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

atrium in heart, endothelial cells in the peribronchial
capillaries (but not bronchial epithelium) in the lung, the
menings, and at the inner surface of the atrium and the
aorta; this expression seemed to be restricted to
endothelial cells in blood vessels and capillaries. Flk-1
was also expressed in capillaries in brain at postnatal day 4
(Millauer et al, 1993).
(iii) The Flt-4 (or VEGF-R3) (for references see Suri et
al, 1996).
Transgenic mouse embryos with targeted disruption of
the VEGF-R1 and R2 genes have unraveled the different
roles these receptors undertake during development: Flt-1
regulates normal endothelial cell-cell or cell-matrix
interactions during vascular development (Fong et al,
1995) and Flk-1 is required for the formation of blood
islands and vascularization of the embryo; disruption of
the flk-1 gene interfered with differentiation of endothelial
cells leading to death of embryos at day 8.5 to 9.5
(Shalaby et al, 1995 ).
The temporal and spatial expression of VEGF exactly
correlated with the expression pattern of Flk-1 in all
tissues and developmental stages in mice examined
suggesting a pivotal role of the growth factor/receptor duet
in development and differentiation of the vascular system;
their expression was high during development and
declined in adulthood (Millauer et al, 1993).
Although VEGF expression showed a moderate
increase during tumor development in pancreatic islets of
Langerhans, the expression of the flt-1 and flk-1 receptor
genes remained unchanged during pancreatic
carcinogenesis in an animal model (transgenic mice
having a targeting expression of the SV40 T antigen gene
under control of the insulin gene regulatory regions in βcells
of the pancreatic islets) (Christofori et al, 1995).
D. Angiopoietin and its receptors
Although the TIE1 and TIE2 receptor tyrosine kinases
were found to be involved in vasculogenesis already in
1992, angiopoietin-1, the natural ligand of TIE2 was only
recognized in 1996 by secretion-trap expression cloning;
according to this method entire cDNA libraries were
transfected into large numbers of cells and the cell that
contained the desired cDNA was uniquely marked on its
surface by expression of the desired ligand; this rare cell
was isolated within a background of millions of cells and
was expanded leading to the isolation of the ligandencoding
cDNA (Davis et al, 1996). This finding
represents a significant milestone in the effort to
understand the molecular mechanisms that govern
formation of blood vessels with important implications for
cancer targeting by inhibition of angiogenesis.
Targeted disruption of the angiopoietin-1 gene in mice
(Suri et al, 1996) gave defects in the blood vessels
Gene Therapy and Molecular Biology Vol 1, page 101
101
reminiscent of those caused by disruption in its receptor
TIE2 (Dumont et al, 1994); these defects were mainly in
the endocardium and myocardium but also generalized
defects in vascular complexity leading to the death of mice
by day 12.5 of gestation. These studies have established
the important role of angiopoietin in mediating reciprocal
interactions between the endothelium and surrounding
matrix and mesenchyme (Suri et al, 1996). However,
angiopoietin-1 does not display the mitogenic activities of
VEGF on endothelial cells. The efficacy of disrupting the
binding of angiopoietin to its ligand in tumor cell growth
remains to be established.
A missense mutation in the receptor tyrosine kinase
TIE2, resulting in a Arg to Trp substitution at position 849
of the kinase domain, was found in patients from two
unrelated families suffering with venous malformations;
this disorder is characterized by the presence of veins with
large lumens lined by a monolayer of endothelial cells but
with thin walls because of a reduction in smooth muscle
layers. Expression of the wild-type and mutant TIE2 in
insect cells has shown that the mutation caused a 6 to 10fold
increase in autophosphorylation activity of TIE2;
thus, this mutation causes a defect in vascular remodeling
and the overproliferation of endothelial cells without a
complementary increase in smooth muscle layers (Vikkula
et al, 1996). This finding demonstrates the significance of
the TIE2 signaling pathway for endothelial cell-smooth
muscle cell communication in venous morphogenesis.
E. Involvement of VEGF in tumor
angiogenesis
Important for tumor growth are alterations in
extracellular matrix. Solid tumors are composed of the
malignant cells and the supportive vascular and connective
tissue stroma whose synthesis is induced by the malignant
cells. Fibrin is an essential component of the connective
tissue stroma upon which tumor cells depend for their
oxygen/nutrient supply and waste disposal. Fibrin gel
provides solid tumors with a matrix which favors the
ingrowth of macrophages, fibroblasts, and endothelial
cells; these compose, along with neoangiogenesis vessels
and elements found in normal connective tissue, the
mature tumor stroma. Fibrinogen and other plasma
proteins are found in increased amounts in tumor stroma
and in healing wounds compared with normal stroma of
most tissues; deposition of fibrin at the extravascular site
requires a previous increase of permeability of the
microvasculature thought to be mediated by VEGF
(Senger et al, 1993).
VEGF is the factor largely responsible for leakiness
and hyperpermeability of tumor blood vessels; leakage of
plasminogen (which is converted to plasmin) and of
fibrinogen and clotting factors II, V, VII, X, and XIII