Side-by-Side Comparison of Culture Media Uncovers Phenotypic and Functional Differences in Primary Mouse Aortic Mural Cells

Abstract

(1) Background: Vascular mural cells reside in the media and outer layers of the vessel wall. Their ability to proliferate and migrate or to change phenotype in response to external cues is a central feature of the vascular response to injury. Genetically engineered mice are used for loss- or gain-of-function analyses or lineage tracing in vivo, their primary cells for mechanistic studies in vitro. Whether and how cultivation conditions affect their phenotype and function is often overlooked. (2) Methods: Here, we systematically studied how the cultivation of primary mural cells isolated from the aorta of adult wild-type mice in either basal medium (DMEM) or special media formulated for the cultivation of fibroblasts or pericytes affects their phenotype and function. (3) Results: Medium composition did not alter cell viability, but the mRNA levels of differentiated smooth muscle cell markers were highest in vascular mural cells expanded in DMEM. Conversely, significantly higher numbers of proliferating and migrating cells were observed in cells expanded in Pericyte medium, and cytoskeletal rearrangements supported increased migratory capacities. Significantly reduced telomere lengths and metabolic reprogramming was observed in aortic mural cells cultured in Fibroblast medium. (4) Conclusions: Our findings underline the plasticity of primary aortic mural cells and highlight the importance of the culture media composition during their expansion, which could be exploited to interrogate their responsiveness to external stimuli or conditions observed in vivo or in patients.

Keywords: fibroblasts; metabolism; migration; pericytes; phenotypic plasticity; proliferation; smooth muscle cells; vascular remodeling.

Figure 1

Experimental workflow and assessment of cell viability. (A), Schematic drawing of the experimental workflow. Of note, aortic mural cells at passage 0 and 1 (for MTS assay, Seahorse metabolic measurements) were examined in parallel in all experiments. (B), Representative brightfield microscopic pictures of mural aortic cells cultivated in different culture media for 4, 5, or 6 days. Scale bars, 10 µm. (C), Results of the MTS assay to assess cell viability. Data shown represent the optical density (OD) at 490 nm. (D–F), the number of total (D), viable (E) and dead (F) cells per mL was automatically determined using the Tecan Spark multimode reader. Data shown represent five (C) or six (D–F) biological replicates per condition. Shapiro–Wilk test was used to test for normal distribution. Statistical significance was assumed if p reached a value < 0.05. ns, non-significant (One-way ANOVA, Sidak’s multiple comparisons test (C,E,F), Kruskal–Wallis, Dunn’s multiple comparisons test) (D).

Figure 2

Analysis of cell proliferation. (A,B), Flow cytometry analysis of Ki-67 was performed to quantify proliferating cells, the gating strategy to detect singlet cells and viable cells as well as representative histogram plots are shown in (A), the summary of findings in seven biological replicates in (B). (C,D), Immunostaining of Ki-67 was performed to visualize proliferating cells. Representative pictures are shown in (C), the quantitative analysis in (D). Data shown represent the mean of eight images (at 60× magnification) collected per sample (five biological replicates), per condition, and are expressed as percentage of total cells. Scale bars, 50 µm. (E,F), Quantitative real-time PCR analysis of proliferation marker genes. The results after qPCR analysis of Ccnd1 (E) and Pcna (F) mRNA transcript levels are shown. Data shown represent 10 biological replicates. (G,H), Immunoblot analysis of PCNA protein expression in aortic mural cells cultivated in DMEM, Fibroblast medium or Pericyte medium for 6 days. Representative findings are shown in (G), the results of the quantitative analysis for PCNA in (H). Note that the representative images for ACTN1 shown in panel (G) are from the same membrane and also shown in Figure 4, panel (H) (the uncropped membranes are provided in the supplement). * p < 0.05, ** p < 0.01, and *** p < 0.001 (One-way ANOVA, Sidak’s multiple comparisons test (B,D–F), Kruskal–Wallis, Dunn’s multiple comparisons in (H)). ns, non-significant.

Figure 3

Detection of biologically aged, apoptotic, and senescent cells. (A,B), Fluorescence in situ hybridization (FISH) was performed to detect the telomere repeat sequence. Representative images are shown in (A), the results of the quantitative analysis of the telomere fluorescent intensity in (B). Data shown represent two biological replicates, with 220 cell nuclei examined (at 60× magnification) per sample, per condition. Scale bars, 50 µm. (C,D), Detection of apoptosis-related DNA fragmentation using the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). Representative fluorescence microscopy images (C) and the results of the quantitative analysis (D). Scale bars, 20 µm. Data shown represent six biological replicates, with five images (at 20× magnification) collected per sample, per condition, and are expressed as percentage of total cells. (E,F), Senescence-associated beta-galactosidase (SA-β-gal) staining was used to assess the presence of senescent cells (arrows). Representative images of two different samples and magnifications are shown in (E), the results of the quantitative analysis in (F). Scale bars, 10 µm (top row) and 20 µm (bottom row). Data shown represent seven biological replicates per condition, with one image (at 10× magnification) collected per sample, per condition. **** p < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparisons test in (B,D), One-way ANOVA, Sidak’s multiple comparisons test in (F)). ns, non-significant.

Figure 4

Expression of vascular mural cell lineage markers. (A–C), Quantitative real-time PCR analysis was used to determine the mRNA transcript levels of the smooth muscle cell marker Acta2 (A), the fibroblast marker Pdgfra (B) and the pericyte marker Cspg4 (C). Data shown represent ten biological replicates. (D–F), Immunofluorescence staining of SMA (red fluorescence) and PDGFRA (green fluorescence). Representative images (D) and quantitative analysis of SMA in (E) and PDGFRA in (F). Data shown represent the integrated fluorescence density in six biological replicates, with mean of eight images (at 60× magnification) examined per sample, per condition. Scale bars, 50 µm. (G), Representative images after immunofluorescence staining of NG2 (red fluorescence). (H–J), Immunoblot analysis of vascular mural markers. A representative membrane (H) and the results of the quantitative immunoblot analysis of ACTA2 (I) and PDGFRB (J) protein expression in five biological replicates per condition are shown. Note that the representative images for ACTN1 shown in panel (H) are from the same membrane and also shown in Figure 2, panel (G) (the uncropped membranes are provided in the supplement). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 (One-way ANOVA, Kruskal–Wallis, Dunn’s multiple comparisons test in (A,I); Sidak’s multiple comparisons test in (B,C,E,F,J)). ns, non-significant.

Figure 5

FACS analysis of vascular mural markers. (A–G), Flow cytometry analyses was used to study the percentage of immunopositive cells (per total, viable cells) and the mean fluorescence intensity (MFI) of vascular mural markers in cells cultivated cells for 6 days under different media conditions. Representative histogram plots of cells stained for smooth muscle alpha actin (SMA), platelet-derived growth receptor alpha (PDGFRA), or neural/glial antigen 2 (NG2) are shown in (A). The percentage and MFI after staining for SMA is shown in (B,C), for PDGFRA in (D,E), and for NGs in (F,G). Data shown represent seven biological replicates per condition. ** p < 0.01 (One-way ANOVA, Sidak’s multiple comparisons test in (B,D–G); Kruskal–Wallis, Dunn’s multiple comparisons test in (C)). ns, non-significant.

Figure 6

mRNA expression of vascular mural differentiation markers. (A–F), Quantitative real-time PCR analysis to determine the mRNA transcript levels of transgelin (Tagln; (A)), calponin (Cnn1; (B)), collagen 1A type 1 (Col1a1; (C)), fibroblast growth factor receptor 1 (Fgfr1; (D)), S100 calcium binding protein A4 (S100a4; (E)), and Krüppel-like factor 4 (Klf4; (F)). Data shown represent findings in six (D) or eight (A–C,E,F) biological replicates. (G,H), Immunofluorescence staining of nuclear KLF4. Representative images (G) and quantitative analysis (H). Data shown represent six biological replicates, with eight images (at 60× magnification) examined per sample, per condition. Scale bars, 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 (One-way ANOVA, Sidak’s multiple comparisons test). ns, non-significant.

Figure 7

Analysis of cytoskeletal remodeling and cell migration. (A–D), Automated quantitative image analysis of cell size and shape. The results of the ImageJ based automated quantification of the single cell area (A), perimeter (B), Feret’s diameter (C) and min Feret’s diameter (D) are shown. Data shown represent three biological replicates, with six images (60× magnification) per sample, per condition. (E,F), Analysis of cytoskeletal rearrangements associated with cell motility. Representative brightfield images of unstained cells (top row) and after staining with fluorescein phalloidin (bottom) (E) and the results of the quantitative analysis (F). Data shown represent six biological replicates, with eight images (60× magnification) per sample, per condition. (G,H), Analysis of cell migration using the scratch wound assay. Representative images immediately after inducing a ‘scratch wound’ on monolayer cells (start) and 5 h later (G). Black, interrupted lines were added to indicate the cell borders. Quantification of the cell-free area at five hours relative to the area at start (H). Data shown represent seven biological replicates, with two images (10× magnification) per sample, per condition. Scale bars: 50 µm. * p < 0.05, *** p < 0.001 and **** p < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparisons test in (A–D); One-way ANOVA, Sidak’s multiple comparisons test in (F,H)). ns, non-significant.

Figure 8

Real-time assessment of cell metabolism and oxidative species generation. (A–F), Cellular bioenergetics were studied using the Seahorse Real-Time Cell Metabolic Analyzer. (A) Extracellular acidification rate (ECAR) as a measure of glycolysis. (B) Oxygen consumption rate (OCR) to determine oxidative phosphorylation, alone and in response to metabolic stress induced by oligomycin, FCCP (fluoro-carbonyl cyanide phenylhydrazone), and antimycin A/rotenone. Data shown are representative of results obtained in three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 for Fibroblast medium vs. DMEM, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 for Fibroblast medium vs. Pericyte medium (Two-way ANOVA, Tukey’s multiple comparisons test B). ns, non-significant. Results after quantification of the basal respiration (C), ATP production (D), maximal respiration (E), and non-mitochondrial respiration (F) are shown. (G,H), Dihydroethidium (DHE) immunofluorescence staining to visualize the formation of reactive oxygen species (ROS). Representative images in (G), results after quantification of the integrated intensity using ImageJ in (H). Data shown represent six biological replicates, with 6 images (at 60× magnification per sample, per condition). Scale bars, 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 One-way ANOVA, Sidak’s multiple comparisons test in (A,C–E); Kruskal–Wallis, Dunn’s multiple comparisons test in (F,H).