Effects of low shear stress and donor sex on human pulmonary artery endothelial cell morphology and gene regulation

Introduction: : One in four individuals are projected to develop heart failure in their lifetime [1]. Left sided heart failure (LHF), the most common manifestation, can lead to pulmonary hypertension in 65% to 85% of patients [2]. Pulmonary hypertension from left heart failure (PH-LHF) is the most common form of PH. Fundamental knowledge gaps in the progression of PH-LHF prevent effective treatment for this deadly disease. Our research group is pursuing the overarching hypothesis that decreased pulmonary vascular wall shear stress due to increased pressure and decreased flow is critical to the progression of LHF to PH-LHF. In particular, we hypothesize that mechanotransduction of low shear stress by endothelial cells in the pulmonary arteries (PA) play a key role in this transition. PA endothelial cells are known to direct vascular cell proliferation, extracellular matrix synthesis and degradation, and smooth muscle cell constriction [3]. Patient sex is also a critical consideration, as there are known sex differences in PH and RV dysfunction [4]. Improved in vitro models are important to isolate the impact of decreased shear stress, understand mechanobiological changes, and identify potential therapeutic targets for intervention. The goal of this work is to investigate the chronic impact of decreased wall shear stress on endothelial extracellular matrix integrity, mechanosensing, and inflammation through an in vitro pulmonary arterial endothelial model.

Materials and

Methods: : Human Pulmonary Artery Endothelial Cells (HPAECs) and Human Umbilical Vein Endothelial Cell (HUVECs) were exposed to low (5 dyn/cm^2) or normal (15 dyn/cm^2) pulsatile shear stress for 120 hours. The HPAECs were obtained from the Pulmonary Hypertension Breakthrough Initiative, and consisted of samples from both male and female healthy subjects. Pooled HUVECS were obtained from Lonza and were used as a control cell line. All endothelial cells were cultured in T75 flasks, and once confluent were seeded into ibidi 0.4 luer slides. After overnight incubation, cells received a media change and then were attached to the ibidi pump system. A static run for 120 hours served as a secondary control condition. All experiments were performed in an incubator at 37 °C. Every 24 hours, phase contrast images were taken with an Olympus IX83 microscope. Serial orientation and cell shape analyses were performed on phase contrast images using Python and Cellsam softwares. At 120 hours, cells were fixed and stained for DAPI, VE-Cadherin, PECAM, WGA (glycocalyx marker), and F-actin. Immunofluorescence stains were imaged using an Olympus FV3000 confocal microscope and the glycocalyx was imaged through confocal Z stacking. RNA was isolated using a Qiagen RNA Minikit and bulk sequencing was done through Illumina sequencing NovaSeq. The mRNA libraries were further analyzed with R, deSeq, and GSEA. Supernatant was collected and spun down and frozen at -80°C for ELISAs, an ELISA for ET-1 (biotechne) and IL-6 (Invitrogen) was performed. Statistical analysis was performed using T-tests and F-tests.

Results, Conclusions, and Discussions:: Based on morphological analysis, HPAECs in the low shear stress condition had decreased orientation to the flow direction, increased circularity, and decreased polarity compared to normal shear stress. HPAEC orientation fluctuated more over time in response to low shear stress. Preliminary semi-quantitative analysis of F-actin confirmed that low shear stress decreased alignment to the flow direction as seen in Figure 1. Low shear stress led to decreased glycocalyx coverage and VE-cadherin staining than normal shear stress, indicating less developed ECM and more limited cell-to-cell communication. HUVECs followed similar trends, except for 50% thinner glycocalyx height under static conditions.
Bulk RNA seq analysis, as seen in Figure 2, identified differential gene expression as functions of shear stress (low vs. normal), sex (male vs. female), and cell line (HUVEC vs. HPAEC). Shear stress differentially impacted genes such as MAGI1, HAPLN3, CD59, and APLNR which are involved with cell junctions and surface proteins. Donor sex impacted genes included SMAD6 (signaling) which discriminated in female samples and genes such as CXCL1 (chemokine), IL33 (inflammatory cytokine), and KLF2 (mechanosensor), which discriminated in male samples. In HUVECs, IL7 and IL32 (inflammatory cytokines) were downregulated compared to HPAECs, while GATA3 was upregulated (T cell differentiation).
Shown in Figure 3, ELISA revealed a nearly 2-fold increase in expression of the potent vasoconstrictor ET-1 by low shear conditions in HPAECs and over 3-fold increase by low shear conditions in HUVECs. ELISA revealed a nearly 80% decrease in IL-6 cytokine by low shear conditions in HPAECs, but no significant changes in HUVECs.
Our results demonstrate that low shear stress leads to a more disorganized endothelium which may have increased permeability and decreased glycocalyx coverage and VE-cadherin, suggesting vulnerability to inflammatory damage. Finally, low shear led to increased expression of ET-1, implicating this stimulus in increased pulmonary vascular resistance via enhanced vasoconstriction. Our results highlight the importance of cell line-specific experimentation, and suggest potential sex differences in mechanotransduction. Overall, our data supports the key role of endothelial mechanobiology in PH-LF progression and highlights the importance of in vitro models that offer insight into the biological consequences of an altered mechanical environment.

Acknowledgements and/or References (Optional):: References
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