Shear stress regulates late EPC differentiation via mechanosensitive molecule-mediated cytoskeletal rearrangement

Abstract

Background: 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.

Methodology/principal finding: 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.

Conclusions/significance: 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.

Figure 1. The shear stress-induced endothelial marker expression was dependent on the cytoskeletal rearrangement in late EPCs.

(A) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm² for 5, 30 or 60 min, and stained with FITC-Phalloidin to detect actin stress fibers. Bars: 100 µm. (B) Stress fibers were quantitated and normalized to the static control group. (C–D) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm²) for 24 h, or cultured in static conditions. The protein levels of vWF and CD31 were determined by FACS. (E) Late EPCs were pretreated with Cyto D (1 µmol/l) for 30 min. The treated cells were then either subjected to shear stress (12 dyne/cm²) for 24 h, or cultured in static conditions. The protein expression of vWF and CD31 were determined by immunoreactivity. Bars: 200 µm. Data represent the mean±SE from three separate experiments. **(P<0.01).

Figure 2. The shear stress-induced differentiation associated with cytoskeletal rearrangement enhanced the reendothelialization capacity in late EPCs.

(A) Fluorescence-labeled EPCs (CM-DiI; red) were located beneath the endothelial layer, visualized by vWF immunostaining (green). Double staining with CM-DiI and vWF indicated the frequencies of EPC differentiation toward the endothelial lineage. Bars: 100 µm. (B) Quantitative analyses of reendothelialization by vWF immunofluoresence in n = 6 rats per group. (C) Vessels were perfusion-fixed 14 days after endovascular injury and EPC seeding. Representative photomicrographs of hematoxylin-eosin-stained carotid arteries. Bars: 100 µm. (D) Hematoxylin-eosin-stained cross-sections were analyzed for neointimal thickening. The intima area/media area ratios were evaluated by computer-assisted histomorphometry in n = 6 rats per group. **(P<0.01).

Figure 3. Integrin β₁ was involved in the shear stress-induced cell differentiation associated with cytoskeletal rearrangement in late EPCs.

(A) Late EPCs were exposed to shear stress (12 dyne/cm²) for 30 min or kept as static controls. The activated integrin β₁ was revealed by immunostaining using HUTS-4 mAb. Bars: 100 µm. (B) Late EPCs were kept in static condition or exposed to shear stress at 12 dyne/cm² for 60 min. F-actin and integrin β₁ were stained with FITC-Phalloidin and anti-integrin β₁, respectively. Bars: 50 µm. (C) Before being exposed to shear stress at 12 dyne/cm² for 60 min, late EPCs were pretreated with anti-integrin β₁ (50 µg/ml) for 30 min. F-actin was stained with FITC-Phalloidin. Bars: 100 µm. (D) Stress fibers were quantitated and normalized to the shear stress-treated EPCs. The results represent the mean±SE from three independent experiments. *(P<0.05).

Figure 4. Ras was essential for the shear stress-induced cell differentiation associated with cytoskeletal rearrangement in late EPCs.

(A) Late EPCs were transfected with RasN17 by the Lipofectamin 2000. The 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. (B) Stress fibers were quantitated and normalized to the shear stress-treated EPCs. (C) Late EPCs were transfected either with control vector or with RasN17. The transfected late EPCs were then subjected to shear stress (12 dyne/cm²) for 3 h. The gene expression of vWF and CD31 was determined by real time RT-PCR. (D) Late EPCs were transfected either with control vector or with RasN17, and the transfected late EPCs were then subjected 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. The results represent the mean±SE from three independent experiments. **(P<0.01) and *(P<0.05).

Figure 5. The shear stress-induced EPC differentiation associated with cytoskeletal rearrangement was mediated via the Ras/ERK1/2– dependent signal pathway.

(A) Western blot was carried out with specific antibody for checking the phosphorylated ERK1/2. The total ERK1/2 served as loading control. (B) Late EPCs were transfected with RasN17. Transfected late EPCs were then subjected to shear stress (12 dyne/cm²) for 5 min. The activation of ERK1/2 was analyzed by Western blot. (C) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min. The cells were then either exposed to shear stress (12 dyne/cm²) for 3 h, or cultured in static condition. After this, the vWF and CD31 mRNA expression was determined using real time RT-PCR. (D) Late EPCs were pretreated with PD98059 (10 µmol/l) for 30 min, and were then 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. The results represent the mean±SE from three independent experiments. **(P<0.01) and *(P<0.05).

Figure 6. Paxillin was necessary for the shear stress-induced differentiation associated with cytoskeletal rearrangement in late EPCs.

(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).

Figure 7. The role of FAK in the shear stress-induced cytoskeletal rearrangement and differentiation in late EPCs.

(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).