From Children's Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Judah Folkman, MD, Julia Dyckman Professor of Pediatric Surgery and Professor of Cell Biology, Children's Hospital and Harvard Medical School, Hunnewell 103, 300 Longwood Ave, Boston, MA 02115.
In this issue of
Circulation, Baumgartner et al1 ). The plasmid DNA was injected
directly into the muscle of ischemic limbs. Anatomic and
functional efficacy was demonstrated by increased serum levels of VEGF,
improved hemodynamic measurements and angiographic
evaluation, reduced pain, increased healing of ischemic ulcers,
limb salvage, and immunohistochemical evidence of proliferating
endothelial cells in tissue specimens. The authors
emphasize that this is the first medical therapy to achieve an increase
in limb perfusion that is equivalent to or greater than successful
surgical or percutaneous intervention.
Direct intramuscular gene transfer of plasmid DNA appears to
effectively stimulate collateral vessel growth, despite the lower
transfection efficiency that is usually associated with gene therapy in
the absence of a viral vector. This result has implications for other
clinical trials of gene therapy that use intramuscular naked DNA. The
fact that Isner's group could prepare the plasmid for human use in a
university medical center laboratory dedicated to this purpose reveals
why gene therapy has moved so rapidly from the laboratory to the
clinic, in contrast to protein therapy, which requires expensive
manufacturing facilities and years of scale-up effort.
Although the plasmid was injected into muscle of the
ischemic limb, VEGF levels were apparently elevated in the
whole circulation, as evidenced by transient peaks of VEGF in the serum
and by edema of the ischemic limb and in some patients, in the
opposite limb. The increased collateral vessels, however, are localized
to the ischemic limb and do not develop in other areas of the
body. This may reflect in part the short half-life of VEGF in the
circulation (minutes) as well as the upregulation of receptors for VEGF
in ischemic tissue.2 3 As the authors
point out, endogenous upregulation of VEGF expression by
hypoxic endothelial cells may provide an amplifying
mechanism that tends to localize the action of exogenous VEGF to the
ischemic limb.4
Because VEGF is a mitogen for vascular
endothelial cells and not for smooth muscle cells, the
increased density of larger (200- to 800-µm) vessels observed by
angiography may be secondary to neovascularization at the level of
microvessels. This possibility is supported by the presence of foci of
proliferating endothelial cells observed in an
amputation specimen obtained 10 weeks after gene therapy. One would not
expect to see endothelial proliferation in normal limb
vasculature. However, the level of endothelial
proliferation that might occur in an ischemic or a gangrenous
limb before therapy is also unknown.
All gene therapy designed to potentiate local angiogenesis
carries the theoretical risk that pathological angiogenesis at a remote
site could be stimulated, ie, ocular angiogenesis or tumor
angiogenesis. The authors cautioned readers about this. In one report,
bFGF administered at very high doses over a prolonged period increased
growth of a pre-existing mouse tumor.4A However, in our own
studies of mice bearing dormant microscopic lung metastases that were
not angiogenic, repeated systemic injections of either basic fibroblast
growth factor (bFGF) or VEGF protein per se did not turn on
angiogenesis in these lesions or stimulate tumor growth (M.S.
O'Reilly, MD, PhD, J. Folkman, MD, unpublished data, 1997). We further
found that corneal neovascularization induced in the mouse by
implantation of a sustained-release pellet of bFGF was not enhanced by
systemic administration of bFGF or VEGF (R. D'Amato, MD, unpublished
data, 1997). The explanation for this result is unclear, except for the
possibility that these sites were not hypoxic or that the VEGF exposure
was too brief. Finally, there remains the question of whether
atherosclerotic plaque neovascularization may be exacerbated by
angiogenic therapy.
A fundamental and still unsolved biological question is, How are
collateral blood vessels induced and sustained?
Schaper5 stated, "Regional tissue
ischemia is still the only situation which leads to collateral
vessel formation in a predictable way." No drug or growth factor has
accomplished this in the absence of ischemia. In fact, chronic
infusion of VEGF into the canine coronary system does not lead
to endothelial proliferation.5 However, it
is not clear whether tissue ischemia is a cause of collateral
vessel formation or only correlates with it. VEGF expression is
upregulated by hypoxia in normal tissues that become
ischemic and in ischemic tumor
cells.6 7 8 9 10 11 Because receptors for VEGF are also
upregulated in hypoxic tissues,2 perhaps these
two processes are accelerated by exogenous VEGF gene therapy.
Nevertheless, in the ischemic limb, the growth of larger
arterioles and arteries, including the necessity of remodeling (without
sprouting), differs from the growth of microvessels in a capillary bed,
in which sprouting has been observed even in the muscle bed that is
exercised.12 Arras et al13
distinguish between "arteriogenesis" and "angiogenesis" in the
ischemic limb. Three days after femoral artery ligation in rabbits,
collateral arterioles in the thigh grew by proliferation of endothelial
cells and smooth muscle cells (arteriogenesis), coincident with the
adherence of monocytes that supplied growth factors, eg, bFGF and
TNF-alpha, to the vessel wall. This process in the thigh appeared to be
independent of perfusion deficiency or hypoxia. Capillary proliferation
(angiogenesis) in the lower limb did not occur until 7 days after
femoral artery ligation and also correlated with monocyte accumulation
as well as with other sources of bFGF. Hypoxia was a major stimulus for
angiogenic growth factors.
Why do collateral vessels form at a considerable distance from the
ischemic capillary bed? No satisfactory mechanism has been
proposed for retrograde diffusion of an angiogenic factor (such as
VEGF) from the site of its overexpression in an ischemic
vascular bed. Therefore, the conventional explanation is that
collateral vessel growth is primarily a result of increased flow and
lateral wall pressure in patent vessels that are carrying blood around
an obstructed main artery to an ischemic vascular bed that is
fully dilated. But this biomechanical hypothesis is inadequate to
explain evidence of a humoral communication between a distal
ischemic bed and its proximal feeding vessels. For example,
after experimental coronary artery occlusion, increased
endothelial thymidine labeling was found not only in
the growing collaterals but also in the coronary venous system
(see Reference 1414 for review). In a model of renal artery occlusion,
there was thymidine labeling in the renal collaterals but also in the
renal vein. Furthermore, endothelial labeling in the
collateral arteries spread from the ischemic zone in a
time-related retrograde gradient. A similar retrograde spread of
endothelial labeling along the spermatic artery was
observed when tumor was implanted in the rat
testis.14 Recent evidence suggests that vascular
endothelial cells in the abdominal aorta of the rat are not stationary
but migrate toward the heart.15 If they originated in a
hypoxic area, could they transport growth factors in a direction
retrograde to blood flow? Thus, the molecular mediation of collateral
arteriolar growth is a potentially fertile field of investigation. The
interdependence of VEGF and bFGF will need to be taken into account in
any molecular model of collateral vessel
development.16 17 18 The recent discovery of
angiopoietin-1,19 which mediates recruitment of
vascular smooth muscle cells by developing vessels, and of
angiopoietin-2, which counteracts
angiopoietin-1,20 will also need to be included
in future models of collateral vessel formation. Finally, the recent
demonstration that VEGF can upregulate nitric oxide production
may play an as yet unrecognized role in collateral
formation.21 22
If the results reported here are confirmed by others or extended,
what would this indicate for the future? Although the authors are
appropriately cautious, this is an important step in the evolving
strategy of angiogenic therapy for severe limb ischemia
refractory to conventional therapy, for which amputation is the only
alternative. It will be of interest to learn whether the intramuscular
injection of phVEGF165 can be repeatedly administered to
those patients who have recurrent pain or tissue loss due to
ischemia. This pioneering study prepares the way for angiogenic
gene therapy of the ischemic human heart. When taken together
with the seminal report by Schumacher et al23 in
the February 24, 1998, issue of Circulation, in which the
angiogenic protein acidic fibroblast growth factor was injected into
the myocardium after coronary bypass graft surgery,
the present article by Baumgartner et al1
suggests that in the future, VEGF gene therapy of the human heart may
be administered by endoscopic thoracotomy or through a limited
thoracotomy.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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© 1998 American Heart Association, Inc.
Editorials
Therapeutic Angiogenesis in Ischemic Limbs
Key Words: Editorials • angiogenesis • endothelium • hypoxia • ischemia
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