Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

Abstract

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.

Transcriptomic analysis of 3D vasculature-on-a-chip reveals paracrine factors affecting vasculature growth and maturation

“…We hypothesized that the temporal morphological changes and the phenotypic transitions of the ECs could be governed at the level of transcriptional regulation. In previous studies, 1,25 the RNA expression levels were measured for ECs cocultured with FBs and ECs monocultured. However, the cells were not cocultured in the distance, making it difficult to independently analyze the expression changes in individual cell types for paracrine communication.…”

“…4D), providing additional evidence that coculturing affects the production of secretory proteins that are involved in cell-cell communication. To investigate if the conventional juxtacrine coculture of ECs and FBs would allow similar observations, 24,25 we performed the same DAVID gene enrichment analysis for the conventional cultures. The results show that only 3 out of the top 75 genes that are differentially expressed between the conventional cocultured ECs and monocultured ECs belong to groups related to secretory proteins (Fig.…”

“…[26][27][28] Importantly, with the uniquely defined spatial positions of ECs and FBs in the microfluidic device, we could specifically infer and isolate non-contact paracrine intercellular communications and interactions, which is traditionally difficult to tease out from other contact-type interactions. 1,25,62 Here we specifically looked at the interactions between the ECs and FBs on day 5, since there is the most dramatic difference between the mono-and cocultured ECs on this day.…”

“…Thus far, data on interactions between these cell types are based on conventional 2D dish cultures, in which both cell types are directly in contact with each other. 1,24,25,62 This confounds the results between juxtacrine (direct contact) and paracrine (non-direct contact) signaling. As such, transcriptional analysis of the cells cultured in a precisely controlled microenvironment while being spatially arranged in a non-contact manner will provide important insight into the paracrine cues of fibroblasts affecting vasculogenesis (Fig.…”

Systems Biology Will Direct Vascular-Targeted Therapy for Obesity

“…Therefore, cell-by-cell qFlow analysis and mixture modeling should be used for identifying cell subpopulations exhibiting distinct receptor expression and VEGFR-mediated cell responses in adipose tissue. qFlow has successfully measured membrane receptor levels on tumor xenograft-derived cells (Imoukhuede and Popel, 2014;Chen et al, 2018), primary mouse skeletal muscle endothelial cells under normal (Imoukhuede and Popel, 2012), and ischemic (Imoukhuede et al, 2013) conditions, and several in vitro cell lines (Chen et al, 2015;Chen and Imoukhuede, 2019). To summarize some of the key qFlow findings, human umbilical vein endothelial cells (HUVECs) present 1800 ± 200 VEGFR1/cell, 4900 ± 400 VEGFR2/cell, and 2800 ± 400 VEGFR3/cell.…”