01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 1, page 107
Figure 32. Representative micrographs showing the characteristics of rabbit carotid arteries 7 days after VEGF or lacZ gene
transfer. A. Control artery transfected with lacZ-plasmid/liposomes using smooth muscle cell (SMC)-specific immunostaining (HHF-35)
showing a typical intimal thickening. B. Artery transfected with VEGF plasmid/liposomes showing a limited intimal thickening after
SMC-specific immunostaining (HHF-35). C. Serial section to A, but stained for endothelium with CD31 monoclonal antibodies; it
shows the presence of endothelium in all vascular segments examined. D. Serial section to B, but stained for endothelium with CD31
monoclonal antibodies, showing an intact endothelium. E. In situ hybridization with a VEGF antisense riboprobe labeled with [ 35 S]UTP
in VEGF-transfected artery; bright spots (arrows) indicate the expression of VEGF mRNA (dark-field image). Control hybridizations
with sense riboprobes were negative (not shown). F. The absence of inflammation was shown in VEGF-transfected arteries with
macrophage-specific immunostaining (RAM-11). G. Neovascularization (arrow) in the adventitia of VEGF-transfected artery 14 days
after gene transfer using endothelium-specific immunostaining (CD31). No neovascularization was detectable in lacZ-transfected
arteries (not shown). H. Nonimmune control for the immunostainings (first antibody omitted); sections were counterstained with
hematoxylin. Magnification, 25X in A-E, G and 50X in F,H. From Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J,
Abedi H, Risau W, Soma M, Laakso M, Martin JF, Ylä-Herttuala S (1997b) VEGF gene transfer reduces intimal thickening via
increased production of nitric oxide in carotid arteries. Hum Gene Ther 8, 1737-1744. Reproduced with the kind permission of the
authors and Mary Ann Liebert, Inc.
Transfer of the bone morphogenetic protein-2 (BMP-2)
gene inhibited serum-stimulated increase in DNA
synthesis and cell number of cultured rat arterial SMCs as
well as injury-induced intimal hyperplasia; the mode of
BMP-2 action differed from that mediated by TGF-β;
BMP-2 had the ability to inhibit SMC proliferation
without stimulating extracellular matrix synthesis
(Nakaoka et al, 1997).
cDNA for hirudin has been delivered to smooth muscle
cells of injured rat carotid arteries using an adenoviral
vector; the coding region for the human growth hormone
107
signal peptide (MATGSRTSLLLAFGLLCLPWLQEGSA)
was engineered upstream of the hirudin cDNA in order to
achieve secretion of the protein in effectively transduced
cells. The therapeutically important levels of hirudin
which were secreted in vivo resulted in 35% reduction in
neointimal hyperplasia as shown on histologic sections of
the carotid arteries (Rade et al, 1996). Significant issues
on toxicity and immunogenicity of the vector remain to be
resolved for application of the method to humans (Rade et
al, 1996).
Table 7. Genes or antisense used to inhibit smooth muscle cell proliferation and neointima formation for the treatment of
arterial injury and restenosis
Gene or antisense Reference
VEGF gene transfer Isner et al, 1996a; Van Belle et al, 1997; Laitinen et al, 1997a,b
HSV-tk gene /GCV Guzman et al, 1994; Ohno et al, 1994
Cytosine deaminase (CD) gene /5-fluorocytosine Harrell et al, 1997
RB gene Chang et al, 1995a; Smith et al, 1997
p21 gene Chang et al, 1995b
Hirudin gene Rade et al, 1996
Dominant-negative mutated form of c-H-ras gene Indolfi et al, 1995; Ueno H et al, 1997b
TGFβ gene Grainger et al, 1995
Nitric oxide synthase gene von der Leyen et al, 1995