Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

Abstract

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.

Keywords: CdM, conditioned medium CCM: complete culture medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule-1; IEL, internal elastic lamina EEL: external elastic lamina; IL-6, interleukin-6 MCP-1: monocyte chemoattractant protein-1; MSC, mesenchymal stem cell GFP: green fluorescence protein; Mesenchymal stem cells; Neointimal hyperplasia; Stem cell-secreted factors; VSMC, vascular smooth muscle cell PDGF: platelet-derived growth factor; Vascular smooth muscle cells.

Fig. 1

In vivo experimental protocol and GFP-MSC characteristics. (a) Protocol of MSC implantation study. MSC localTx, local MSC administration onto the adventitial sites. MSC ivTx, systemic MSC administration via tail vein. (b) Cultured green fluorescence protein (GFP)-MSCs. Nuclei were stained with DAPI (blue). (c) Flow cytometric analysis for MSCs. GFP rat MSCs expressed the mesenchymal marker CD90 (Thy 1), but not markers of hematopoietic or endothelial cells (i.e. CD45, CD34, CD31). Blue = Cell surface epitope-specific antibodies, PE-conjugated and per-titered for FACS. Red = Non-specific isotype control antibodies, also PE-conjugated and per-titered for FACS.

Fig. 2

Local MSC therapy in a rat vascular injury model. (a) Transient engraftment of MSCs without differentiation. A few GFP-positive MSCs (green) were detected in the adventitia 3 days after the perivascular administration of MSCs. Nuclei were stained with DAPI (blue). SMA (red), alpha-smooth muscle actin. DAPI, 4′,6-Diamidino-2-phenylindole. L, lumen of artery. Bar scale, left= 100 µm, right (3 panels) = 20 µm. (b) Prevention of neointimal formation by the perivascular MSC administration. Representative images of rat carotid arteries 16 days after the injury (14 days after the treatment). Con, controls. MSC, perivascular MSC administration. MSCiv, intravenous systemic MSC administration. I, intima. M, media. Bar scale, HE, hematoxylin-Eosin staining. EVG, elastica van Gieson staining. Bar scale, upper= 200 µm, lower= 50 µm. (c) Quantitative morphometric analyses. By day 14 after treatment, local perivascular administration of MSCs (MSC, n = 10) significantly suppressed neointimal hyperplasia (the intima/media ratio and the max intimal thickness) compared with controls (Con, n = 10). Intravenous MSC administration (MSCiv, n = 4) did not limit neointimal hyperplasia. *, p < 0.05.

Fig. 3

Inhibition of VSMC proliferation by local MSC therapy. (a) Left images, Immunohistochemistry was performed with antibodies to Ki67 to evaluate cell proliferation. Brown staining in the nuclei indicate the presence of Ki67. Bar= 20 µm. Right graphs, Local MSC administration significantly decreased the number of proliferating VSMCs (Ki67-positive cells) in the arterial wall. Con, n = 10; MSC, n = 10. I, intima. M, media. *, p < 0.05. (b) Left images, Immunostaining for p27^Kip1 protein. Brown staining in the nuclei indicate the presence of p27^Kip1. Bar= 20 µm. Right graphs, Local MSC administration significantly increased the number of p27^Kip1-positive VSMCs. Con, n = 10; MSC, n = 10. #, p < 0.01. (c) Phenotypic changes in VSMCs after local MSC therapy. Local MSC administration down-regulated the expression of SMemb and RM-4 in neointimal VSMCs. Brown staining indicates positive cells. SMemb, embryonic isoform of myosin heavy chain. RM-4, membrane protein of inflammatory cells. Bar= 20 µm.

Fig. 4

Effects of factors secreted by MSCs on cultured-VSMC proliferation and phenotype. (a) Conditioned medium from MSCs (MSC-CdM) significantly attenuated the growth of rat VSMCs exposed to PDGF-BB. Base, baseline before the treatment. Con, the controls. n = 5 in each group. #, p < 0.01. (b) By western blot, we observed increased p27 expression and decreased RM-4 expression in PDGF-BB-stimulated rVSMCs treated with MSC-CdM compared with controls. Three independent experiments were performed. rVSMC, rat vascular smooth muscle cells. (c) Real-time RT-PCR analysis demonstrated that MSC-CdM significantly decreased gene transcription for IL-6, MCP-1, ICAM-1, and cyclin D1 in the rVSMCs exposed to PDGF-BB. n = 3 in each group. * , p < 0.05. #, p < 0.01.