Fig 1.
Effects of EC after exposure to shear stress.
ECs were exposed to 4 dyn/cm2 (pathological flow), 12 dyn/cm2 (physiological flow) or were cultured under static conditions. (A) Morphological changes induced by mechanical force leading to re-orientation of cells. (B) The same image with demarcation of endothelial cell length in static culture or when exposed to SS. (C) Graphical representation of endothelial cell length in static culture or when exposed to SS (μm). 40 X magnification. Scale bar = 20 μm. (D) Adhesion assay on fibronectin (0, 2.5, 5, 10, 15 or 20 μg/mL). Values are expressed as absorbance at 540 nm. *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The assays were performed in triplicate. (E) Transwell® migration assay. *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The assays were performed in triplicate.
Fig 2.
Angiogenesis-related assays of ECs exposed to shear stress.
(A) Capillary-like tube formation assay on reconstituted basement membrane was performed on ECs exposed to 4 dyn/cm2, 12 dyn/cm2 or ECs in static conditions, to assess the ability of these cells to form an organized tubular network. The images were acquired under an inverted light microscope at 40 X magnification. Scale bar = 50 μm. (B) The total length of capillary segments connected forming tubular structures on the GeltrexTM was measure using ImageJ® software as “mm” tube length/cm2 area. (C) Analysis of gene expression in qPCR of VEGF-A; (D) Analysis of gene expression in qPCR of TGFβ1 and (E) Analysis of gene expression in qPCR of TGFβ3. *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The assays were performed in triplicate.
Fig 3.
Analysis of sulfated glycosaminoglycans synthesized by ECs exposed to shear stress.
After exposure to 4 dyn/cm2, 12 dyn/cm2 or maintained in static conditions, ECs were metabolically radiolabeled for 18 hours with F12 medium containing [35S]-sulfate (150 μCi/ml). After the incubation period, GAGs from the culture medium and cell fraction were extracted, analyzed and quantified. (A) Electrophoretic behavior of the sulfated GAGs present in the culture medium and cell fraction of ECs after exposure to shear stress. (B) Analysis of the absolute values of the HS and CS secreted to the culture medium. (C) Analysis of total GAGs content synthesized by the cells. The results are expressed as scintillation counts per minute normalized by protein quantification of the sample (cpm/100 μg protein). The gels were cut and organized in this image for a better understanding of the results. CS: chondroitin sulfate; HS: heparan sulfate; *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The experiments were performed in duplicate.
Fig 4.
Proteoglycans and connexin immunofluorescence and gene expression of ECs exposed to shear stress (4 dyn/cm2 or 12 dyn/cm2) or ECs in static conditions.
(A) Location of syndecan-4 by immunofluorescence analyzed with confocal microscopy. (B) Analysis of the gene expression of syndecan-4 core protein by qPCR. (C) Location of perlecan by immunofluorescence analyzed with confocal microscopy. (D) Analysis of the gene expression of perlecan core protein by qPCR. (E) Location of Connexin 43 by immunofluorescence using confocal microscopy. (F) Analysis of the gene expression of Connexin 43 by qPCR. Red: Alexa Fluor 594 secondary antibody for staining of syndecan-4 or perlecan; Green: Alexa Fluor 488 secondary antibody for staining of Connexin 43; Blue: DAPI, nuclei staining. 630 X magnification. Scale bar = 50 μm. Sdc4: Syndecan-4. Con43: Connexin 43. RE: Relative expression to GAPDH. *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The assay was performed in triplicate.
Fig 5.
Analysis of gene expression by qPCR from ECs after exposure to shear stress (4 dyn/cm2 or 12 dyn/cm2) or ECs in static conditions.
Analysis of gene expression by qPCR of Decorin core protein, Fibronectin and Collagen IIIα1. RE: Relative expression compared to GAPDH. *p < 0.01; **p < 0.001; ***p < 0.0001 (ANOVA, Bonferroni test). The assay was performed in triplicate.