Poster BB8 - Cell derived extracellular matrix substrate modulates endothelial cell response to fluid shear stress

Introduction: Endothelial cells (EC) morphology and alignment are important factors in vascular health [1, 2]. ECs aligned with the direction of laminar flow are atheroprotective, while disorganized ECs are associated with atheroprone factors [1, 3]. Additionally, substrate mechanics and architecture modulate the EC response to hemodynamic flow. ECs on stiff substrates align to the direction of flow at lower magnitudes of wall shear stress [4] and are more spread [1] in comparison to ECs cultured on more compliant substrates. At the microscale, vascular basement membrane is anisotropic [1], and substrate structure has been shown to govern EC alignment and migration [1, 3]. Since changes in vessel wall mechanics and structure are linked to multiple vascular diseases [1], substrate mechanics and architecture are important factors when building vascular disease models. Current methods of replicating these features in vitro focus on applying a pattern to collagen [1]. These methods do not account for the additional matrix composition, such as laminin, collagen IV, nidogen, and fibronectin, which contribute to matrix structure and assembly [2].
Here, we use the naturally formed microstructure of cell derived extracellular matrix (hCDM) as a substrate for human aortic endothelial cells (HAECs). We use a custom 3D printed cone and plate rheometer to apply physiological shear stress to a 10 cm dish of HAECs cultured on hCDM, and we demonstrate a resulting HAEC morphology that does not change significantly with the application of flow and appears more similar to HAECs on fibronectin exposed to shear.

Materials and

Methods: The 3D printed components of the cone and plate rheometer were designed in Fusion 360 and printed in tough PLA on an Ultimaker Cura S5. The PDMS cone sleeve was made by first 3D printing a mold with an mSLA resin printer (Anycubic Mono X 6K), then vacuum formed (Vaquform DT2) in PETG to create a PDMS compatible mold.
Tissue culture plates (10 cm) were prepared with a 50 μm thick Teflon disk glued with cyanoacrylate superglue into the center before adding substrate. A 5μg/mL fibronectin coating was prepared by allowing the diluted fibronectin to incubate on the plate for at least an hour. The cell derived matrix (hCDM) substrate was prepared by growing human dermal fibroblasts (HDF) on the dish for a week, with media supplemented with 50 μg/ml ascorbic acid. After allowing the HDFs to grow matrix for a week, the cells were removed from the plate, leaving the hCDM behind.
Human aortic endothelial cells (HAECs) were plated on these different substrates and allowed to adhere to the plate for at least 24 hours before being added to the cone and plate rheometer. The rheometer ran at a set shear of 10 dynes/cm2 for 24 hours. Cells were imaged with a widefield microscope with phase contrast before and after application of flow.
Sections of the dish were removed and stained with DAPI, phalloidin, VE cadherin, and GM130 (golgi).
Phase images were analyzed in MATLAB using connected components. Cell shape index was calculated as perimeter2/4π*area.

Results, Conclusions, and Discussions: Results and

Discussion:
A 10 dynes/cm2 shear was applied to HAECs plated on a hCDM substrate using a cone and plate rheometer with a rotation rate of 16.67 rpm (Figure 1A). The point of the cone rested on a Teflon disk to reduce friction and provide a known gap height. The cone consisted of a 3D printed insert, within a molded PDMS sleeve with a 1˚ cone angle (Figure 1B). An aluminum ball bearing supported the weight of the cone, and the hexagon standoff caused the cone to rotate.
Before application of flow, HAECs plated on fibronectin demonstrated a cobblestone morphology and random orientation (Figure 1C), while HAECs plated on hCDM demonstrated an elongated and semi-aligned morphology before the application of flow. After the application of flow, HAECs on fibronectin were more elongated and aligned, while HAECs on hCDM did not significantly change elongation or alignment (Figure 1D). HAECs on hCDM had a significant increase in cell shape index compared to HAECs on fibronectin before the application of flow, but this difference decreased after the application of flow. When comparing the size of the regions of locally aligned cells, HAECs on hCDM were aligned in significantly larger regions than HAECs on fibronectin. This difference was less significant after the application of flow. HAECs on hCDM appeared elongated in the direction of the underlying hCDM fibrils (Figure 1E). Despite the reduced morphological change in HAECs on CDM after application of flow, RNA sequencing indicated an upregulation of flow sensitive genes such as KLF2.

Conclusions:
The data contained in this study suggest that HAECs align parallel to the fibrillar orientation on hCDM substrates, and that flow has less of an effect in directing cell alignment on hCDM substrates as compared to fibronectin. Furthermore, cell elongation and local correlation of alignment direction for HAECs under static conditions on hCDM phenocopies HAECs on fibronectin and exposed to flow. Despite the flow-like morphology in the static hCDM condition and the minimal change after application of flow, RNA sequencing indicates that HAECs on hCDM continue to sense and respond to flow.

Acknowledgements (Optional): Sarah Kubik is supported by the Comparative Molecular Medicine Training Program (T32 GM133393), and the work is supported by the National Institutes of Health (1R35 GM142944). Additional funding for this project comes from NIH F31HL162462 (ELD) and NIH T32HL69768 (ELD&CPW)

References
1. Dessalles, C.A., et al., Integration of substrate- and flow-derived stresses in endothelial cell mechanobiology. Communications Biology, 2021. 4(1).
2. Leclech, C., C.F. Natale, and A.I. Barakat, The basement membrane as a structured surface – role in vascular health and disease. Journal of Cell Science, 2020. 133(18): p. jcs239889.
3. Nakayama, K.H., et al., Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress. Nano Letters, 2016. 16(1): p. 410-419.
4. Galie, P.A., et al., Application of multiple levels of fluid shear stress to endothelial cells plated on polyacrylamide gels. Lab on a Chip, 2015. 15(4): p. 1205-1212.