 min, 30 min, 1 h and 24 h respectively (K-N).</p

late EPCs treated with jasplakinolide displayed impaired tube formation ability.

<p>(A) Representative images of capillary networks formed by late EPCs treated with jasplakinolide or DMSO. The averages of the length (B) and total area (C) of complete tubes formed by cells were compared by computer software. Data represent the fold increase by comparison to the untreated cells (arbitrarily = 100). **P<0.01.</p

Role of NO signaling in jasplakinolide down-regulated EPCs.

<p>(A) After incubation of late EPCs with jasplakinolide or DMSO for 1 h. Phosphorylation of eNOS and total eNOS were assessed by western blot. (B) NO in culture medium was measured by ELISA. (C) Late EPC proliferation was analyzed by the EdU incorporation assay after culture in DMSO or jasplakinolide medium without VEGF in the absence or presence of SNP (NO donor, 25 µmol/l) and L-NAME (NOS inhibitor, 100 µmol/l). Data represent the mean±SE of four different experiments. *P<0.05.</p

Characterization of late EPCs derived from bone marrow.

<p>A: MNCs were isolated and plated on fibronectin-coated culture dish on the first day (100×). B: Seven days after plating (100×). C: After 3–4 weeks, the third-fifth passage cells, namely late EPCs (100×). D-F: DiI-acLDL (red) and fluorescein isothiocyanate UEA-1(green) staining (100×). G: Flow cytometry analysis using several markers. H: Representative image of tubuli like structures formed on Matrigel by late EPCs (40×).</p

Jasplakinolide decreased late EPC proliferation.

<p>(A-B) Late EPCs were incubated in the presence or absence of VEGF with either jasplakinolide or DMSO for 1 h. The cells were then washed to remove the jasplakinolide or DMSO, and cultured in EGM-2 with or without VEGF for 12 (A) or 24 h (B). Cell proliferation was assessed by CCK-8 assay. (C) Late EPCs were incubated in the absence of VEGF with either jasplakinolide or DMSO for 1 h. The cells were then washed to remove the jasplakinolide or DMSO, and cultured in EGM-2 without VEGF for 12 h. Cell proliferation was assessed by the EdU incorporation assay. More than five random fields per well were captured at 200× magnification, and IPP 6.0 was used to calculate the percentage of EdU-positive cells (identified by Apollo® 567 fluorescence) in total cells (identified by Hoechst33342 nuclei staining). Data represent the mean±SE of four different experiments. *P<0.05, **P<0.01.</p

Shear Stress Regulates Late EPC Differentiation via Mechanosensitive Molecule-Mediated Cytoskeletal Rearrangement

<div><p>Background</p><p>Previous studies have demonstrated that endothelial progenitor cells (EPCs), in particular late EPCs, play important roles in endothelial maintenance and repair. Recent evidence has revealed shear stress as a key regulator for EPC differentiation. However, the underlying mechanisms regulating the shear stress–induced EPC differentiation have not been understood completely. The present study was undertaken to further investigate the effects of shear stress on the late EPC differentiation, and to elucidate the signal mechanism involved.</p><p>Methodology/Principal Finding</p><p>In vitro and in vivo assays revealed that cytoskeletal remodeling was involved in the shear stress-upregulated expression of endothelial markers vWF and CD31 in late EPCs, with subsequently increased in vivo reendothelialization after arterial injury. Moreover, shear stress activated several mechanosensitive molecules including integrin β₁, Ras, ERK1/2, paxillin and FAK, which were all involved in both cytoskeletal rearrangement and cell differentiation in response to shear stress in late EPCs.</p><p>Conclusions/Significance</p><p>Shear stress is a key regulator for late EPC differentiation into endothelial cells, which is important for vascular repair, and the cytoskeletal rearrangement mediated by the activation of the cascade of integrin β₁, Ras, ERK1/2, paxillin and FAK is crucial in this process.</p></div

The role of FAK in the shear stress-induced cytoskeletal rearrangement and differentiation in late EPCs.

<p>(A) Western blot was carried out with specific antibody for checking the phosphorylated FAK. The total FAK served as loading control. (B) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h. The cells were then either exposed to shear stress (12 dyne/cm²) for 60 min, or cultured in static condition. After this, F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (C) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. (D) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and were then either exposed to shear stress (12 dyne/cm²) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (E) Late EPCs were pretreated with PF-573,228 (2 µmol/l) for 1 h, and the cells were then either exposed to shear stress (12 dyne/cm²) for 24 h, or cultured in static condition for the same duration. The protein levels of vWF and CD31 were determined by FACS. The results represent the mean±SE from three independent experiments. **(P<0.01) and *(P<0.05).</p

Paxillin was necessary for the shear stress-induced differentiation associated with cytoskeletal rearrangement in late EPCs.

<p>(A) Western blot was carried out with specific antibody for checking the phosphorylated paxillin. The total paxillin served as loading control. (B) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm² for 60 min. Paxillin was stained with specific antibody. Bars: 50 µm. (C) Late EPCs were transfected either with scrambled siRNA or with paxillin siRNA by the Lipofectamin 2000. The cells were then either exposed to shear stress (12 dyne/cm²) for 3 h, or cultured in static condition. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) The cells were either exposed to shear stress (12 dyne/cm²) for 24 h, or cultured in static condition. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were transfected either with scrambled siRNA or paxillin siRNA by the Lipofectamin 2000. Transfected late EPCs were then subjected to shear stress (12 dyne/cm²) for 60 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (F) Stress fibers were quantitated and normalized to the shear stress treated-EPCs. The results represent the mean±SE from three independent experiments. **(P<0.01) and *(P<0.05).</p