Dynamic shear stress regulation of inflammatory and thrombotic pathways in baboon endothelial outgrowth cells

Abstract

Endothelial outgrowth cells (EOCs) have garnered much attention as a potential autologous endothelial source for vascular implants or in tissue engineering applications due to their ease of isolation and proliferative ability; however, how these cells respond to different hemodynamic cues is ill-defined. This study investigates the inflammatory and thrombotic response of baboon EOCs (BaEOCs) to four hemodynamic conditions using the cone and plate shear apparatus: steady, laminar shear stress (SS); pulsatile, nonreversing laminar shear stress (PS); oscillatory, laminar shear stress (OS); and net positive, pulsatile, reversing laminar shear stress (RS). In summary, endothelial nitric oxide synthase (eNOS) mRNA was significantly upregulated by SS compared to OS. No differences were found in the mRNA levels of the inflammatory markers intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) between the shear conditions; however, OS significantly increased the number of monocytes bound when compared to SS. Next, SS increased the anti-thrombogenic mRNA levels of CD39, thrombomodulin, and endothelial protein-C receptor (EPCR) compared to OS. SS also significantly increased CD39 and EPCR mRNA levels compared to RS. Finally, no significant differences were detected when comparing pro-thrombotic tissue factor mRNA or its activity levels. These results indicate that shear stress can have beneficial (SS) or adverse (OS, RS) effects on the inflammatory or thrombotic potential of EOCs. Further, these results suggest SS hemodynamic preconditioning may be optimal in increasing the efficacy of a vascular implant or in tissue-engineered applications that have incorporated EOCs.

FIG. 1.

Shear stress waveforms. Four hemodynamic conditions were applied in this study: steady laminar shear stress (A, SS, 10±0 dynes/cm²), pulsatile, nonreversing laminar shear stress (B, PS, 10±10 dynes/cm²), oscillatory laminar shear stress (C, OS, 0±10 dynes/cm²), and net positive, pulsatile, reversing oscillatory laminar shear stress (D, RS, 2±10 dynes/cm²).

FIG. 2.

Baboon endothelial outgrowth cells (BaEOC) elongation under shear stress. Bright-field image at an original objective of 5×and immunocytochemistry staining of VE-Cadherin at an original objective of 10×of BaEOCs after exposure to SS (A, E), OS (B, F), PS (C, G), RS (D, H), and static (I). Cellular elongation of the cells was determined through immunocytochemical staining for VE-Cadherin (E–I) and quantification can be seen in (J). BaEOCs aligned in response to flow in SS, PS, and RS conditions, while OS maintains the cobblestone-like morphology of the static BaEOCs. S, static. Values are mean±standard error of the mean (SEM) with an n_donor=3. *p<0.05 in S and OS versus SS, PS, and RS. Scale bars represent 200 μm for (A–D) and 100 μm for (E–I).

FIG. 3.

Laminar and pulsatile shear stress increased eNOS mRNA while not affecting inflammatory markers of BaEOCs. eNOS (A), PECAM-1 (B), intercellular adhesion molecule-1 (ICAM-1) (C), vascular cell adhesion molecule-1 (VCAM-1) (D), P-Selectin (E), E-Selectin (F), and MCP-1 (G) mRNA levels were determined by using quantitative real-time polymerase chain reaction (RT-PCR). mRNA was quantified and compared between shear conditions. Values are mean±SEM with an n_donor=5. *p<0.05 in OS versus SS.

FIG. 4.

Shear stress does not affect the protein levels of the inflammatory markers VCAM-1 and E-Selectin. VCAM-1 (A) and E-Selectin (B) were determined by western blotting. Actin was used as a loading control. Protein levels were determined by densitometry. n=4.

FIG. 5.

Laminar shear stress decreased the inflammatory potential of BaEOCs. Monocyte binding assay was performed and the number of bound monocytes was quantified (A) and normalized by static levels of monocyte binding. (B–F) are representative images of fluorescently labeled monocytes. Values are mean±SEM with an n_donor=5. S, static. *p<0.05 in SS versus OS. Original magnification of 10×. Scale bars represent 200 μm.

FIG. 6.

Laminar shear stress increased the anti-thrombogenicity of BaEOCs. Thrombomodulin (TM) (A), endothelial protein-C receptor (EPCR) (B), CD39 (C), and TFPI (D) mRNA levels were determined by using quantitative real-time PCR. mRNA was quantified and compared between shear conditions. Values are mean±SEM with an n_donor=5. *p<0.05.

FIG. 7.

Shear stress had no significant effect on tissue factor mRNA or activity in BaEOCs. Tissue factor mRNA (A) was determined by using quantitative real-time PCR. mRNA was quantified and compared between shear conditions. Tissue factor activity was determined by conversion of Factor X to Factor Xa through Spectroenzyme FXa and was normalized by total DNA by a PicoGreen^® assay. Values are mean±SEM with an n_donor=5 (A) or n_donor=4 (B).