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Angiogenesis and Vasculogenesis
Angiogenesis involves the sprouting of capillaries from preexisting
capillary beds, resulting in an increased capillary density. In
vasculogenesis, angioblasts are differentiated from mesodermal cells and
organized to form a primitive vascular network.
Vasculogenesis occurs in limited embryonic sites, while angiogenesis,
the formation of new blood vessels by sprouting from existing ones,
occurs in many situations containing embryonic development and pathological
conditions (1). Therapeutic angiogenesis is the use of angiogenic strategies
to promote increased capillary development, improving blood flow to ischemic
tissue.
Regulation of vascular growth is a complex phenomenon, and various
authors have proposed many putative mechanisms of angiogenesis (2). The three
main stimuli most commonly considered are: 1) mechanical factors 2) energy
imbalance due to hypoxia and 3) inflammatory processes.
The most prominent mechanical factors are a) an increased red blood
cell-endothelial cell interaction as in policytemia, b) an increased
wall tension as a result of an increased capillary pressure, and c) an
increased shear stress which occurs with increased flow velocity.
The energy imbalance hypothesis regards various situations of
prolonged imbalances between the perfusion capabilities of blood vessels and
the metabolic requirements of tissue.
Changes in oxygenation have been implicated as a major triggering and
controlling element.
Finally, Schaper stressed the importance of inflammatory processes
associated with activation of endothelial cells and angiogenic growth factors
(3). A survey of the literature shows that over fifty putative factors may be
involved in angiogenesis. The list extends from simple ions like copper,
magnesium and selenium to complex growth factors.
The most often cited angiogenic growth factors are basic fibroblast
growth factor (bFGF) and vascular endothelial growth factor (VEGF) (4-7). In
addition to the angiogenic factors above, vascular growth can be further
modified and regulated by local geometric conditions: endothelial size and
shape and the location of other cell types such as smooth muscle cells,
pericytes, platelets, macrophages and mast cells.
The arrangement and composition of the extracellular matrix can also
influence the final response. Therapeutic angiogenesis is rapidly ongoing in
the clinical arena of cardiovascular diseases. We will briefly review:
1. Evidences supporting the functional impact of Arterial Collateral
Circulation (ACC)
2. Evidences (molecular; cellular; experimental; and clinical)
suggesting a role for cardiovascular drugs as a ACC promoter
3. Promising strategies to improve blood flow using new treatment
approaches such as recombinant angiogenic growth factors and human gene
transfection to induce growth factors production.
Development
of collateral circulation: brief overview of mechanisms and meaning
Coronary collateral circulation
Chronic myocardial ischemia as occurs with coronary artery stenosis
leads to the development of collateral circulation.
This type of growth of new blood vessels has been demonstrated in
heart and skeletal muscle and results from a reduction in the oxygen
supply/demand ratio (8-10). Collaterals develop from preexisting anastomotic
channels, as a result both of the establishment of a pressure gradient
between their origin and termination and of chemical mediators; they also
develop from newly formed microvessels, which establish new communications
between adjacent vascular beds as a result of tissue ischemia. The functional
role of collaterals is to provide blood flow that protects against necrosis
and ischemia (11,12).
The protection of collaterals against ischemia is achieved through
gradually increasing poststenotic pressure in the collateralized coronary
arterial bed, eventually providing some flow reserve. Six months after
circumflex coronary artery occlusion in exercising dogs, myocardial blood
flow, measured by the microsphere technique, can increase to the same extent
(about 300%) in normal and collateral-dependent myocardium. In man, coronary
collaterals have the potential to ameliorate myocardial ischemia (13,14). Moreover,
myocardial viability after acute myocardial infarction has been correlated
with the extent of collateral blood flow within the territory of the
infarct-related vessel (15). Coronary collaterals also may limit enzymatic
estimates of infarct size (16) and may prevent formation of left ventricular
aneurysm in acute myocardial infarction (17).
Peripheral circulation.
The peripheral vasculature is a complex and fascinating organ that is
associated with a very unique disease: the course of peripheral artery
disease is one of slow progression of symptoms over time. Approximately 70%
of patients will remain unchanged or become even less symptomatic after 5 to
10 years, <30% will progress to require intervention, and <10% will
need amputation.
The development of collateral circulation after ligation of the left iliac
artery was studied in rats by means of microsphere distribution in muscles of
both lower limbs (18). Flow in the thigh increased from 43% of control value
20 min after ligation to 70% after 26 days of recovery; flow in calf muscles
increased correspondingly from 4% to 33%. In dogs, ischemia was created in
the leg by ligation of the proximal and peripheral arteries (19); in one
month, intermittent claudication improved in accordance with improvement in
muscle tissue perfusion, angiographic evidence of distal runoff became
visible six months after surgery. Plethysmographic course controls in
patients at the age between 70 and 76 years showed that the maximum of the
collateral blood supply in arteriosclerosis in the femoral region is reached
2 years at the latest after vascular occlusion (20). Therefore, although a
functional role of collaterals has been debated, conclusive experimental and
consistent clinical evidences suggest that collateral development occurs as
an adaptive response to arterial occlusive disease (16,17); however,
individuals vary widely in their propensity for collateral growth, and the
possibility exists that collateral development could be enhanced
pharmacologically (4-7, 21,22).
 CARDIOVASCULAR
DRUGS AND ANGIOGENESIS
The Heparin Factor in Arterial Collateral
Circulation.
Evidences favoring a role of heparin in arterial collateral
circulation (ACC) development stem from experimental and clinical evidences.
Experimental evidences. A large number of mast cells gather around
rapidly growing capillaries, but mast cells alone do not cause angiogenesis. Azizkhan
et al demonstrated that mast
cells increase endothelial cell migration as the earliest event in the
formation of a capillary sprout, and this endothelial migration is
accelerated by heparin (23). In 1982, Taylor and Folkman demonstrated that
heparin could facilitate angiogenesis induced by tumor extracts from human
hepatoma cells implanted in the chorioallantoic membrane of chick embryos
(24). Thus these data suggested a new function of heparin as a positive
regulator of angiogenesis (25) (Table I). This probably occurs through
interactions with a family of polypeptide growth factors mitogens that
stimulate endothelial cell proliferation. Basic fibroblast growth factor
(bFGF) is one such peptide. This factor is now recognized to be a mitogen for
the three principal-vascular cell types (endothelial cells, smooth muscle
cells, and fibroblasts) and is chemotactic for endothelial cells in vitro. The
bFGF produced in endothelial cells is sequestered in the extracellular matrix
and basement membranes and bonds to heparin or heparan sulfate
glycosaminoglycans, which may protect FGF from degradation and serve to
chaperone bFGF through different cellular compartments (26-28).
Another heparin-binding growth factor is vascular endothelial growth
factor (VEGF), whose mitogenic activity appears to be specific for
endothelial cells and not for vascular smooth muscle (29,30). VEGF
receptors have been identified on the cell surface of endothelial cells. VEGF
is a potent stimulus to angiogenesis in the ischemic limb of rabbits and in
the ischemic myocardium (29,30). The binding of VEGF to the VEGF receptors of
vascular endothelial cells was potentiated by heparin or heparan sulfate, but
not by other glycosaminoglycans. The bFGF may promote angiogenesis both by
direct effect on endothelial cells and also indirectly by the upregulation of
VGEF in vascular smooth muscle cells (31,32). Heparin induces a
conformational change in the growth factor that allows it to interact with
its receptor on the cell surface, protects it from inactivation, potentiates
its activity, and releases it from the extracellular matrix, thereby making
this factor available for interaction with endothelial cells.
Heparin-binding growth factors are postulated to remain, under normal
physiologic conditions, sequestered in myocytes, extracellular matrix, or the
endothelial cell membrane. Ischemia, which is associated with cellular
hypoxia, acidosis, and an increase in adenosine, triggers the release of
these heparin-binding growth factors (31). Once these factors are activated,
they act locally to initiate and/or to mantain angiogenesis. Heparin and/or
heparan sulfate per se are unable to trigger angiogenesis but act as a
positive modulator (enhancer or amplifier) of angiogenetic processes
triggered by other stimuli.
Clinical evidences: coronary circulation. Fujita et al evaluated
16 patients, 10 of whom were heparin-treated, who had at least one obstructed
major coronary artery and chronic angina on effort (Table II). All patients
exercised following the standard Bruce protocol twice a day for ten days
(33). The 10 heparin-treated patients were given a single intravenous dose of
heparin 5000 IU, ten to twenty minutes before each exercise test, and the
remaining 6 patients exercised without the heparin treatment. Total exercise
duration and maximal rate pressure product were increased by about 35% by
heparin treatment. Further coronary arteriography revealed a significant
increase in the extent of collateral circulation to the region perfused by
the completely obstructed coronary artery in patients treated with heparin. In
another study from the same group, it was shown that heparin treatment alone,
without exercise (34), was unable to improve ischemia on exercise (Table II).
Quyyumi et al studied 23 patients with stable coronary artery disease who
received low-molecular-weight heparin or corresponding placebo (35). During
the first two weeks, patients were exercised to ischemia three times a day. Eight
(80%) of the 10 heparin-treated patients compared with the 4 (31%) of 13
placebo-treated patients (p<0.02) had an increased rate-pressure product
at the onset of one minute of ST segment depression.
The number and duration of episodes of ST segment depression during
ambulatory monitoring decreased by 30% and 35%, respectively (p<0.05) in
the heparin-treated group but were unchanged in the placebo-treated group.
Clinical evidences: peripheral circulation
Mannarino et al evaluated 44 patients, 22 of whom were heparin
treated, who had chronic intermittent claudication. The 22 heparin-treated
patients were given for six months a single daily subcutaneous dose (15,000
UaXa) of LMW heparin (36). Heparin treatment improved walking capacity (by
lengthening the pain-free walking time by 25%) in comparison with the absence
of changes occuring in patients treated with placebo.
Andreozzi et al evaluated 40 patients suffering from peripheral
arteriopathies of the lower limbs: patients were randomly allocated to one of
two treatment groups, receiving either 12,500 IU/day of subcutaneous calcium
heparin or 250 mg/day of oral ticlopidine (37), each given for three months:
both calcium heparin and ticlopidine induced at the end of treatment an
improvement in pain free walking distance (51% and 32% respectively), less
effective for ticlopidine than for calcium heparin.
Cina et al studied 374 patients suffering from chronic arteriopathy,
instructed to follow a programme of physical training and randomly allocated
to calcium heparin (12,500 IU daily) for 6 months (38). An improvement
in the claudicatio parameters (free gait interval, absolute gait interval and
recovery time) measured at constant speed and in the resting Winsor
ankle-to-arm index of the most severely damaged limb were observed in both
groups. These improvements were significantly greater in the group receiving
heparin treatment (80% vs 28% of exercise alone, p<0.01) and efficacy
increased in line with deambulatory impairment at baseline, before study
entry.
Altogether, these findings suggest that heparin facilitates collateral
development stimulated by exercise-induced ischemia in humans.
Since heparin in itself and by itself has no angiogenetic properties,
it should be evaluated more appropriately as an”add-on” therapy, on the top
of a promoter stimulus such as increase in endogenous adenosine, obtained by
regular exercise program and/or by drugs determining accumulation of
endogenous adenosine such as dipyridamole.
Calcium antagonists and angiogenesis: an experimentally supposed
stimulating effect with poor clinical demonstration
Some experimental findings support a positive modulation of
angiogenesis by calcium antagonists. Rakusan (1) described quantitative
evaluations of vascular growth in cardiac muscle with examples of substantial
angiogenesis in the early postnatal stages, together with examples depicting
a more moderate stimulation of capillary growth in adult rat hearts treated
with nifedipine. Treatment with nifedipine resulted in a moderate capillary
growth along the entire pathway, as evidenced by smaller capillary domains,
longer segment lenghts, and unchanged proportion of proximal and distal
capillaries in tissue cross sections (2). The Authors suggested that in
this angiogenic response the major effect of nifedipine is due to
mechanical stimuli resulting from chronically increased coronary blood
flow.
Saito (39) in a placebo controlled study suggested that increased
syntesis of bFGF and their receptors may be related to the patogenesis of
nifedipine induced gingival hyperplasia in humans.
Calcium antagonists are a wide class of drugs, with different
concentration-related effects on voltage operated calcium channels,
receptor operated calcium channels and store operated calcium channels
(40). Although some reports exist about the role of calcium-mediated
signal transduction and angiogenesis (41), only in the work of Saito (39)
there is a direct evidence of relation between nifedipine therapy and
specific mRNA bFGF expression in humans. A possible link between
calcium channel blockade and vascular tissue remodeling has been recently
found with the demonstration of interleukine 6 induction by calcium
antagonists (42) ; interleukine 6 essentially partecipates in the control of
the cell proliferation and induces production of VEGF. All 3 subclasses of
calcium antagonists increased interleukine-6 promoter activity.
Amlodipine, diltiazem, and verapamil, at nanomolar concentrations,
stimulated interleukine-6 promoter activity to 214%, 282%, and 292% of
unstimulated controls, respectively. Interleukine-6 induction by calcium
antagonists was independent of intracellular calcium concentrations,
suggesting that calcium antagonists affect expression by a different,
calcium-independent mechanism. Further study are certainly needed to support
the observed relationship between angiogenetic factors and concomitant
drug therapy with calcium antagonists.
Nitrates and angiogenesis: nitric oxide promotes
proliferation through endogenous bFGF
Myocardial vascularity increases in response to chronic alveolar
hypoxia (eg, altitude hypoxia) or cardiac hypermetabolism (eg,
hyperthyroidism, augmented preload or afterload, increased inotropy) and
after coronary ischemia.
A common denominator in all these chronic conditions is the sustained
elevation of coronary blood flow in the regions that will later exhibit
neovascularization. A number of molecules (eg, adenosine and vascular
endothelial growth factor) released from hypoxic tissue have received
attention as potential linkages between angiogenesis and myocardial function
(43). However, the rate of coronary blood flow itself may be an important
physical determinant of coronary angiogenesis (44).
Continuous administration of coronary vasodilators leads to augmented
myocardial vascularity, and a role for the endothelium-derived relaxing
factor, nitric oxide (NO), has been postulated. Coronary postcapillary
venular endothelium exposed to the NO showed increased DNA synthesis. The
expression of the angiogenic factor bFGF was increased in endothelial cells
treated with sodium nitroprussiate and with the NO dependent vasodilating
peptide substance P. It was shown that the expression of the growth factor is
under the control of the NO pathway via an autocrine/paracrine loop
(45).
In microvascular endothelium the elevation of intracellular NO induces
the endogenous production of angiogenic factors that makes the capillary
endothelium acquire the feature of an “angiogenic endothelium” (as for
increased proliferative and degradative capacity). These findings indicate
that elevation of NO levels in coronary endothelium increases endogenous bFGF
production: vasodilation is coupled to angiogenesis, revealing a feedback
loop controlled by the endothelium via NO and bFGF. Clinical demonstration of
these experimental data is completely lacking.
 Chronic
Oral Dipyridamole as a “Novel” Antianginal Drug: the collateral hypothesis
Background: dipyridamole is an adenosine transport blocker that
produces elevation of tissue adenosine levels.
The oral formulation has long been used as a “coronary vasodilator”,
but inappropriate vasodilation can lead to a proischemic effect. However, in
an hypoxic milieu increased interstitial adenosine can promote
angiogenesis.
Experimental data suggest that chronic treatment with dipyridamole
increases collateral flow and decreases exercise-induced left ventricular
dysfunction in the territory dependent upon a critical coronary stenosis.
The rationale for its antiischemic effect was even too obvious:
myocardial ischemia is believed to result from imbalance between oxygen
supply and demand, dipyridamole markedly increases oxygen supply without
significantly augmenting oxygen demand, thus it should re-establish the
normal oxygen balance (46). Although initial uncontrolled studies were
encouraging, dipyridamole has been subsequently discared for this application
on the basis of inconsistent results in placebo controlled trials, mainly
showing a lack of an acute or short-term protective effect.
Oral dipyridamole therapy in myocardial ischemia: the time
factor.
Chronic myocardial ischemia as occurs with coronary artery stenosis
leads to the development of collateral circulation. This type of growth of
new blood vessels has been demonstrated in heart and skeletal muscle and
results from a reduction in the oxygen supply/ demand ratio . It has been
shown that adenosine is involved in this angiogenic effect of the reduced
oxygen supply (47). In support of this concept, it has been demonstrated that
low oxygen concentrations (2%) increased proliferation of endothelial cells
in culture (47) by stimulating A1 and A2 adenosine receptors present on the
endothelial cells surface (48). Very recent studies showed that endogenous
adenosine produced by ischemia induces Vascular Endothelial Growth Factor
mRNA in the heart. Furthermore, dipyridamole increased the formation of
capillaries in the rat and rabbit hearts (49). In the presence of a critical
coronary stenosis, adenosine acts as a regulator of endogenous growth factors
triggered by repeated episodes of ischemia.
The beneficial antischemic effect of coronary collateral circulation
should require several weeks to become detectable, and probably a few months
to plateau. More recently, it has been experimentally shown that chronic
long-term treatment with dipyridamole increased collateral flow and improved
transmural blood flow and systolic wall thickening during exercise-induced
ischemia, with a beneficial protective antischemic effect more marked than
the one exerted by diltiazem (50).
From the clinical viewpoint, a 1988 meta-analysis of all 11 published
randomized placebo-controlled trials evaluating the efficacy of dipyridamole
for prophylaxis of angina pectoris showed that there was evidence of a
statistically significant benefit from the drug (51). Therefore, the final
result of the meta-analysis of all published trials documents a beneficial
effect of dipyridamole in treating ischemia. If the collateral circulation
pathway is indeed activated, one should expect that studies with longer
duration of treatment show the greatest benefit. In dogs, collaterals often
reach their full development and remain available for at least 4 months after
the obstruction.
Once the benefit of collateral circulation has been developed, weeks
and months of therapy withdrawal are not enough to lose it.
Other non-pharmacological treatments, such as enhanced external
counterpulsation, thought to act by improving collateral circulation are
characterized by a beneficial effect emerging over several weeks but long
lasting, even for years after discontinuation of treatment .
THE GENE THERAPY REVOLUTION: RECOMBINANT VEGF, BFGF
AND GENE TRANSFECTION TO INDUCE ANGIOGENESIS IN HUMANS
Numerous animal and human trials are currently underway to assess the
safety and efficacy of angiogenic growth factors in the treatment of
ischemic heart disease (52). While many growth factors have been
identified, the mayority of research has involved vascular endothelial growth
factor (VEGF) and fibroblast growth factor (FGF).
Recombinant growth factors delivery in humans
Initial experiences with local FGF delivery during CABG. Twenty-four
patients were randomized in a trial comparing local delivery of recombinant
FGF with placebo at the time of cononary artery by pass surgery: high dose
FGF (100 mcg) were administered.
The efficacy data appeared promising. There seemed to be improved
myocardial perfusion, determined by nuclear an magnetic resonance imaging. FGF
delivery was safe, feasible and well tolerated, and a phase II trial is
currently underway.
Phase I Intracoronary and Intravenous FGF. To assess the safety and
efficacy of intracoronary (IC) and intravenous (IV) recombinant FGF, 66
patients with severe CAD unsuitable candidates for angioplasy or bypass
surgery, received either IC FGF (n=52) or IV FGF (n=14). The efficacy data
appear promising. Patients increased their treadmill exercise time compared
with baseline testing. Regional wall motion and perfusion improved in a
subgroup of patients undergoing magnetic resonance imaging.
Phase I recombinant VEGF IC administration. The Phase I intracoronary
VEGF administration trial showed that while recombinant VEGF was safe and
well tolerated, no significant difference in exercise time or angina scores
is present comparing VEGF to control arms.
Growth Factors: Unanswered Questions. It is unclear which growth
factor is the best. The optimal mode of delivery remains to be
determined.
Researchers are also struggling with determining the best endpoint in
these angiogenesis trials. Finally, whether delivering growth factor protein
versus gene therapy is superior awaits further investigation.
Gene transfection in humans
to induce angiogenesis
Because no recombinant protein formulation of any of the three
principal VEGF isoforms is currently approved or available for human clinical
application, arterial gene transfer of VEGF has been investigated as an
alternative strategy for accomplishing therapeutic angiogenesis in patients
with ischemia.
Administration of VEGF in this fashion is particularly appealing
because the first exon of the VEGF gene includes a signal sequence that
permits the protein to be naturally secreted from intact cells.
Previous studies (53) suggested that arterial gene transfer of cDNA
encoding for a screted protein could potentially yield meaningful biological
outcomes despite a low transfection efficacy.
Phase I intracoronary gene therapy trial. In a double-blind study
randomized patients received VEGF plasmids (n=10) or placebo (n=5) following
coronary angioplasty: an infusion perfusion catheter was used for the 10
minutes intracoronary administration.
This phase I trial did show that gene transfer after angioplasty
appears to be safe and well tolerated.
Despite these encouraging findings candidates for growth factors gene
therapy has exposed certain potential limitations of arterial gene transfer:
in atherosclerotic patients, even in the absence of a thickened neointima,
extensive calcific deposits at the intimal medial interface, may limit gene
transfer to the smooth muscle cells of the arterial media.
Intramuscolar gene therapy trials. IM gene transfer, which was
pioneered by Wolff and colleagues, represents a less invasive
alternative to arterial transfection. Striated muscle has been shown to take
up and express foreign genes transferred in the form of “naked” plasmid DNA,
ie, DNA unassociated with viral or other adjunctive vectors.
IM gene transfer of naked plasmid DNA would be potentially
advantageous because it obviates immunological concerns associated with
adenoviral vectors (54).
Because naked plasmid DNA injected IM remains in a nonreplicative,
unintegrated, circular form, this strategy also is unlikely to be complicated
by insertional mutagensis.
Accordingly, studies was undertaken to test the hypothesis that IM
injection of naked plasmid DNA encoding the 165 amino acid isoform of VEGF
could augment collateral development and tissue perfusion in the setting of
experimentally induced hind limb ischemia.
In a initial number of patients with hind limb ischemia IM plasmid
VEGF transfection was successefully attempted by the group of Isner and
coworkers in Boston (55).
This study was the first to demonstrate the feasibility of the IM gene
trasfection of naked DNA encoding VEGF for therapeutic angiogenesis in
particular and the first to show bioactivity of naked DNA after IM
transfection for cardiovascular therapy in general.
VEGF gene transfer works as sole therapy for medically resistant
angina. Recently performed experiments in a porcine model of myocardial
ischemia have shown how direct intramyocardial gene transfer of VEGF
can be safely and successeful achieved via a minimally invasive chest wall
incision.
Accordingly, a phase I, dose escalating, open-label clinical study to
determine the safety and bioactivity of direct myocardial gene tranfer of
naked plasmid DNA encoding VEGF was undertaken (56). Gene transfer was the
sole therapy, performed without concomitant angioplasty, stenting or bypass
graft, in patients with symptomatic myocardial ischemia. All the 16 enrolled
patients experienced marked symptomatic improvement and objective evidence of
improved myocardial perfusion.
At the latest follow-up of 90 days, 6 of 11 patients were free of
angina. Thirteen out of 14 patients showed evidence of reduced ischemia by
SPECT-sestamibi myocardial perfusion. Coronary angiography showed evidence of
new collateral vessel development in 12/13 patients at 60 day
follow-up.
This early clinical experience, although encouraging, leaves several
issues unresolved. Optimizing the anatomic site, number, and dose of
intramyocardial injections will require further investigation.
The strategy of gene therapy alone administered via a mini-thoracotomy
does not permit randomization against placebo (untreated controls).
We anticipate that incorporation of a lacebo group as well a clinical
testing of alternative dosing regimens — including multiple treatments — will
be addressed upon availability of a catheter-based systems for reliable
percutaneous myocardial gene delivery.
Such systems are currently under investigation in several
laboratories.
Tonino Bombardini
Servizio Tecnologie Biomediche
Policlinico S.Orsola - Bologna
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