Basic research study
Differential effects of orbital and laminar shear stress on endothelial cells

Presented in part at the American College of Surgeons Surgical Forum, New Orleans, La, Oct 10–14, 2004, at the Annual Meeting of the Association for Academic Surgery, Houston, Texas, Nov 11–13, 2004, and at the Annual Meeting of the American Society for Cell Biology, San Francisco, Calif, 2003.
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Objective

Laminar shear stress is atheroprotective for endothelial cells (ECs), whereas nonlaminar, disturbed, or oscillatory shear stress correlates with development of atherosclerosis and neointimal hyperplasia. The effects of orbital and laminar shear stress on EC morphology, proliferation, and apoptosis were compared.

Methods

ECs were exposed to orbital shear stress with an orbital shaker (210 rpm) or laminar shear stress (14 dyne/cm2) with a parallel plate. Shear stress in the orbital shaker was measured with optical velocimetry. Cell proliferation was assessed with direct counting and proliferating cell nuclear antigen staining; apoptosis was assessed with transferase-mediated deoxyuridine triphosphate nick end labeling staining. Cell surface E-selectin and intercellular adhesion molecule expression were assessed with fluorescence-activated cell sorting. Akt phosphorylation was assessed with Western blotting.

Results

Orbital shear stress increased EC proliferation by 29% and 3[H]thymidine incorporation two-fold compared to 16% and 38% decreases, respectively, in ECs treated with laminar shear stress (P < .0001 and P = .03, analysis of variance). Cells in the periphery of the culture well aligned to the direction of shear stress similar to the shape change seen with laminar shear stress, whereas ECs in the center of the well appeared unaligned similar to ECs not exposed to shear stress. Shear stress at the bottom surface of the culture well was reduced in the center of the well (5 dyne/cm2) compared to the periphery (11 dyne/cm2); the Reynolds’ number was 2066. ECs were seeded differentially in the center and periphery of the wells. ECs in the center of the well had increased proliferation, increased apoptosis, reduced Akt phosphorylation, increased intercelluar adhesion molecule expression, and reduced E-selectin down-regulation, compared with ECs in the periphery of the well.

Conclusion

Although the orbital shaker does not apply uniform shear stress throughout the culture well, arterial magnitudes of shear stress are present in the periphery of the well. ECs cultured in the center of the well exposed to low magnitudes of orbital shear stress might be a model of the “activated” EC phenotype.

Clinical relevance

The perfect in vitro model to study and assess treatments for atherosclerosis and neointimal hyperplasia does not exist. An extensive body of literature describing effects of laminar shear stress on endothelial cells has contributed to our understanding of the interactions between shear stress and blood vessels. Laminar shear stress is atheroprotective, whereas oscillatory or disturbed shear stress correlates with areas of atherosclerosis and neointimal hyperplasia in vivo. This study describes the orbital shear stress model, its effects on endothelial cell proliferation and apoptosis, and suggests that activation of the intracellular Akt pathway is associated with these differing effects of laminar and orbital shear stress on endothelial cells.

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Competition of interest: none.

Supported by an American College of Surgeons Faculty Research Fellowship (A.D.), the Pacific Vascular Research Foundation, San Francisco (A.D.), the Dennis W. Jahnigen Career Development Scholarship Program, which is administered by the American Geriatrics Society through an initiative funded by The John A. Hartford Foundation of New York City and The Atlantic Philanthropies (A.D.), a National Institutes of Health grant R01-HL47345-05 (B.E.S.), and a VA Merit Award (B.E.S.).