Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis

Abstract

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of 'growth' factors with 'maturation' factors like PDGF-BB, angiopoietin-1 or TGF-beta. Myoblast-mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long-lasting expression, and can be considered as a relatively rapid form of 'adult transgenesis' in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different 'growth' and 'maturation' factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline-inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell-based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

Figure 1

Following injection into the muscle, transduced myoblasts expressing the beta-galactosidase reporter gene either fuse with each other to form myotubes or fuse with pre-existing muscle fibers. Hematoxylin/eosin staining (right panel) shows that the tissue architecture is fully preserved, the area of engraftment being recognizable only by the centralization of the muscle nuclei (yellow arrows). Note the absence of inflammatory cells. Enzymatic beta-galactosidase staining on a neighboring section (left panel) shows the expression of the reporter gene by the transgenic muscle fibers (blue arrows). With permission from Dekker Publishing group: Ozawa et al. (2000) in Templeton & Lasic eds.: Gene Therapy: therapeutic mechanisms and strategies.

Figure 2

Large vascular structures with blood pools develop in skeletal muscle implanted with primary myoblasts expressing VEGF. Tissue was harvested 44–47 days following myoblast implantation (hematoxylin/eosin staining). The control leg shows normal size and morphology (left panel), whereas the leg injected with VEGF-expressing myoblasts shows hemangioma leading to a more than two-fold increase in leg size (right panel). With permission from Elsevier Science: Springer et al. (1998). VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol. Cell, 2, 549-558.

Figure 3

Tibialis anterior muscle injected with VEGF-expressing myoblasts: a few myoblasts have leaked back through the wound created by the needle during injection and fused with muscle fibers along the track as shown by the beta-galactosidase expression (blue fibers in the right panel). No hemangioma formation is seen, but areas of higher capillary density of normal morphology are apparent. The left panel shows immunofluorescence for PECAM/CD31 (red) and alpha-smooth-muscle actin (green). Around these areas, numerous arterioles have formed, which replace the capillary pattern typically present in skeletal muscle. Bar 100 μm. With permission from Elsevier Science: Springer et al. (2003). Localized arteriole formation directly adjacent to the site of VEGF -induced angiogenesis in muscle. Mol. Ther., 7, 441-449.

Figure 4

Confocal microscope generated three-dimensional reconstruction of an arteriole near an implantation site (immunofluorescence for the endothelial cell marker PECAM/CD31 (red) and alpha-smooth-muscle actin (green)). Green-stained smooth-muscle cells are seen wrapping around a red-stained endothelial tube. Bar 10 μm. With permission from Elsevier Science: Springer et al. (2003). Localized arteriole formation directly adjacent to the site of VEGF -induced angiogenesis in muscle. Mol. Ther., 7, 441-449.