Single cell mechanical properties of PIK3CA activated mutant endothelial cells contribute to altered mechanotransduction under fluid shear stress

Introduction: Vascular malformations (VMs) are a class of genetic disorders that cause vessel abnormalities, typically presenting as structural lesions in the endothelium. Most VMs are caused by somatic activating mutations in the PIK3CA gene, which encodes the p110a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) [1,2]. PI3K is a major downstream effector of receptor tyrosine kinases and is essential for the transduction of growth factors and cytokines into cell growth, metabolism, proliferation, survival, and angiogenesis [3,4]. Recent studies demonstrated that mutant endothelial cells (ECs) expressing PIK3CA activating mutations (H1047R, E545K, or E542K) are hyperproliferative and develop into dilated vessels with lesion-like structures when cultured within fibrin hydrogels [1,5]. However, these studies, conducted under static conditions, do not indicate how mechanical forces such as fluid shear stress, distending pressure, and stretch that are sensed by ECs regulate proliferation, quiescence, vessel diameter, and vascular permeability [6,7,8]. Our preliminary findings demonstrate that expression of the PIK3CA activating mutation E542K alters endothelial cytoskeletal alignment and barrier function, hallmarks of vascular mechanotransduction, in response to physiologically relevant hemodynamic shear stress. This study seeks to further investigate changes in the intrinsic mechanical properties of endothelial cells expressing PIK3CA mutations, which will provide insight into the time-dependent viscoelastic changes underlying PIK3CA-driven VMs [9]. Understanding the role of PI3K signaling in EC compliance will provide novel therapeutic methods for targeting cell mechanics defects in VMs.

Materials and

Methods: Endothelial cells expressing PIK3CA activating mutation E542K and wildtype PIK3CA (PIK3CAE542K and PIK3CAWT, respectively) were generated as previously described [1]. P6 to P14 ECs were seeded at 150,000 cells/well on collagen type I-coated 6-well plates. Flow was applied by culturing ECs for 3 days on an orbital shaker at 210 rpm [10]. Cells were fixed and stained with anti-VE-cadherin (1:200, Santa Cruz), 4’,6-Diamidino-2-phenylindole (DAPI, Life Technologies) and rhodamine-phalloidin (1 mg/mL, Life Technologies). Samples were imaged using a wide-field microscope (10X/NA 0.4 objective, Olympus IX83). Silicon wafers for the micropipette device were fabricated using standard SU-8 multi-layer photolithography. Devices were designed with two inlet channels converging on an array of 18 pockets with a length, width, and height of 25x20x15 microns, respectively. The pockets trapped and strained cells into 30x3x5 micron micropipette channels under an applied pressure gradient [9]. Devices were molded from silicon wafers using soft lithography with polydimethyl siloxane (PDMS, Sylgard 184, Dow Corning) at a 10:1 base:crosslinker ratio. Devices were plasma treated, bonded to glass slides, and immediately passivated with sterile bovine serum albumin (BSA, 20 mg/mL, Sigma). Cells were stained with Hoechst-33342 (10 mg/mL, Thermo Scientific) and CellMask DeepRed plasma membrane stain (1:2000, 5 mg/mL, Invitrogen) and resuspended in BSA at 5,000,000 cells/mL. Constant-pressure pumps (Flow-EZ, Fluigent) applied 7 kPa of cell suspension and 1.4 kPa of PBS at opposing inlets, with the outlet port open to the atmosphere. Nucleus and cytoplasm deformation were imaged at 0.3407 Hertz using a wide-field microscope (20X/NA 0.45 objective, Olympus IX83).

Results, Conclusions, and Discussions: Flow generated by an orbital shaker creates distinct flow profiles, as fluid shear stress (FSS) changes from multidirection to laminar from the center to the outermost radius of the well [12]. In response to laminar wall shear stress at the outermost radius of the well, PIK3CAWT ECs elongate and align to flow (Fig. 1A, C, & D). As FSS becomes increasingly multidirectional towards the center of the well, orientation of PIK3CAWT ECs becomes more random. In contrast, PIK3CAE542K ECs remain randomly oriented across all radial positions within the well. These results indicate a normal mechanotransduction and cytoskeletal modeling response in PIK3CAWT ECs but not in PIK3CAE542K ECs. Additionally, while PIK3CAWT ECs became less eccentric and less dense in response to lower magnitude multidirectional FSS towards the center of the well, the eccentricity and density of the PIK3CAE542K ECs remained unchanged across all radial positions within the well. Collectively, these results indicate that mechanotransduction of FSS is impaired in PIK3CAE542K ECs.
To distinguish changes in cell intrinsic mechanical properties from mechanics within an EC monolayer, we next analyzed the compliance of PIK3CAWT and PIK3CAE542K ECs on a single cell basis using a microfluidic micropipette aspiration assay. After 85 seconds, PIK3CAE542K ECs demonstrated increased cytoplasm and nucleus deformation, undergoing a greater stretch and budding response through the microfluidic micropipette channels as compared to PIK3CAWT ECs. Thus, PIK3CAE542K ECs were characterized by increased compliance as seen in Figure 2.
Together, these results suggest that PIK3CAE542K ECs have altered mechanical responses to FSS in a monolayer, which can be attributed to their mechanical properties when analyzed autonomously. Through single cell micropipette analysis, PIK3CAE542K ECs demonstrate increased cytoplasm and nuclear compliance, contributing to their decreased alignment to flow and unchanging eccentricity under FSS, as membrane remodeling and barrier function properties are disrupted in the mutant type.
These results suggest that therapeutics normalizing changes in cell compliance in PIK3CAE542K ECs could be a promising strategy for treating the structural and functional abnormalities present in VMs.

Acknowledgements (Optional): References
1. Aw, W.Y. Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation. Sci Adv. 2023 Feb 15;9(7):eade8939. doi: 10.1126/sciadv.ade8939.
2. Castillo, Sandra. PIK3CA mutations in vascular malformations. Current Opinion in Hematology 26(3):p 170-178, May 2019. | DOI: 10.1097/MOH.0000000000000496
3. Fruman D.A. The PI3K Pathway in Human Disease. Cell. 2017 Aug 10;170(4):605-635. doi: 10.1016/j.cell.2017.07.029.
4. Zhu Y.F. PI3K expression and PIK3CA mutations are related to colorectal cancer metastases. World J Gastroenterol. 2012 Jul 28;18(28):3745-51. doi: 10.3748/wjg.v18.i28.3745.
5. Le Cras T.D. Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation. Angiogenesis. 2020 Aug;23(3):425-442. doi: 10.1007/s10456-020-09722-0.
6. Chatterjee S. Mechanotransduction in the endothelium: role of membrane proteins and reactive oxygen species in sensing, transduction, and transmission of the signal with altered blood flow. Antioxid Redox Signal. 2014 Feb 20;20(6):899-913. doi: 10.1089/ars.2013.5624.
7. Abdelilah-Seyfried S. Recalibrating vascular malformations and mechanotransduction by pharmacological intervention. J Clin Invest. 2022 Apr 15;132(8):e160227. doi: 10.1172/JCI160227.
8. Davies P.F. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995 Jul;75(3):519-60. doi: 10.1152/physrev.1995.75.3.519.
9. Davidson, Patricia. (2019). High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells. Lab on a Chip. 19. doi: 10.1039/C9LC00444K.
Jackson, Molly. (2022). Mechanobiology of the endothelium in vascular health and disease: in vitro shear stress models. Cardiovascular Drugs and Therapy. 37. 1-14. doi: 10.1007/s10557-022-07385-1.