Mechanisms of endothelial cell coverage by pericytes: computational modelling of cell wrapping and in vitro experiments

Abstract

Pericytes (PCs) wrap around endothelial cells (ECs) and perform diverse functions in physiological and pathological processes. Although molecular interactions between ECs and PCs have been extensively studied, the morphological processes at the cellular level and their underlying mechanisms have remained elusive. In this study, using a simple cellular Potts model, we explored the mechanisms for EC wrapping by PCs. Based on the observed in vitro cell wrapping in three-dimensional PC-EC coculture, the model identified four putative contributing factors: preferential adhesion of PCs to the extracellular matrix (ECM), strong cell-cell adhesion, PC surface softness and larger PC size. While cell-cell adhesion can contribute to the prevention of cell segregation and the degree of cell wrapping, it cannot determine the orientation of cell wrapping alone. While atomic force microscopy revealed that PCs have a larger Young's modulus than ECs, the experimental analyses supported preferential ECM adhesion and size asymmetry. We also formulated the corresponding energy minimization problem and numerically solved this problem for specific cases. These results give biological insights into the role of PC-ECM adhesion in PC coverage. The modelling framework presented here should also be applicable to other cell wrapping phenomena observed in vivo.

Keywords: cell wrapping; cellular Potts model; pericyte; vascular morphogenesis.

Figure 1.

Structure and molecular interactions of PCs and ECs. (a) Schematic of the circumferential cross section of a PC-covered blood vessel. BM, basement membrane. (b,c) Confocal images of mouse retina at postnatal day 5. Immunohistochemistry against PDGFRβ (PC, green), CD31 (EC, magenta) and type IV collagen (BM, white). Isolectin B4 (EC, magenta) and Hoechst 33342 (nuclei, blue). The white line ((c), left) corresponds to the orthogonal cross sections ((c), right). Arrows and *, PC; #, EC. Scale bars, 10 μm (b), 5 μm (c). (d) Schematic of major molecular interactions between PC and EC. (Online version in colour.)

Figure 2.

Schematic of the CPM. (a) Schematic lattice image. Colours represent cell types σ. σ = 1 in the green cell region and σ = 2 in the red cell region. The ECM region (σ = 0) is shown in black. ΔH, the energy change upon the transition from σ(a) to σ(b). (b) The relationship between ΔH and the acceptance probability p. (c) Definitions of the energy terms and the model parameters. λ_V and λ_S represent volume and surface elasticity of the cells. V* and S* represent target volume and surface area of the cells, respectively. J represents the adhesivity between cell types. (Online version in colour.)

Figure 3.

PCs can wrap around ECs in vitro in 2D and 3D. (a) Time lapse images of 2D monolayer coculture of PCs and ECs. Green, CellTracker Green (PC); red, Rhodamine-UEA-I lectin (EC). Scale bars, 50 μm. (b) Confocal images of 3D coculture in type I collagen. Green, CellTracker Green (PC); red, CellTracker Red (EC); blue, Hoechst 33342. Scale bars, 5 μm. x–y and x–z represent single z slice images and reconstructed orthogonal cross-sectional images along white lines in x–y, respectively. (Online version in colour.)

Figure 4.

An example simulation reproducing PC coverage. (a) Snapshots from the successful simulation reproducing PC coverage. MCS, Monte Carlo step. (b) Time course of the coverage index in the simulation (a). (c) The definition of the coverage index. (Online version in colour.)

Figure 5.

The combination of PC surface softness and preferential ECM adhesion of PCs reproduced PC wrapping in CPM. (a) A heatmap of the coverage index obtained by changing the PC surface elastic coefficient (λ_S,PC) and the PC–ECM contact energy parameter (J(PC, Medium)). (b) Representative simulation results corresponding to i–vi in (a). (Online version in colour.)

Figure 6.

The combination of PC surface softness and strong PC–EC adhesion reproduced PC wrapping in CPM. (a) A heatmap of the coverage index obtained by changing the PC surface elasticity (λ_S,PC) and the PC–EC contact energy parameter (J(PC, EC)). (b) Representative simulation results corresponding to i–ix in (a). (Online version in colour.)

Figure 7.

The effects of PC and EC surface softness on PC wrapping in two different adhesive conditions in CPM. Heatmaps of the coverage index and the representative simulation results obtained by changing PC and EC surface elastic coefficient (λ_S,PC and λ_S,EC). (a) Preferential ECM adhesion of PC condition, corresponding to figure 5, i and ii. (b) Strong PC–EC adhesion condition, corresponding to figure 6, i–iii. (Online version in colour.)

Figure 8.

PCs have stiffer surface properties than ECs in vitro. (a) Young’s moduli of PCs and ECs measured by AFM. Median values: 3.4 kPa (PC, 461 points from 30 cells), 2.2 kPa (EC, 201 points from 13 cells). p = 5.2 × 10⁻⁶ (Mann–Whitney U test). (b) Actin cytoskeleton distribution in 2D cultured PCs and ECs. Green, phalloidin; blue, Hoechst 33342. Scale bars, 10 μm. (c) Quantification of actin cytoskeleton intensity in supranuclear regions. p = 8.3 × 10⁻³ (Mann–Whitney U test, 34 (EC) and 50 (PC) microscopic fields). (Online version in colour.)

Figure 9.

The relative effects of cell–cell and cell–ECM adhesion under the experimentally measured elasticity ratio in CPM. (a) A heatmap of the coverage Index obtained by changing the PC–EC contact energy parameter (J(PC, EC)) and the PC–ECM contact energy parameter (J(PC, Medium)). (b) Representative simulation results corresponding to * in (a). The vertical and horizontal boxes correspond to each other in (a) and (b). (Online version in colour.)

Figure 10.

Experimental and bioinformatic analyses of PC–ECM interaction. (a) Adhesion assay. Adhered cells in ECM-coated wells were stained by Crystal Violet. The absorbance of eluted Crystal Violet is shown. Data are presented as mean±s.e.m. *p < 0.05; n.s., not significant (Student’s t-test). (b) Expression profiles of genes of the integrin gene family between EC and PC in [31]. Significantly differentially expressed genes are shown in blue. (c) Comparison of mRNA expression level between PC and EC by RT-qPCR. Data are shown as mean and 95% confidence interval based on Ct value. (d,e) Effect of integrin β₁ inhibition on PC–EC spheroid morphology. (d) Representative confocal images of spheroids without ECM (n = 4), and without (n = 10) or with (n = 13) inhibition under the presence of ECM. Green, GFP-PCs; red, RFP-ECs; blue, DAPI. Scale bars, 50 μm. (e) Quantification of the proportion of spheroid surface occupied by ECs to the total. Data, mean ± s.d. *p < 0.001; n.s., not significant (post hoc Tukey test after one-way ANOVA (p = 2.3 × 10⁻⁹)). (Online version in colour.)

Figure 11.

PCs were larger than ECs in vitro. Top: Representative confocal images. Green, CellTracker Green; blue, Hoechst 33342. Scale bars, 10 μm. Bottom: Quantification of cell area (a) and volume (b). (a) 2D monolayer culture. Median values, 1943 μm² (PC), 1508 μm² (EC). p = 0.022 (Mann–Whitney U test, 163 (PC) and 79 (EC) cells). (b) 3D culture in Matrigel. Median values, 4952 μm³ (PC), 1987 μm³ (EC). p = 1.4 × 10⁻³⁵ (Mann–Whitney U test, 461 (PC) and 201 (EC) cells). (Online version in colour.)

Figure 12.

The size asymmetry between the cell types can contribute to the wrapping in CPM. Heatmaps of the coverage index and representative simulation results obtained by changing PC size and adhesion parameters. (a) Strong PC–EC adhesion condition, corresponding to the horizontal boxes in figure 9. (b) Preferential ECM adhesion of PCs, corresponding to the vertical boxes in figure 9. (Online version in colour.)

Figure 13.

Comparison between CPM and the mechanical model. (a) The definitions of the cell domains D₁ and D₂, the cell volumes (areas) V₁ and V₂, and the surface areas (perimeters) S₀, S₁ and S₂. (b) A heatmap of the coverage index for the mechanical model using the perimeter scaling parameter ξ. J_CPM is shown. The contact energy parameters for PC–EC (J(PC, EC)) and PC–ECM (J(PC, Medium)) were changed the same as in figure 9a. (c) The correlation between the coverage index for CPM (figure 9a) and the mechanical model (figure 13b). Pearson’s r = 0.95. (Online version in colour.)