Differentiation of Murine Bone Marrow-Derived Smooth Muscle Progenitor Cells Is Regulated by PDGF-BB and Collagen

Abstract

Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor-BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.

Fig 1. Bone marrow cells express smooth muscle cell markers upon short-term culture.

qRT-PCR analysis of SMC marker expression in cultured bone marrow cells and murine aortic smooth muscle cells (SMCs) after the indicated culture period. Expression of α-SMA and SM22-α (A) and SM-MHC (B) was normalized relative to GAPDH (* p < 0.05, ** p < 0.01 compared to 0 d, Dunnett’s test, n = 3). The number of α-SMA⁺ cells was quantified for the indicated time (C). Western blot detection of α-SMA (D) and SM22-α (E) expression of mouse bone marrow cells cultured for the indicated period.

Fig 2. Bone marrow cells express smooth muscle cell markers at different time points.

Immunofluorescence microscopy measurements of α-SMA+, SM22-α+, and SMMHC+ bone marrow cells in culture on days 1, 4, and 14. 20 X magnification, scale bar 20 μm.

Fig 3. Cells committed to SMPC lineage in early bone marrow cultures are marked by a high proliferative index.

BMCs were pulse-labelled with BrdU for a 24 h period starting at days 2, 5, and 7 and subsequently immuno-stained for BrdU and α-SMA at day 10, a subgroup was labelled with BrdU for 2 hours immediately prior to staining, percentages of α-SMA⁺ cells during culture periods are indicated (A). At day 10 cells were stained for BrdU and α-SMA, the percentage of α-SMA⁺ and α-SMA^- cells labelled with BrdU during the indicated time points are plotted (B and C) (*p < 0.05, Bonferroni multiple comparisons test, n = 3).

Fig 4. Effects of PDGF-BB on smooth muscle cell marker expression.

qRT-PCR analysis of α-SMA and SM22-α expression in BMCs cultured for 10 d in the specified media (A) († p < 0.001, Tukey-Kramer multiple comparisons test, n = 3). All BMCs were cultured in 10% α-MEM with additional PDGF-BB (50 ng/mL) for 8 d before replacing the media as indicated for 2 d. qRT-PCR analysis of α-SMA and SM22-α expression at 10 d, normalized against GAPDH (B). (* p < 0.05, ** p < 0.01, Bonferroni multiple comparisons test, n = 3). At 10 d, cells were immunolabelled for α-SMA and BrdU (** p < 0.01, Bonferroni multiple comparisons test, n = 3) (C). SMPCs were re-plated at day 10 in 10% α-MEM ± supplemental PDGF-BB (50 ng/mL) for 1 and 5 days, followed by immunofluorescence labelling of α-SMA (D) († p < 0.001, Bonferroni multiple comparisons test, n = 3). SMPC culture media was replaced with: control (no media change), media plus PDGF-BB (50 ng/mL), media plus PDGF-BB (50 ng/mL) and AG9 (10 μM), media plus PDGF-BB (50 ng/mL) and AGL2043 (10 μM), as indicated. Expression of α-SMA was detected by immunofluorescence microscopy after 5 d incubation (E) († p <0.001, Tukey-Kramer multiple comparisons test, n = 3).

Fig 5. Effects of collagen polymerization on SMPC proliferation and viability.

Representative SEM images depicting the topology of surfaces coated with fibrillar (A) or non-fibrillar collagen preparations (B, scale bars are 10 μm). BMCs cultured for 10 d in 10% α-MEM, and then re-plated onto substrates as indicated, at 24, 48 and 72 h, cell density (C) and BrdU incorporation (D) were measured. Indices of Apoptosis were assessed in SMPCs (E) and primary cultures of murine SMCs (F) by Annexin V/PI co-staining. (* p < 0.05, ** p < 0.01, (C, D, n = 4), (E, F, n = 3), Tukey-Kramer multiple comparisons test).

Fig 6. SMPC differentiation and focal adhesion formation are suppressed on monomeric collagen.

Bone marrow cells were cultured for 10 d in 10% α-MEM media on TCPS to allow expression of SMC markers and re-plated onto fibrillar or non-fibrillar collagen for 72 h. (A, E) TEM images of transverse sections through SMPCs on collagen matrices. Magnification, (A) 10,000 X; (E) 15,000 X. Scale bars 2 μm. (B, F) Representative images for SMA expression at 72 h. (C, G) Phalloidin-Alexa 546 labelling of SMPC F-actin. Magnification, 20 X. Scale bars, 100 μm. (D, H) Co-labelling with phalloidin and mouse anti-vinculin antibody. 60 X magnification, scale bars 20 μm.

Fig 7. Effects of monomeric collagen on SMPC differentiation and cell shape.

qRT-PCR analysis of expression of α-SMA and SM22-α mRNA in BMCs cultured for 10 d on the indicated substrates (A) (* p < 0.01, Bonferroni multiple comparisons test, n = 3). Bone marrow cells were cultured for 10 d in 10% α-MEM media on TCPS followed by re-plating on indicated collagen surfaces for 24 to 72 h. Quantification of α-SMA⁺ cells at indicated period via immunofluorescence microscopy using mouse anti-αSMA antibody and DAPI (B).(* p < 0.05, † p < 0.001, Bonferroni multiple comparisons test, n = 3). Morphometric analysis of cell area (C) and roundness (D) after culture for 24 and 72 h on collagen was achieved. #: roundness is proportional to the ratio of the perimeter squared over the radius squared of the cells, with increasing values representing further deviation from circular.