Blood-derived smooth muscle cells as a target for gene delivery

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Blood-derived smooth muscle cells as a target for gene delivery Zhe Yang, MD, PhD, Hongwei Shao, MD, PhD, Yaohong Tan, MD, PhD, Darwin Eton, MD, Hong Yu, PhD Journal of Vascular Surgery Volume 47, Issue 2, Pages 432-440 (February 2008) DOI: 10.1016/j.jvs.2007.10.039 Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 1 In vitro differentiation of peripheral blood mononuclear cells (PB-MNC) into smooth muscle cell (SMC)-like cells. A few attached PB-MNCs grew out (a) and formed colonies after 2 weeks in culture (b). They were differentiated into SMC-like cells (c) after cultured in medium containing PDGF-BB for 14 days. The outgrowth colony cells were immunostained with fluoresceinisothiocynate conjugated antibody against CD34 (d). Fluorescent Dil-ac-LDL uptake (e) and lectin binding (f) showed EPC characteristic of the PB-MNC (original magnification: ×100). Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effect of statin and stromal cell-derived factor (SDF)-1 on mononuclear cells (MNCs). A, Peripheral blood mononuclear cells (PB-MNCs) were cultured in MNC culture medium with additional of (a) control: no additive, (b) SDF-1, and (c) fluvastatin. The attached MNCs were stained with Dil-ac-LDL at the 10th day of culture and visualized under fluorescence microscopy. B, Relative MNC number in the three groups described in (A). Asterisk indicates P < .05 vs control (n = 3) (original magnification: ×200). Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 3 Characterization of blood-derived smooth muscle cells (BD-SMC) by immunostaining. The outgrowth colony cells from MNC culture (a), (e), and (i), and the differentiated BD-SMCs (b), (f), and (j) were immunostained with antibodies against SMC markers: smooth muscle α-actin (α-SMA, upper panel), calponin (middle panel), and smooth muscle heavy chain (SMHC, lower panel). Vascular SMC (c), (g), and (k), and EC (d), (h), and (l) isolated from jugular vein were similarly stained as a positive and negative control, respectively (original magnification: ×100). Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 4 Proliferation and migration of smooth muscle cells (SMC). A, Cell proliferation was measured by counting the cell number daily after the culture medium was switched from serum free to 20% FBS. B, Migration in a Boyden chamber. The percentage of migration was expressed as the ratio of migrated cells on bottom side to the total number of cells on both sides. No significant difference in either proliferation or migration was observed between blood-derived smooth muscle cells (BD-SMC) and vascular derived SMC (VSMC) (P > .05, n = 3). Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 5 SMC apoptosis. A, 4’-6-Diamidino-2-phenylindole (DAPI) staining was used to detect apoptotic cells (arrow points at white condensed pyknotic nuclei) after blood-derived smooth muscle cells (BD-SMC) (a) and (c), or vascular derived SMC (VSMC) (b) or (d) were passed for three (top panel) and 10 (bottom panel) passages (original magnification: ×400). B, The percentage of apoptotic BD-SMCs at passage 10 (11 ± 2%) is significantly higher than that at passage 3 (4 ± 2%, n = 3, P < .05). The increase of apoptotic cells in BD-SMCs at different passages is similar to that of VSMCs. Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 6 Retroviral transduction efficiency and stability of transgene expression. A, Blood-derived smooth muscle cells (BD-SMCs) (left) and vascular derived SMC (VSMCs) (right) have similar transduction efficiency with MuLV vector bearing lacZ gene. The cells with blue-stained nucleus are the ones successfully transduced at passage 3 (P3) (original magnification: ×100). B, The number of β-gal positive cells in the LacZ transduced BD-SMCs and VSMCs decreased slightly after passages, but no significant difference between the groups was observed (P > .05, n = 3). Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

Fig 7 Transgene expression in vitro and in vivo. A, Secreted alkaline phosphatase gene (SEAP) concentration in the cell culture media of blood-derived smooth muscle cells (BD-SMCs) and vascular derived SMC (VSMCs), which have been retrovirally transduced with SEAP gene. B, SEAP concentrations in blood after i.v. injection of BD-SMC/SEAP (open squares) and VSMC/SEAP (filled squares) into rabbits. Journal of Vascular Surgery 2008 47, 432-440DOI: (10.1016/j.jvs.2007.10.039) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions