ErbB2 promotes endothelial phenotype of human left ventricular epicardial highly proliferative cells (eHiPC)

Abstract

The adult human heart contains a subpopulation of highly proliferative cells. The role of ErbB receptors in these cells has not been studied. From human left ventricular (LV) epicardial biopsies, we isolated highly proliferative cells (eHiPC) to characterize the cell surface expression and function of ErbB receptors in the regulation of cell proliferation and phenotype. We found that human LV eHiPC express all four ErbB receptor subtypes. However, the expression of ErbB receptors varied widely among eHiPC isolated from different subjects. eHiPC with higher cell surface expression of ErbB2 reproduced the phenotype of endothelial cells and were characterized by endothelial cell-like functional properties. We also found that EGF/ErbB1 induces VEGFR2 expression, while ligands for both ErbB1 and ErbB3/4 induce expression of Tie2. The number of CD31posCD45neg endothelial cells is higher in LV biopsies from subjects with high ErbB2 (ErbB2high) eHiPC compared to low ErbB2 (ErbB2low) eHiPC. These findings have important implications for potential strategies to increase the efficacy of cell-based revascularization of the injured heart, through promotion of an endothelial phenotype in cardiac highly proliferative cells.

Keywords: Cell biology; Cell differentiation; Myocardium; Neuregulin.

Copyright © 2018 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Isolation and expansion of human epicardial eHiPC

Single cell suspensions prepared from LV epicardial biopsies were plated into 48 well plates at low cell density (500 cell per cm2) and analyzed for growing colonies twice weekly. A. Number of viable cells per mg of LV biopsies. B-E. Micrographs of highly-proliferative colony-forming cells (B, C) and cells characterized by non-colony expansion (D, E). F. Comparison of proliferative potentials of colony-forming and non-colony forming cardiac cells. Cumulative number of population doublings was calculated by repeated cell number recordings in triplicate cultures at ~90% confluence and plating at a fixed number of 104 cells per cm2 in 6 well plates. G. Average number of eHiPC colonies isolated from individual LV biopsies. H. Representative flow cytometric histograms showing cell surface expression of ErbB1-4 receptors; open histograms represent specific ErbB1-4 staining, shaded represent the isotype-matched IgG control. I. Graphical representation of ErbB1-4 receptor expression on HiPC; Data are expressed as ΔMFI and presented as scatter dot plots. ΔMFI was calculated by subtraction of the MFI corresponding to cells stained with isotype-matched control antibody from the MFI of the specific ErbB antibody. The median values (indicated by the horizontal lines) and individual values are shown. J. Expression of ErbB receptors was measured at passage 1 and passage 10 in five eHiPC clones as described in the text. Significance was calculated using a paired t test. P values are indicated. K. Effects of recombinant ErbB ligands, 100 nM epidermal growth factor (EGF), 100 nM of neuregulin-1 (NRG) and 100 nM glial growth factor 2 (GGF2) on the proliferation of eHiPC. L. Effects of ErbB antagonists, 100 nM AST1306 (AST, a pan-ErbB inhibitor), 300 nM AG1478 (AG, ErbB1 inhibitor) and 300 nM TAK-165 (TAK, ErbB2 inhibitor) on the EGF-induced proliferation of HiPC; n=15, One-way ANOVA, p-values for the Bonferroni post hoc test are indicated.

Figure 2. Analysis of cell surface marker expression on eHiPC

A. Representative flow cytometric histograms demonstrating expression of mesenchymal stem cells and hematopoietic cell markers; open histograms represent antigen-specific IgGs and shaded ones represent isotype-matched IgGs. B. Graphical representation of flow cytometric data; horizontal lines indicate median values. C. Correlations between expression of ErbB1-4 receptors and CD31 (PECAM-1) on eHiPC; Pearson correlation coefficient (rp), Spearman correlation coefficient (rs), and p-values are shown. D. Cell surface expression of endothelial cell markers in groups of ErbB2high and ErbB2low eHiPC.

Figure 3. Assessment of endothelial cell-like properties of ErbB2high and ErbB2low eHiPC

In vitro angiogenic properties were examined using growth factor reduced Matrigel (A-B) and after stimulation of eHiPC with collagen I (C-G). Barrier function was measured by paracellular permeability of fluorescently labeled 4kDa and 70kDa dextrans (H-L). Representative microscopic fields (A) and graphical representation (B) of data showing Matrigel-based tube formation by ErbB2high (ErbB2hi, upper panel) and ErbB2low (lower panel) eHiPC, unpaired t test. C,D. Representative microscopic fields demonstrating cell retraction and reorganization of the monolayer of ErbB2high eHiPC into cord-like structures at 12 hours after addition of 500 μg/ml collagen I. E, F. In contrast, ErbB2low eHiPC did not undergo morphogenesis. Bar = 200 μm. G. Graphical representation of total empty area after collagen I–induced morphogenic activity within cultures shown in C-F. Graphs are presented as scatter dot plots and the horizontal lines indicate the median values for each group. Each dot represents averaged total empty area from four fields per well (two wells per colony) normalized to mm2. Significance level (p-value) calculated by Mann Whitney test. H. Calcein AM and 7-AAD were used to determine number of dead cells (7-AAD positive/Calcein AM negative); the upper left panel represents viable cells, the lower right panel represents dead cells (0.5% Tween 20). I. Number of dead cells under basal (Bas) and collagen I (Col)-induced conditions; ns is not significant, Mann Whitney test. J-L. Diffusive paracellular permeability of 4 kDa (J) and 70 kDa (K) dextrans. Permeability was measured at a 3 hour time point in triplicate per individual eHiPC colony and averaged. Empty filter indicates no cells. Horizontal lines demonstrate mean values. One-way ANOVA, p-values determined by Bonferroni’s posttest. L. Paracellular diffusion of 4 kDa and 70 kDa dextrans through eHiPC monolayers was normalized to diffusion across empty filters, unpaired t test.

Figure 4. ErbB ligands, EGF, NRG-1 and GGF2 upregulate cell surface expression of endothelial cell markers

Progenitors were serum-starved for 24 hrs and then incubated in the absence (Basal) or presence of 100 ng/ml of EGF, 100 ng/ml of NRG-1 (NRG) or 100 ng/ml of GGF2 for 48 hrs. A. Representative flow cytometric histograms showing the effect of EGF (red) on cell surface expression of CD31. B,C. Graphical representation of the effects of ErbB ligands on CD31 expression in ErbBhigh (n=8) (B) and ErbB2low (n=7) (C) eHiPC, paired t test. D. Effect of ErbB receptor antagonists on CD31 expression in five colonies with high response to EGF, one-way ANOVA, p-values for the Bonferroni post hoc test are indicated. E, F. Effect of ErbB agonists on cell surface expression of VEGFR2 in ErbBhigh (E) and ErbBlow (F) eHiPC; paired t test. G. Effect of ErbB receptor antagonists on VEGFR2 cell surface expression; one-way ANOVA, p-values are indicated (Bonferroni’s multiple comparison test). H,I. Effect of ErbB agonists on cell surface expression of Tie2 in ErbBhigh (H) and ErbBlow (I) eHiPC; paired t test. J-L. Effect of ErbB receptor antagonists on Tie2 cell surface expression in cells stimulated with EGF (J), NRG-1 (K) or GGF2 (L); one-way ANOVA, p-values are indicated (Bonferroni’s multiple comparison test).

Figure 5. Endothelial cell number is higher in LV epicardial biopsies obtained from subjects with ErbB2high eHiPC compared to subjects with ErbB2low eHiPC

A. Representative flow cytometric dot plots demonstrating the percentage of endothelial cells in cell suspensions prepared from LV epicardial biopsies obtained from subjects with ErbB2high eHiPC (upper panel) and ErbBlow eHiPC (lower panel). Microbeads were used to gate events with size higher than 6 μm (SSC-A/FSC-A, upper panel, left plot). Dead cells and cell debris were excluded using amine-reactive dye (VioDye, LIVE/DEAD® Fixable Dead Cell kit) and cell aggregates were excluded using FSC-H/FSC-A. Endothelial cells (blue gate, right plot) were defined as CD31posCD45neg cells. B. Number of viable cells obtained from LV epicardial biopsies of subjects with ErbB2high and ErbB2low eHiPC; unpaired t test. C. Number of EC in the epicardium of subjects with high and low expression of ErbB2 on eHiPC; number of EC was calculated using percent of CD31posCD45neg cells and total number of cells; unpaired t test.