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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session E

Poster U6 - Role of Uniaxial Stretch on Endothelial von Willebrand Factor and Angiopoietin-2 Regulation

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Patients supported on Continuous Flow Ventricular Assist Devices (CF-VAD) develop a condition called acquired von Willebrand Syndrome (AVWS), marked by a reduction in total von Willebrand Factor (vWF) and significant degradation of available vWF into low molecular weight multimers, impairing coagulation. Consequently, these patients face a high risk of non-surgical bleeding. Our prior studies have focused on the shear-induced production and degradation of vWF under continuous- and pulsatile-flow conditions, and Angiopoietin 2 (ANGPT-2) as a biomarker for loss of pulsatility. However, the effect of loss of pulsatility in the context of uniaxial stretch remains unexplored. To evaluate pulsatile versus constant stretch, adult human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) were seeded into in vitro platforms to impose either pulsatile or constant stretch.

Materials and

Methods:  Base setup consists of a polydimethylsiloxane (PDMS) stretch 'device' that serves as a housing for the cell monolayer. Devices are treated with a round of low temperature, oxygen plasma cleaning to enhance protein binding affinity. Then, devices are coated with human fibronectin for 45 minutes and washed thoroughly after to remove poorly bound fibronectin. Devices are then seeded with ~5 x 10^5 human aortic endothelial cells (HAECs) or human umbilical vein endothelial cells (HUVECs) to act as arterial and venous vessels, respectively. Devices are left to incubate for 1-2 days for cells to spread into a uniform monolayer. Once confluent, devices are divided into two groups: static control and preliminary stretch. The preliminary stretch devices are placed on vacuum conditions (60 bpm, ~ 50 mmHg, 24 hours) that will account for a 10-12% stretch of the membrane. Once done, the preliminary group will be divided in two groups: cyclic and continuous. Cyclic group will have the same conditions as the preliminary group for 72 hours. Continuous stretch group will undergo constant stretch (~ 12 mmHg) accounting for a ~6% stretch of the membrane for 72 hours. Media collection take place at 24- and 96- hour timepoints for release kinetics analysis using ELISA kits. Cells are either lysed for intramembrane protein analysis or gene expression through qRT-PCR, or cells are fixed for Immunofluorescent imaging.

Results, Conclusions, and Discussions: 

Results:
Results indicate that under cyclic, physiological stretch, vWF expression and production increase in HAECs but remain unchanged in HUVECs compared to the continuous stretch group. Conversely, ANGPT-2 production and expression are upregulated in both HAECs and HUVECs under continuous stretch. Analysis of the cell culture medium shows no significant difference in the release of vWF or ANGPT-2 between cohorts.

Conclusions:
Results suggest that stretch on arterial vessels is a crucial stimulus for maintaining basal levels of vWF, while loss of pulsatile stretch upregulates ANGPT-2 production in both vascular cell types. Nonetheless, flow-mediated shear stress remains essential for the release of these proteins as stretch seems to not play a crucial role on release kinetics.

Discussion:
This study underscores the importance of simulating both stretch and flow conditions in vitro to better understand the vascular complications seen in patients with CF-VADs. The introduction of both pulsatile and constant stretch as variables allows for a more comprehensive examination of the mechanical forces impacting endothelial function. The findings demonstrate that cyclic, physiological stretch significantly increases vWF production in HAECs, highlighting the role of pulsatile stretch in maintaining coagulation homeostasis. This response was not observed in HUVECs, indicating a potential difference in how various endothelial cell types respond to mechanical stimuli. Furthermore, the upregulation of ANGPT-2 under constant stretch in both cell types suggests that loss of pulsatility could contribute to endothelial dysfunction in CF-VAD patients. These results emphasize the necessity of incorporating both stretch and shear stress in vitro to accurately replicate the physiological conditions affecting these patients. Understanding these dynamics could inform the development of improved CF-VADs that preserve pulsatile flow, potentially reducing the risk of acquired von Willebrand Syndrome and associated bleeding complications.

Acknowledgements (Optional):