ABSTRACT
Objective To examine the effects of fluid shear stress (FSS) on epithelial-mesenchymal transition (EMT) in Hep2 cells. Methods Hep2 cells were exposed to 140 mPa FSS. The morphologic changes of Hep2 cells exposed to FSS at different durations were observed using inverted microscope. The migration ability of Hep2 cells after FSS loading was investigated using scratch wound assay. The distribution and expression of cytoskeleton protein F-actin were examined by confocal microscope. The expression of the EMT marker proteins were detected by Western blotting. Results Most of Hep2 cells changed their morphology from polygon to elongated spindle with well-organized F-actin under FSS. After removing FSS, Hep2 cells recovered their initial morphology with flat polygon. FSS regulated Hep2 cells to enhance their migration capacity in a time-dependent manner. FSS promoted the rearrangement of cytoskeletal protein F-actin，which enhanced the migration behavior of Hep2 cells. In addition, FSS induced a time regularity of expression of the EMT marker proteins in Hep2 cells. Conclusions FSS as an important physical factor can induce EMT in Hep2 cells.
ABSTRACT
Objective To investigate the effect of focal adhesion kinases (FAK) inhibitor with different concentration on the adhesion and migration of endothelial cells (ECs) and the expression of downstream Rac1 protein, and to explore the role of FAK in adhesion and migration of ECs by using FAK inhibitor to inhibit the phosphorylation of Y397 site of FAK. Method Scratch wound migration assay was performed to examine the effect of FAK inhibitor with different concentration (from 0 nmol/mL to 250 nmol/mL) on ECs migration at 2, 4, 8 and 24 h, respectively. Western blot combined with immunofluorescence analysis were performed to determine the effect of FAK inhibitor with different concentration on distribution and expression of Rac1 protein. Results With the concentration of FAK inhibitor increased, ECs migration distance and the Rac1 protein expression decreased. Conclusions The inhibition of FAK phosphorylation could inhibit cell adhesion and migration with the decrease in downstream Rac1 protein, and ECs adhesion/migration was related to FAK Rho GTPases signaling pathways.
ABSTRACT
Objective To elucidate the mechanical chemical interaction and its mechanobiological mechanism on the migration of endothelial cells. Method RT PCR, Western blot and immunofluorescence were applied to detect the expression of CXCR1 and CXCR2 and their distributions under three levels of shear stress; anti-IL8RA and anti-IL8RB were used to inhibit CXCR1 and CXCR2 to evaluate endothelial cell migration under shear stress; ECs were transfected to obtain the wild type Rac1(Rac1WT) or RhoA (RhoAWT), the constitutively active forms of Rac1(Rac1Q61L) or RhoA (RhoA63L), and the dominant negative forms of Rac1(Rac1T17N) or RhoA (RhoA188A) respectively, with lipofectamine 2000 reagent. ECs transfected with three plasmids of Rac1 were exposed to three levels of shear stress and IL-8, respectively; ECs transfected with three plasmids of RhoA were stimulated by IL-8. Results CXCR1 and CXCR2 are novel mechano sensors mediating laminar shear stress induced endothelial cell migration. High expression of Rac1 and RhoA can promote EC migration, while their low expression inhibits EC migration. Conclusions CXCR1, CXCR2, Rac1 and RhoA are critical signaling molecules in mechanical chemical interaction of EC migration.