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