Inhibition of apoptosis prevents shear induced detachment of endothelial cells

Abstract

Introduction. The mechanism of shear-induced detachment of endothelial cells (ECs) from biomaterials is undefined. Either healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induce apoptosis leading to cell loss. Previously published work by others has identified that shear stress can “rescue” endothelial cells from apoptosis, utilizing a model in which apoptosis is first induced by serum starvation. We question the relevance of that model. We hypothesized that shear-induced apoptosis is a critical event leading to EC detachment and if apoptosis could be prevented, detachment from a biomaterial would be inhibited. Methods. Human Aortic ECs (passages 5–8) were established on Dacron membranes and subsequently were cultured in the presence or absence of Z-VAD-FMK (25 μM), a pan-caspase inhibitor, in medium for 2 h. Membranes were placed in a parallel plate bioreactor and exposed to 0, 1, 10, and 30 dyn/cm 2 of shear stress for 24 h. Following flow, cells were stained with ethidium bromide and Hoeschst 33258. The ECs remaining on the membrane, percentage of cell retention on the membrane (normalized to no flow controls), and percentage of cells undergoing apoptosis by identification of nuclear morphology were determined by image analysis ( n = 7–13 per group). Results. TABLE—ABSTRACT 54 Mean ± SEM Dynes/cm 2 EC Retention (%) Apoptosis (%) Z-VAD+ Z-VAD− Z-VAD+ Z-VAD− 1 96.7 ± 6.0 97.1 ± 1.1 2.6 ± 0.9 1.7 ± .03 10 †65.5 ± 8.3 †69.3 ± 7.4 ∗2.5 ± 0.5 †8.9 ± 2.2 30 ∗†78.1 ± 2.8 †45.5 ± 7.9 ∗2.3 ± 0.4 †9.0 ± 0.6 ∗ P < 0.05 for Z-VAD+ versus Z-VAD−, † P < .05 as compared with 1 Dyne/cm 2 Increasing shear stress in the absence of Z-VAD-FMK induced apoptosis and detachment as compared with the 1 dyn/cm 2 group. However, treatment with Z-VAD-FMK effectively inhibited apoptosis and resulted in significantly improved EC retention following exposure to high shear stress of 30 dyn/cm 2. Conclusion. These data demonstrate that apoptosis induced by mechanical loading is a critical element responsible for flow-induced cell detachment from biomaterials, and can be modulated pharmacologically.