Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells

Abstract

Aims: Bone marrow-derived smooth muscle cells (BM-SMCs) have high potential as an autologous cell source of vascular progenitors but normal cell function and turnover frequency may decline with age. In this study we set out to study the effects of organismal ageing on the molecular and functional properties of BM-SMCs.

Methods and results: To address this issue, we employed a smooth muscle alpha-actin promoter (alphaSMA) driving expression of enhanced green fluorescence protein (EGFP) to isolate SMCs from bone marrow of neonatal (nBM-SMCs) or adult (aBM-SMCs) sheep and examined their proliferation potential and contractility. Compared with nBM-SMCs, aBM-SMCs exhibited lower clonogenicity and proliferation potential that could be improved significantly by addition of basic fibroblast growth factor. Vascular constructs from aBM-SMCs showed reduced ability to generate force and contract fibrin hydrogels and this function could be partially restored by addition of transforming growth factor-beta1. They also exhibited lower receptor- and non-receptor-mediated vascular contractility and mechanical strength, which was comparable to that of tissue constructs prepared with vascular SMCs from neonatal umbilical veins. In agreement with the contractile properties and mechanical strength of vascular constructs, aBM-SMCs displayed significantly lower expression of alphaSMA, smoothelin, desmin, type I collagen, and tropoelastin transcripts compared with nBM-SMCs.

Conclusion: Understanding the effects of organismal ageing on BM-SMCs and the properties of the resulting vascular constructs may lead to innovative ways to facilitate application of these cells in the treatment of cardiovascular disease which is especially prevalent in the elderly.

Figure 1

nBM-SMCs showed higher proliferation and clonogenic potential compared with aBM-SMCs. Cells were seeded at 1.8 × 10⁴ cells/cm² and grown to near confluence, trypsinized, and counted at the indicated times. (A) Bright-field photomicrographs of n1 or a3 BM-SMC (bar = 100 µm). (B) The results are plotted as cumulative cell number over time for nBM-SMCs (n1, n2, and n3) or aBM-SMCs (a1, a2, and a3). (C) Cells were plated onto 100 mm dishes at clonal density (400 cells/dish) and cultured for 10 days. Photographs of cell culture dishes after 10 days in culture display colonies that originated from single cells. (D) Colony size distribution for nBM-SMCs and aBM-SMCs. *P < 0.05. (E) Representative colonies originating from nBM-SMCs or aBM-SMCs (bar = 1 mm).

Figure 2

Vascular contractility of BM-TEVs from nBM-SMCs vs. aBM-SMCs. (A) BM-SMCs were embedded in fibrin, which was allowed to polymerize in 24-well plates to form disks. One hour after polymerization, the gels were detached from the walls and allowed to compact. At the indicated times, the gels were photographed and their area was measured using Image J software. The ratio of the area of each gel (A) at the indicated times over the initial area (A₀) was plotted as a function of time. (B) Representative pictures of fibrin hydrogels containing BM-SMCs at t >76.5 h. Dotted circles indicate initial hydrogel perimeter. (C) BM-SMCs were embedded in fibrin hydrogels and cultured around 6 mm mandrels for 2 weeks to form cylindrical tubes. Vascular reactivity (kPa) was measured in response to endothelin-1 (20 nM), U46619 (10⁻⁶ M), or KCl (118 mM). (D) Representative graphs of isometric contraction in response endothelin-1 and U46619. *P < 0.05.

Figure 3

nBM-SMCs expressed higher level of SMC contractility-related genes. Total RNA was isolated from nBM-SMCs or aBM-SMCs cultured to near confluence in the presence of TGF-β1 (2 ng/mL) and ascorbic acid (300 µM) and reverse transcribed to cDNA. Expression of the indicated transcripts was measured by real-time RT–PCR and normalized relative to GAPDH. Each experiment was done with triplicate samples. *P < 0.05.

Figure 4

Mechanical properties of BM-TEV and ECM synthesis in BM-SMCs. (A) Stress–strain curves generated with TEV from nBM-SMCs or aBM-SMCs. (B) Ultimate tensile stress (kPa). The dotted line denotes the value of UTS for V-SMCs from umbilical veins of neonatal lambs, which served as a control. (C–E) Total RNA was isolated from nBM-SMCs or aBM-SMCs cultured to near confluence and expression of the indicated transcripts was measured by real-time RT–PCR and normalized relative to GAPDH. The relative expression level was defined as the ratio of the normalized expression level of each gene in BM-SMCs to that in V-SMCs from umbilical veins of neonatal lambs. RT–PCR experiment was repeated with RNA from three independent experiments. (C) Collagen type I, (D) collagen type III, and (E) elastin.

Figure 5

FGF augmented proliferation of aBM-SMCs to a larger extent than nBM-SMCs. (A) nBM-SMCs or aBM-SMCs were cultured with the indicated bFGF concentrations and cell number was determined 3 days later and normalized each to cell number without bFGF. (B) Phase contrast microscopy showed that bFGF induced significant morphological changes to aBM-SMCs and nBM-SMCs (bar = 100 µm). (C) aBM-SMCs and nBM-SMCs expressed similar level of FGFR1 as determined by RT–PCR. GAPDH served as a loading control.

Figure 6

TGF-β1 increased fibrin gel contractility in nBM-SMCs and aBM-SMCs. Cells were embedded and polymerized in fibrin and allowed to compact gels in the absence (A) or presence (B) of TGF-β1. At the indicated times, the gels were photographed and their area was measured using Image J software. The ratio of the area of each gel (A) at the indicated time over the initial area (A₀) was plotted as a function of time.