Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity under Homeostatic Laminar Flow

Abstract

ObjectiveEndothelial cells that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to endothelial cell quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level endothelial cells regulate overall expression heterogeneity during this transition is poorly understood. Here we profiled endothelial cell transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow.Approach and ResultsSingle-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human endothelial cells under homeostatic laminar flow compared to non-flow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous. Analysis of a subset of genes for relative protein expression revealed that most protein profiles showed decreased heterogeneity under flow. In contrast, the magnitude of expression level changes in RNA and protein was coordinated among endothelial cells in flow vs. non-flow conditions.ConclusionsEndothelial cells exposed to homeostatic laminar flow showed increased cohort heterogeneity in RNA expression levels, while cohort expression heterogeneity of selected cognate proteins decreased under laminar flow. These findings suggest that EC homeostasis is imposed at the level of protein translation and/or stability rather than transcriptionally.

Adaptation of peristaltic pumps for laminar flow experiments

“…In vitro systems have been built to mimic individual types of flow forces or patterns felt by ECs along different regions of the vascular tree, including oscillatory, pulsatile, and laminar. Consistent with what is observed under physiological conditions, ECs cultured in vitro under laminar flow conditions align parallel to the direction of flow [5,6,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Similarly, ECs experiencing pulsatile or oscillatory flow conditions remain more haphazardly organized, or with a perpendicular alignment to the flow direction [5,6,14,15,[17][18][19][20][21][22][23][24][25][26][27][28][29].…”

“…To help maintain cell health, at the start of the experiment the rate of flow was stepwise increased 10% every 10 minutes until reaching the desired final experimental flow rate. The copyright holder for this preprint this version posted October 18, 2021. ; https://doi.org/10.1101/2021.10.17.464751 doi: bioRxiv preprint 7 [5,6,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], validating that our system does indeed reliably model flow stimuli to elicit the expected changes in cellular behavior.…”

“…Perfusing 2D or 3D cultures of endothelial cells (EC) with fluid is a popular technique to model the effects of blood flow and shear stress forces on the vasculature [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. In vitro systems have been built to mimic individual types of flow forces or patterns felt by ECs along different regions of the vascular tree, including oscillatory, pulsatile, and laminar.…”

Large Vessel Cell Heterogeneity and Plasticity: Focus in Aortic Aneurysms

“…In various in vitro models, flow-induced shear stress has been shown to affect signaling of ECs and SMCs, either directly or via cellular crosstalk, and to induce SMC phenotype modulation, altered gene expression or re-organization of the cellular cytoskeleton. 50–53 Laminar flow-induced EC polarization and alignment to the direction of flow is evident, and recently Kant et al 53 demonstrated involvement of PGC1α - TERT - HMOX1 pathway in this response. Also, for example, PGI2 and other factors secreted by ECs under laminar flow can control phenotypic modulation of SMCs towards contractile stage.…”

The Aging Aorta: Are We Only as Old as Our Endothelium?

“…3 EC heterogeneity in the normal vasculature is driven by factors, such as anatomic location and shear stress. 4,5 Single-cell RNA-sequencing (scRNA-seq) is a powerful tool for dissecting the transcriptional signatures of individual cell populations. Prior scRNA-seq studies of the murine aorta have identified EC subpopulations with functionally distinct marker genes that suggest functional specialization.…”