Project 2

Background and preliminary work: Endothelial cells (ECs) are among the cell types of the cardiovascular system with the highest degree of cellular and molecular plasticity, as they form the dynamic interface between the blood stream and each tissue in the human body. The Karen Hirschi and Eckhard Lammert laboratories have a long-standing research interest in EC plasticity and signals involved in EC development and maturation. More specifically, the Hirschi lab found that the cell cycle state of ECs (early G1 versus late G1) has a major impact on their developmental fate and can be endogenously (e.g, via retinoic acid; RA) and pharmacologically (e.g., via palbociclib) altered ^{1, 2}. The lab further showed that shear flow forces trigger arterial EC specification through a Notch-Cx37-p27-mediated cell cycle arrest ³. Like the Hirschi lab, the Lammert lab has been working on the impact of chemical signals (e.g., free fatty acids, FFA, like palmitic acid, PA, and oleic acid, OA) and mechanical forces (e.g. mechanical stretching) on vascular endothelium ^4-6. For example, the lab showed that oscillatory stretching of ECs (as it occurs during vasodilation) alters the angiocrine secretome of human hepatic ECs and results in the release of angiokines (such as IL-6 and TNFα), HGF and cardioprotective myeloid-derived growth factor, MYDGF ^{5, 6}. A major question still to be solved is how the expression of angiocrine signals change in response to mechanical forces when the ECs are at different G1 states. These investigations are now possible at UVA due to the availability of so-called Fluorescent Ubiquitination Cell Cycle Indicator (FUCCI) reporter transgenes that can be expressed in ECs and allow flow cytometric sorting of ECs (from both cell culture and mouse tissue) into EC populations at the early (FUCCI-negative) versus late (FUCCI-Red) G1 state ⁷, as previously explored by the Hirschi lab ¹. Cell sorting with subsequent bulk and scRNAseq, bioinformatic analyses (such as PCA and PHATE mapping) as well as subsequent gene knockdown or knockout of differentially expressed genes are in place for functional analyses at both laboratories ^{1, 8, 9}. A molecular analysis of EC plasticity and limits thereof in response to mechanical and chemical stimuli is urgently needed to better understand how ECs develop and shape their cellular environment within the cardiovascular system under physiologic as well as pathologic situations. 

Hypothesis: The hypothesis of this collaborative research project is that (i) adult human vascular ECs maintain a high degree of plasticity, as to differentially respond to mechanical and chemical cues by altering their cell fate and angiocrine secretome and that (ii) this EC plasticity depends on the EC cycle state. Here the molecular alterations in human (and mouse) ECs in response to mechanical forces (shear flow and vasodilatory stretch) and biological chemicals (RA and FFA) will be dissected to better understand EC plasticity that partially depends on the cell cycle state. 

Work program: The collaborative research project wishes to identify how ECs change their phenotypes. Chemically and mechanically treated versus untreated human microvascular ECs (applied to the 10x Genomics Chromium platform for generation of single cell libraries) will be compared by using scRNAseq (using the HiSeq 3,000 system from Illumina) with subsequent filtering and PCA analyses to identify alterations in EC populations (e.g., by using UMAP dimension reduction plots) (N = 5,000 transcriptional profiles per condition: stretch vs. non-stretch; PA vs. OA, RA vs. no RA; In sum: 30,000 scRNAseq). Cell cycle scoring and cell-cell communication via angiocrine signals will be analyzed by using the Seurat and CellPhoneDB software. FUCCI-transfected HUVECs (with and without stretch and PA vs. OA treatment) will be sorted into S/G2/M (FUCCI-Green), early G1 (unstained) and late G1 state (FUCCI-Red) and subjected to bulk RNA sequencing ^{1, 8}. The goal is to identify alterations in gene expression depending on the early vs. late G1 cell cycle state (N = 8 bulk RNAseq). Identified genes will be analyzed by bioinformatic tools, including PHATE mapping ^{1, 2}. It will be studied whether (i) FFA treatment or mechanical stimuli reduce or enlarge the EC population with a growth-promoting angiocrine secretome (with expression of e.g., IL6, TNFα, HGF and MYDGF) and whether (ii) this angiocrine signature depends on the cell cycle state. ECs will also be sorted from mice in order to perform scRNAseq in order to correlate the human in vitro data with mouse in vivo data (N = 20,000 scRNAseq). Finally, EC morphologies alter upon mechanical stretch and FFA treatment ^{4, 6}; and scanning electron microscopy (SEM) will be applied to obtain high resolution images of ECs that are subsequently analyzed by using a machine learning-based, unbiased quantification. The latter was recently developed by the Lammert lab in collaboration with the Institute of Computer Science and Helmholtz AI at HHU and Forschungszentrum Jülich ⁴. The overall objective is to identify shifts in EC populations, angiocrine secretomes and EC morphologies using a combination of “wet work” and bioinformatics. 

Cellular plasticity and omics methods: All experiments are designed to better understand microvascular EC plasticity and the limits thereof. Bulk and scRNAseq experiments will be performed in collaboration with the BMFZ at HHU and Bioinformatics Core Units at HHU and UVA.

Added value through the collaboration between HHU & UVA: The Hirschi and Lammert laboratories both work on vascular endothelium and its development. They work on human cell systems as well as mice to investigate changes in vascular endothelial phenotypes. Both labs have worked on aspects of mechanical and chemical stimulation and RNAseq. The advantage of the proposed collaboration is that the Hirschi lab brings in the whole knowledge on retinoic acid (RA) signaling and EC cycle state, while the Lammert lab brings in the technology of oscillatory mechanical stretching, FFA treatment and machine-based image analyses. The collaboration will add significantly to Hirschi’s research on human hemogenic EC phenotypes and Lammert’s research on adult human microvascular EC. More specifically, a PhD student from HHU will join the Hirschi lab to perform the FUCCI experiments to analyze the impact of FFA treatment on ECs at different G1 states. In turn, a PhD student from the UVA will join the Lammert lab to perform cell stretching experiments to analyze the impact of RA on hemogenic development of stretched vs. unstretched ECs.