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. 2018 Jun 29:4:20-35.
doi: 10.1016/j.isci.2018.05.003. Epub 2018 May 9.

Human Organ-Specific Endothelial Cell Heterogeneity

Raluca Marcu  1 , Yoon Jung Choi  1 , Jun Xue  1 , Chelsea L Fortin  2 , Yuliang Wang  3 , Ryan J Nagao  1 , Jin Xu  1 , James W MacDonald  4 , Theo K Bammler  4 , Charles E Murry  5 , Kimberly Muczynski  6 , Kelly R Stevens  7 , Jonathan Himmelfarb  8 , Stephen M Schwartz  2 , Ying Zheng  9
Affiliations

Human Organ-Specific Endothelial Cell Heterogeneity

Raluca Marcu et al. iScience. .

Abstract

The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs-the heart, lung, liver, and kidneys-in individual fetal tissues at three months' gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells.

Keywords: Biology of Human Development; Developmental Biology; Stem Cells Research.

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Figures

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Graphical abstract
Figure 1
Figure 1
Human Fetal Endothelial Cells Show Organ-Specific Heterogeneity Ex Vivo (A) (A) Representative immunofluorescence images of frozen tissue sections from fetal human kidney, lung, liver, and heart stained with antibodies against CD144 (i) and vWF (ii) with merged (iii) and zoomed (iv) views, and (v) CD31 and vWF. Scale bar: 50 μm (i–iii, v) and 25 μm (iv). (B–D) Quantification of vWF fluorescence area normalized to the number of CD31-positive cells for all four organs (B), within lungs (C), and kidneys (D). **** p ≤ 0.0001. (E) Representative immunofluorescence images of frozen tissue sections stained with antibodies against PV1 and Cav1. Scale bar: 50 μm. (F) Quantification of PV1 fluorescence area normalized to the number of CD31-positive cells for all four organs. ** p ≤ 0.01. (G) Representative flow cytometry profiles of fresh total cell tissue suspension from fetal human heart, lung, liver, and kidney stained with antibodies against CD45 and CD144. Endothelial population is gated as CD144-positive and CD45-negative cells (left panels). The percentage of endothelial cells is compared among tissues against the total cell number (right panel). Bar plots were made from n = 3 images per donor for two donors (B–D, F) or three donor sets (G). Data are presented as mean ± SEM *p ≤ 0.05. SEM, standard error of the mean.
Figure 2
Figure 2
Human Fetal ECs Retain the Ex Vivo Heterogeneity upon In Vitro Expansion (A) Representative flow cytometry and sorting profiles of enriched cell suspension from fetal human heart, lung, liver, and kidney, stained with antibodies against VECad, PDGFRb. (B and C) Representative immunofluorescence images of CD31 and vWF (B) and PV1 and Cav1 (C) fluorescence in cultured heart, lung, liver, and kidney ECs (scale bars: 50 μm). (D) Representative western blot analysis (i) and densitometric quantification (ii) of vWF, PV1, and caveolin expression on cell lysates obtained from four ECs, using GAPDH as the loading control (n = 3 donors). Data are presented as mean ± SEM ** p ≤ 0.01, **** p ≤ 0.0001. SEM, standard error of the mean.
Figure 3
Figure 3
Ultrastructural Heterogeneity of Human Fetal ECs upon In Vitro Expansion (A) Projected immunofluorescence images and cross-sectional views of engineered perfusable, collagen-embedded, three-dimensional (3D) microvessel networks generated using the four types of organ-specific ECs (scale bar: 100 μm). (B) Transmission electron microscopy of 3D microvessel networks showing distinctive structural signatures. Black arrows indicate the presence of fenestrae, and red circles denote clathrin-coated alveolae.
Figure 4
Figure 4
Global RNA Sequencing Reveals Heterogeneous Gene Expression Profiles among Four EC Types (n = 3 Donors) (A) 2D principle component analysis of RNA sequencing data for cultured organ-specific ECs showing clustered heart ECs compared with the kidney, lung, and liver ECs. (B) Venn diagram of differentially expressed heart-specific ECs genes with respect to kidney, lung, and liver ECs. (C and D) Gene Ontology terminology analysis (C) and volcano plot (D) showing different gene clusters for heart versus kidney ECs. (E) Heatmaps of bona fide markers for the heart-, kidney-, and lung-specific EC transcription factors and co-factors (i), angiocrine and signaling factors (ii), and metabolism-related and other genes (iii). (F) PCR validation of selected heart- and kidney-specific genes for additional three donor sets.
Figure 5
Figure 5
Validation of Heterogeneous Gene Expression Profiles in Freshly Isolated ECs and Ex Vivo Tissue Sections (n = 3 Donors) (A) Organ-specific genes identified from cultured sets (heart versus kidney) were validated in freshly isolated EC sets. (B) Immunostaining of selected genes for heart and kidney in human fetal tissues at 120 days. Scale bar: 100 μm. (C) Selected organ-specific gene expression in mouse embryo for heart and kidneys at E14.5 from Genepaint (genepaint.org).
Figure 6
Figure 6
Cultured Organ-Specific Human Fetal ECs Display Distinct Vascular Functions and Bioenergetics and Support Specific Parenchyma Function (A) Left panel: Raw data of transendothelial electrical resistance measurement versus time for four types of ECs in monolayer culture. Cell index is calculated as CI(t)=R(fn,t)−R(fn,t0)Zn, where fn is the frequency at which the impedance measurement is carried out (fn = 10 kHz), R(fn,t) is the measured impedance at frequency fn at time point t, t0 is the time when the background is measured, and Zn is the corresponding frequency factor of fn (Zn = 15 Ω). Right panel: Electrical impedance measurements (10 kHz frequency) for confluent monolayers of organ-specific ECs (n = 5 cultures). (B) Spheroid sprouting assay measurements of organ-specific EC angiogenic potential. (i) Representative bright-field images of heart and kidney EC sprouting spheroids, collagen-embedded, upon 24 hr of VEGF stimulation (40 ng/mL) (scale bar: 75 μm) and (ii) quantification of sprouts number at baseline and after VEGF stimulation (40 ng/mL) for organ-specific EC spheroids embedded in collagen matrix (24 hr) (n = 3 donors). (C) Seahorse measurements of total cellular oxygen consumption rate (OCR) in organ-specific ECs: (i) time course of cellular oxygen consumption upon sequential addition of oligomycin (1 μM), CCCP (1 μM), and rotenone/antimycin A (1 μM each); (ii) quantification of mitochondrial OCR obtained upon electron transport chain inhibition with rotenone and antimycin A; and (iii) quantification of mitochondrial OCR due to ATP synthase activation obtained upon ATP synthase inhibition with oligomycin (n = 3 donors). (D) Seahorse measurements of total extracellular acidification rate (ECAR) in organ-specific ECs (n = 3 donors): (i) time course of extracellular acidification upon sequential addition of oligomycin (1 μM), CCCP (1 μM), and rotenone/antimycin A (1 μM each) and (ii) ECAR quantification. (E) ELISA measurement of albumin production from rat hepatocytes when cultured alone and when co-cultured with heart, lung, liver, and kidney ECs after 7 days (n = 4 replicates). Data information: data are presented as mean ± SEM *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. SEM, standard error of the mean.
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References

    1. Abrahamson D.R., Chen F., Chen M., Mecham R., Nguyen N., Jarad G. Development of kidney glomerular endothelial cells and their role in basement membrane assembly. Organogenesis. 2009;5:275–287. - PMC - PubMed
    1. Adams R.H., Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 2007;8:464–478. - PubMed
    1. Aird W.C. Phenotypic heterogeneity of the endothelium I. Structure, function, and mechanisms. Circ. Res. 2007;100:158–173. - PubMed
    1. Aird W.C. Phenotypic heterogeneity of the endothelium II. Representative vascular beds. Circ. Res. 2007;100:174–190. - PubMed
    1. Aird W.C., Edelberg J.M., WeilerGuettler H., Simmons W.W., Smith T.W., Rosenberg R.D. Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment. J. Cell Biol. 1997;138:1117–1124. - PMC - PubMed