Brain pericytes in culture display diverse morphological and functional phenotypes

Abstract

Pericytes play several important functions in the neurovascular unit including contractile control of capillaries, maintenance of the BBB, regulation of angiogenesis, and neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with the pericyte subtypes in vivo, numerous recent publications have used a primary human brain vascular pericytes (HBVP) cell line where this pericyte heterogeneity has not been considered. Here, we used primary HBVP cultures, high-definition imaging, cell motility tracking, and immunocytochemistry to characterise morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exists in cultures. We identified five distinct morphological subtypes that were defined using both qualitative criteria and quantitative shape analysis. The proportion of each subtype present within the culture changed as passage number increased, but pericytes did not change morphological subtype over short time periods. The rate and extent of cellular and membrane motility differed across the subtypes. Immunocytochemistry revealed differential expression of alpha-smooth muscle actin (αSMA) across subtypes. αSMA is essential for cell contractility, and consequently, only subtypes with high αSMA expression contracted in response to physiological vasoconstrictors endothelin-1 (ET1) and noradrenaline (NA). We conclude that there are distinct morphological subtypes in HBVP culture, which display different behaviours. This has significance for the use of HBVP when modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered.

Keywords: Alpha-smooth muscle actin; Cell culture; Contractility; Morphology; Pericytes.

Fig. 1

In vitro pericyte morphological heterogeneity changes with passage number. A Representative DIC images of five different morphologies observed in three different HBVP cultures: (i) standard, (ii) circular, (iii) sheet, (iv) spindle, and (v) balling morphologies. Images from cultures between P6-P8. Scale = 10 μm. B Percentage of each morphology within passage 6 cultures was calculated. N = 4 cultures and 360–400 cells analysed per culture (1528 total cells analysed). Data points are presented as individual cultures overlayed with mean ± SD. C Percentage of each morphology within cultures was calculated from passages 3–12. N = 4 cultures and 583–4625 cells analysed per passage number (20,173 total cells analysed). Residuals were normally distributed, and data for each individual subtype were compared using one-way ANOVA with Dunnett’s multiple comparisons test (comparing back to P3). Error bars are not presented on this figure for clarity

Fig. 2

Morphological subtypes of cultured pericytes possess different shape parameters. Tracing around each cell provided four different shape parameters that were quantified across different pericyte morphologies. Analysis from N = 1219 cells in images of cultures between P6 and P8. (A) Cell area was the area (µm²) within the traced region of each cell. (B) Cell perimeter was calculated as the length (µm) of the trace around each cell. (C) Feret’s diameter was determined by the longest distance between any two points along the selection boundary divided by the cell perimeter, an indicator of elongation. (D) Circularity Index was defined by 4π × [Area]/[Perimeter]² with a value of 1.0 indicating a perfect circle while a value approaching 0.0 indicating an increasingly elongated shape. Data points are presented as individual cells overlayed with mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparisons test or Ordinary One-Way ANOVA with Dunnett’s multiple comparisons test was used to compare groups relative to standard morphology. *** p < 0.001, **** p < 0.0001. (E) Radar chart describing the relative correlation of each shape descriptor to each morphological subtype. Numbers (1–5) represent very low, low, moderate, high, and very high correlation, respectively

Fig. 3

Pericyte morphology remains consistent but pericytes are motile over 2 h. (A) Representative DIC images showing morphological subtypes at T = 0 and T = 20 min. Images from cultures between P6 and P8. Scale = 10 μm. (B–E) The change in cell area, perimeter, feret, and circularity of each pericyte subtype was calculated over 20 min. Data points are presented as individual cells overlayed with mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparison test (B, C, and E) or Ordinary One-Way ANOVA with Dunnett’s multiple comparisons test (D) was used to compare groups. 96 cells were analysed in total. (F) Representative DIC images showing pericytes at T = 0 Hr (i) and T = 2 Hr (ii). Scale = 20 μm. White tracks indicate movement of individual pericytes at 20 min intervals over a 2 h period. Images from cultures between P6 and P8. (G) Percentage of four pericyte morphologies (standard, circular, sheet, and spindle) at baseline (T = 0 h) and 2 h (T = 2 h) calculated from DIC images of P6–P8 cultures. N = 256 cells analysed

Fig. 4

Pericyte cell velocity and process velocity differs across morphological subtype. (A) Representative DIC images showing morphological subtypes at T = 0 and T = 20 min. Tracks indicate 1 min interval movement of the cell nucleus (pink, 1) and most active membrane projection (cyan, 2). Images from cultures between P6 and P8. (B) Mean cell velocity as calculated by the distance of the pink tracks over time in different pericyte morphological subtypes. (C) Process velocity in different pericyte morphological subtypes was assessed in the most active process determined by the highest velocity process per cell out of the two primary processes measured as indicated by the cyan tracks. 106 cells were analysed in total. Data points are presented as individual cells overlayed with mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare groups. **** p < 0.0001, *** p < 0.001 compared to Standard

Fig. 5

Standard morphology pericytes contract in response to NA and ET1. (A) Representative panel of DIC images of standard (i), circular (ii), sheet (iii), and spindle (iv) pericytes at T = 0 and T = 20 min treated with NA. The white dotted outline indicates tracing of the cell area. Images from cultures between P6 and P8. (B–C) Relative change in cell area compared to baseline area following exposure to NA (B) and ET1 (C). Control indicates treatment with a DMSO vehicle. 233 cells were analysed in total. Data points are presented as individual cells overlayed with mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare groups. **** p < 0.0001, *** p < 0.001, ** p < 0.001, * p < 0.05 compared to Standard in either control or treatment group

Fig. 6

αSMA expression in pericytes varies across morphological subtype. (A) Representative confocal microscopy images of pericyte subtypes with αSMA-positive (green) and DAPI-positive nuclei (blue). White dotted outlines represent cell membrane boundaries. Images from P6 cultures. (B) Quantification of corrected total cell fluorescence (CTCF) of αSMA expression between pericyte subtypes. 1219 cells were analysed in total. Data points are presented as individual cells overlayed with mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare groups. **** p < 0.0001, * p < 0.05 compared to standard