Pro-elastogenic effects of bone marrow mesenchymal stem cell-derived smooth muscle cells on cultured aneurysmal smooth muscle cells

Abstract

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non-surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and -9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM-MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM-MSC-derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM-MSC-derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow-up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long-term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd.

Magnetically Responsive Bone Marrow Mesenchymal Stem Cell-Derived Smooth Muscle Cells Maintain Their Benefits to Augmenting Elastic Matrix Neoassembly

“…10 ) on human fibronectin-coated plastic (BD Biosciences), differentiated in a medium containing 2.5 ng/mL transforming growth factor-beta1 (TGF-b1; Peprotech) for 6 days, and then propagated for 5 days in high-glucose (4.5 g/L) DMEM (Invitrogen) containing 10% v/v FBS (Invitrogen), 1% v/v Penstrep, 2.5 ng/mL of TGF-b1, and 5 ng/ mL of platelet derived growth factor (R&D Systems). The derived BM-SMCs were propagated in DMEM-F12 containing 10% v/v FBS and then passaged.…”

“…10 EaRASMCs were seeded within wells of six-well plates (3 · 10 4 cells/well) and maintained for 21 days in standalone culture (control) or in noncontact coculture with BM-SMCs and SPION-BM-SMCs similarly seeded within PET-transwell inserts (pore size = 1 mm; Greiner Bio-One). The EaRASMCs were subsequently harvested for analysis.…”

“…10,17 Primary antibodies for SMA, SM22, Smoothelin, MHC, MMP2 (Abcam), MMP9 (Millipore), and the housekeeping protein b-actin (Sigma-Aldrich) were used. Secondary antibodies were conjugated with either IRDye Ò 680LT or IRDye Ò 800CW (LI-COR Biosciences).…”

“…Previously, 10 we showed that rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (BMSMCs) significantly stimulate synthesis of crosslinked elastic matrix and attenuate matrilysis in rat aneurysmal SMCs (EaRASMCs). 10 We now seek to enable their targeted delivery to AAA tissue for regenerative benefit.…”

“…11 As the first step, in this study, we assessed viability of SPIONlabeled BM-SMCs, their responsiveness to an externally applied magnetic field for improved uptake into a matrixdisrupted aortic wall, and their ability to maintain their previously demonstrated functional benefits upon SPIONlabeling. 10 …”