Schwann cells promote endothelial cell migration

Abstract

Directed cell migration is a crucial orchestrated process in embryonic development, wound healing, and immune response. The underlying substrate can provide physical and/or chemical cues that promote directed cell migration. Here, using electrospinning we developed substrates of aligned poly(lactic-co-glycolic acid) nanofibres to study the influence of glial cells on endothelial cells (ECs) in a 3-dimensional (3D) co-culture model. ECs build blood vessels and regulate their plasticity in coordination with neurons. Likewise, neurons construct nerves and regulate their circuits in coordination with ECs. In our model, the neuro-vascular cross-talk was assessed using a direct co-culture model of human umbilical vein endothelial cells (HUVECs) and rat Schwann cells (rSCs). The effect of rSCs on ECs behavior was demonstrated by earlier and higher velocity values and genetic expression profiles different of those of HUVECs when seeded alone. We observed 2 different gene expression trends in the co-culture models: (i) a later gene expression of angiogenic factors, such as interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), and (ii) an higher gene expression of genes involved in actin filaments rearrangement, such as focal adhesion kinase (FAK), Mitogen-activated protein kinase-activated protein kinase 13 (MAPKAPK13), Vinculin (VCL), and Profilin (PROF). These results suggested that the higher ECs migration is mainly due to proteins involved in the actin filaments rearrangement and in the directed cell migration rather than the effect of angiogenic factors. This co-culture model provides an approach to enlighten the neurovascular interactions, with particular focus on endothelial cell migration.

Keywords: cell migration; human umbilical vein endothelial cells; neurovascular; poly(lactic-co-glycolic acid); rat Schwann cells.

Figure 1.

Imaging platform. After staining with a green fluorescent cell tracker and imaging, the original images are cropped into a region of interest (A) and converted into greyscale images (B). The images are then filtered using a Gaussian filter (C) and a 3-class threshold applied to distinguish the foreground from the background (D). The tissue assumed as foreground is measured in terms of its area and compared against the total area of the cropped image (E). The resulting images are then outlined (white line) and overlaid with the cropped image (F). Scale bar: 1 mm; x and y axxis in pixels

Figure 2.

Scaffold characterization and cell morphology. (A) SEM micrographs of PLGA 50:50 ESP scaffolds showed a good fiber alignment (scale bar 1 µm). (B) Box diagram of fiber diameter (Whiskers Tukey, mean ± SD, n=12). (C) Cell circularity box diagram, cells seeded on fibrous substrates showed a more elongated morphology (1 is a perfect circle, while approaching 0 the cell become more elongated; unpaired t test ** P<0.05, mean ± SEM, n≥89 ). (D and E) Rose plot diagramsof HUVECs directionality response after one day in culture in fibers and coverslips, respectively (Rose plots are in degrees). (F and G) Immunofluorescence images show HUVECs morphology after one day in culture in ESP fibers and coverslips, respectively. The cells seeded in the fibrous substrates exhibited a more orientated alignment. (In red actin filaments are labeled using AlexaFluor 594 conjugated phalloidin, while in blue cell nuclei are labeled using DAPI, scale bar 50 µm). ESP: electrospun.

Figure 3.

Analysis of cell viability by PrestoBlue ® (A) Each single culture model was individually tested for their viability potential. rSCs have a higher viability rate when compared to HUVECs. (B) For each time-point used in the migration studies the viability potential of the HUVECs single culture model was measured. Viability is plotted against the covered area. The percentage of covered area in fibrous substrates is mainly due to cell migration rather than increased cell viability. (mean ± SD, n = 3, linear regression). ESP: electrospun.

Figure 4.

Effect of rSCs on HUVECs migration. Co-culture models with low ratios of rSCs have a positive effect on HUVECs migration, with increased percentage of covered area over time and higher values of cell migration speed. (A) Wound closure over time. The 5% ratio is the one that showed a higher covered area over time, with higher displacement of the cell sheet (One-way ANOVA, mean ± SD, n ≥ 2 # p < 0.05 (5% versus 10 %)). (B) Velocity profile of HUVECs using several rSCs:HUVECs ratios (One-way ANOVA, mean ± SD, n≥2 , second order polynomial (qudratic), #. p < 0.001 (5% vs. 20 %), # p < 0.05 (0% versus 20 % and 0% vs. 20 %), # p < 0.05 (10% versus 20 %)). (C) Results of the modified scratch wound healing assay; each color represent a different tissue that software will assume and measure as a cell sheet or single cell. The dashed gray vertical line represents the width of the initial gap. Scale bar 1 mm.

Figure 5.

Influence of rSCs on angiogenic factors' gene expression expressed by HUVECs. The later up-regulation of IL-8 and VEGF in co-culture models seems to be insufficient to decrease HUVECs migration, being masked by significant higher expression of other genes involved in the actin filament rearrangement. PLGA 50:50 ESP fibers used as substrate. (Fold increase was calculated using ΔΔCt method, mean ± SD, n = 3, One-way ANOVA Tukey's multiple comparison Test, *p < 0.05, **. p < 0.01, *** p < 0.001, comparisons between single culture model vs. co-culture model).

Figure 6.

Influence of rSCs on actin turnover proteins' gene expression expressed by HUVECs. In both graphics is exhibited a constant increase in gene expression in the co-culture model, peaking at 36 hours, suggesting a higher actin filaments rearrangement in those culture models. The setup used was the same as previously stated (Fold increase calculated using ΔΔCt method, mean ± SD, n = 3, One-way ANOVA Tukey's multiple comparison Test, *p < 0.05, **. p < 0.01, *** p < 0.001, comparisons between single culture model versus co-culture model).

Figure 7.

Influence of rSCs on protein kinases' gene expression expressed by HUVECs. The constant upregulation of these protein kinases suggested an increased substrate phosphorylation and an enhanced directed cell migration in co-culture models. The setup used was the same as previously stated. (Fold increase calculated using ΔΔCt method, mean ± SD, n = 3, One-way ANOVA Tukey's multiple comparison Test, *p < 0.05, **. p < 0.01, *** p < 0.001, comparisons between single culture model vs. co-culture model). Note: non-significant differences were observed for FAK between 48 hours versus 2 hours.