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Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia  Muhammad I. Niagara, MSc, Husnain.

Published byMaria do Mar Rodrigues Modified over 5 years ago

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Presentation on theme: "Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia  Muhammad I. Niagara, MSc, Husnain."— Presentation transcript:

1 Autologous skeletal myoblasts transduced with a new adenoviral bicistronic vector for treatment of hind limb ischemia  Muhammad I. Niagara, MSc, Husnain Kh. Haider, MPharm, PhD, Lei Ye, MD, Valerie S.W. Koh, BSc, Yean T. Lim, FRCP, Kian K. Poh, MRCP, Ruowen Ge, PhD, Eugene K.W. Sim, FRCS  Journal of Vascular Surgery  Volume 40, Issue 4, Pages (October 2004) DOI: /j.jvs Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

2 Fig 1 Ligation of left femoral artery for hind limb ischemia model creation. Blue cross, Point of ligation. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

3 Fig 2 A-J, Angiographic assessment of ischemic limbs 10 days after ligation (before) and 6 to 8 weeks after treatment (after) with DMEM injection, myoblasts, Ad-Null transduced myoblasts, Ad-VEGF transduced myoblasts, or Ad-Bicis transduced myoblasts transplantation. *Ligation sites. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

4 Fig 3 A, Immunostaining of skeletal muscle–derived myoblast for expression of desmin (magnification ×300). B, Phase contrast photomicrograph shows cells at confluence (magnification ×60x). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

5 Fig 4 A, X-gal staining of LacZ expressing myoblasts in vitro (magnification ×400). B, BrdU-incorporated myoblasts detected with immunostaining (magnification ×300). C, DAPI-labeled myoblasts observed under fluorescence (magnification ×300). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

6 Fig 5 A, FITC fluorescence of VEGF165 expressing myoblasts after bicistronic adenoviral transduction in vitro; counterstained with DAPI (B). Corresponding TRITC fluorescence of Ang-1 expressing myoblasts after bicistronic adenoviral transduction in vitro; counterstained with DAPI (C) FITC and TRITC overlayed fluorescence of VEGF165 and Ang-1 expression, respectively (magnification ×300). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

7 Fig 6 ELISA for in vitro secretion of VEGF from Ad-VEGF transduced myoblasts (A) and Ad-Bicis transduced myoblasts (B) over 28 days. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

8 Fig 7 A, LacZ expressing myoblasts (green nuclei) in host tissue 6 weeks after autologous myoblast transplantation. Tissue section was counterstained with hematoxylin to visualize the host nuclei (purple) (magnification ×600). B, Immunostaining for BrdU-positive myoblasts (magnification ×300). C, DAPI-labeled myoblasts at site of grafting 6 weeks after transplantation (magnification × 150). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

9 Fig 8 Angiographic score from quantification of collateral vessels in various treatment groups (P = .022 vs DMEM; P = .024 vs myoblasts; P = .057 vs Ad-Null myoblasts; P = .181 vs Ad-VEGF myoblasts). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

10 Fig 9 A, D, G, J, and M, FITC fluorescence of von Willebrand factor expression from ischemic limb tissue of groups treated with DMEM, myoblasts, Ad-Null transduced myoblasts, Ad-VEGF transduced myoblasts, and Ad-Bicis transduced myoblasts. B, E, H, K, and N, Corresponding TRITC fluorescence of smooth muscle actin expression from respective treatment groups. C, F, I, L, and O, FITC and TRITC overlayed fluorescence of von Willebrand factor and smooth muscle actin expression, respectively (magnification ×200). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

11 Fig 10 Capillary density from von Willebrand factor–positive vessels in various treatment groups. Capillary density was highest in group 5. P < .001 vs normal, DMEM, Ad-Null myoblasts, and myoblasts groups. P = .009 vs Ad-VEGF myoblasts group. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

12 Fig 11 Proximal localization of LacZ-expressing myoblasts to smooth muscle actin–positive vessels in tissue of Ad-Bicis transduced myoblast–treated ischemic limb. A, Site of extensive LacZ expression with concomitant enhanced number of smooth muscle actin–positive vessels. B, Distal site of minimal LacZ expression with corresponding low number of smooth muscle actin–positive vessels (magnification ×100). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

13 Fig 12 In vivo VEGF expression in site of LacZ-expressing Ad-Bicis transduced myoblasts graft (A) and even in areas adjacent and distal to transplanted region (B) compared with faint VEGF signal in DMEM group (C). (A and B, magnification ×200; C, magnification ×300). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

14 Fig 13 Tissue sections from Ad-Bicis transduced myoblast–treated ischemic limb demonstrates extensive expression of rhodamine fluorescence of myosin heavy chain in DAPI-labeled myoblasts vicinity in viable mature host muscle fiber region (A) and ischemic area (B). (Magnification ×150). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

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