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Tissue Engineering

Tissue Engineering - Poster Session A

Poster L20 - The Sexually Dimorphic Effect of Shear Stress on Aortic Valve Endothelial Cells

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Congenital heart valve defects (CHVD) and their concomitant valvular diseases are increasingly being considered sexually dimorphic. Bicuspid aortic valve disease (BAV), the most common CHVD, affects 1.5% and 0.5% of 46,XY and 46,XX patients respectively. Aortic stenosis, the chronic narrowing of the aortic valve is more common in biological women than men with BAV. Stenotic degeneration of the valve in patients with BAV also progresses differentially with respect to biological sex. Valvular calcifications and regurgitation is more common in biological men, while fibrosis is more common in biological women. In-vitro experiments have also shown that female aortic valve interstitial cells (VICs) have a higher sensitivity to myofibroblast activation with respect to hydrogel stiffness but were less susceptible to calcific induction than male VICs. This suggests the X-chromosome has a regulative role in AV homeostasis and supports the need to incorporate biological sex as a control to study aortic valve physiology and pathology in-vitro. Aortic valve endothelial cells (VECs) are an important mediator in AV hemostasis as they can respond to mechanical stimuli, such as shear stress, by secreting signals for inflammation, permeability, and tissue remodeling. Literature suggests that pathological shear stress on VECs leads to transcriptional changes towards an invasive phenotype known as the endothelial-to-mesenchymal transition (EndMT). VECs lose their endothelial markers, restructure their cytoskeleton and can promote fibrosis and inflammation. It is unknown if the X-chromosome is responsible for the regulation of EndMT in response to shear stress.

Materials and

Methods:  To investigate the role of shear stress on EndMT with respect to biological sex, we implemented a tissue engineering model that uses a cone-plate bioreactor to apply uniform shear stress on porcine aortic valve endothelial cell monolayers (PAVECs). We first isolated PAVECs from fresh hearts of male and female young adult pigs (6-9 months old) and purified the endothelial cell line twice through magnetic activated cell sorting using a CD31 porcine antibody. Passage 3 male and female PAVECs were seeded at approximately 10k cells/cm^2 on gelatin coated 60 mm petri dishes and incubated for 5 days at standard cell culture conditions before being exposed to either physiological (15 dynes/cm^2) or pathological (25 dynes/cm^2) shear stress magnitudes for 24hrs. Brightfield images were taken at 4 positions of the petri-dish before and after shear stress stimulation to quantify cellular alignment. rt-qPCR was also performed to measure relative gene expressions of endothelial (vWF), inflammatory (VCAM-1), End-MT (SNAI-1) markers. Baseline cellular alignment and gene expressions were obtained from control samples not exposed to shear stress (0 dynes/cm^2).

Results, Conclusions, and Discussions: Assessment of female PAVECs sorted twice showed 94% purity, confirming the effectiveness of the magnetic-activated cell sorting process. Brightfield images of the female PAVEC monolayer acquired before and after the application of shear stress showed no consistent alignment. However, the high amount of variability in the later time point suggests moderate changes in cellular alignment, but not in a consistent manner. Meanwhile, the male PAVECs demonstrated a small peak around 45 degrees, where 90 degrees is perpendicular to the direction of flow. Additionally, the peak became larger with increasing shear stress, indicating more cellular alignment with a higher shear rate.
The relative gene expression of vWF increased in the male PAVEC 15 dynes/cm^2 group when compared to the control group, however, there was no difference in the male 25 dynes/cm^2 group vs. the control group and the physiological shear group. When the male PAVECs were tested for VCAM-1, the 25 dynes/cm^2 group was significantly greater than both the control and physiological groups, while the 15 dyne group showed no difference with the static control. In comparison, the expression of vWF in the female PAVECs trended downward with increasing shear rate, with statistical significance only between the static control and 25 dynes/cm^2. Additionally, VCAM-1 had the highest expression in the 15 dynes/cm^2 group, but only showed significance with the pathological shear rate. Ongoing work will determine the relative gene expression of SNAI-1 for both the female and male PAVECs. When comparing the gene expression results between the female and male groups, it is clear that both cell types respond differently to different levels of shear stress. More research will need to be conducted in order to fully understand the relationship between shear stress and the end-MT pathway in this sexually dimorphic disease state of calcific aortic valve disease.

Acknowledgements (Optional):