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Therapeutic angiogenesis: Translating experimental concepts to medically relevant goals
Vascular Pharmacology 45 (2006) 334 – 339 www.elsevier.com/locate/vph
Therapeutic angiogenesis: Translating experimental concepts to medically relevant goals Costanza Emanueli a,⁎, Paolo Madeddu a,b,⁎ a
Experimental Cardiovascular Medicine, Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW Bristol BS2 8HW, United Kingdom b Multimedica Research Institute, Via Fantoli, Milan, Italy Received 5 August 2006; accepted 5 August 2006
Abstract Angiogenesis is central to many physiological and pathological phenomena. In physiological angiogenesis, new vessels are well shaped and their growth is finely tuned to match the metabolic needs of tributary tissues. Accordingly, neovascularization is activated by physical exercise and destabilized by non-use. In contrast, pathological blood vessels that are observed in retinal neovascularization, cancer or in ischemic tissues are leaky, irregularly shaped, and tend to form arterial–venous fistulae. A great deal of attention is focused on new approaches for medical manipulation of vascular growth. These methods are aimed at facilitating the reperfusion of ischemic tissues or eradicating pathological vasculature. In this position paper, we challenge the rationale of therapeutic angiogenesis for the cure of myocardial and peripheral ischemia. Therapeutic angiogenesis aims at combating the insufficiency of, or insensitivity to angiogenic factors in the setting of atherosclerotic-induced arterial occlusion. However, clinical evidence indicates that such a defect is not common among patients with ischemic disease, as a whole. Genetic and environmental factors could account for the great heterogeneity in the expression of the master angiogenic factors. Future improvements in the strategy would require the introduction of in vitro assays and in vivo imaging systems for assessing human angiogenesis. Finally, the promise is to find individualized angiogenesis-based therapies for a genuine cure of ischemia and prevention of organ failure. © 2006 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Neovascularization; Myocardial and peripheral ischemia
1. Introduction Tissues depend on oxygen and nutrients for their metabolic needs. Because there is a threshold limit for the diffusion of these elements, vessels need to grow whenever cells increase in number or size. Accordingly, muscular hypertrophy is associated to physiologic neovascularization, while vessels tend to regress under conditions of non-use (Sandri et al., 2005). The healing of damaged tissues represents one prototypical situation in which the rapid formation of new vessels is urgently required. However, the process is not invariably working for health ⁎ Corresponding authors. Experimental Cardiovascular Medicine Bristol Heart Institute, University of Bristol Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW Bristol BS2 8HW, United Kingdom. Tel.: +44 117 928 3582; fax: +44 117 928 3581. E-mail addresses: [email protected] (C. Emanueli), [email protected] (P. Madeddu). 1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2006.08.012
benefit (Fig. 1). In fact, under pathological circumstances, excessive or disordered vascular growth can facilitate fluid extravasation and hemorrhage, which constitute major causes of retinal damage and blindness in diabetic patients. Furthermore, tumor cells use leaky neovessels to spread and metastasize (Jain and Munn, 2000). The other way round, defective neoangiogenesis may jeopardise the healing of ischemic tissues. The rationale behind the anti-angiogenic therapy for the cure of cancer or retinopathy is that if an angiogenic factor and/or its receptors are over-expressed, then suppressing pathological vessels could arrest tumoral growth in synergy with chemotherapy. This led to the construction of vascular endothelial growth factor (VEGF)-based new drugs such as Lucentis, a Fab humanized monoclonal anti-VEGF antibody (Husain et al., 2005) or Macugen, an anti-VEGF aptamer (Rakic et al., 2005). A novel corollary strategy envisions to transform the leaking cancerous vessels into stable and mature vessels.
C. Emanueli, P. Madeddu / Vascular Pharmacology 45 (2006) 334–339
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Fig. 1. Graphic illustrates the central role of angiogenesis in physiological and pathological processes. Medical manipulation of angiogenesis enables us to control or modify the course of ischemic or cancer disease.
The other way round, in patients with limb ischemia, supply side of angiogenic factors is thought to benefit tissue repair by compensating a putative endogenous deficit in production/ release (Fig. 2). More recently, transplantation of progenitor vascular cells has emerged as a powerful method to provide the substrate for revascularization and regenerate the injured heart (Laflamme and Murry, 2005). These concepts were initially tested in experimental animal models and are now under scrutiny in the clinic. Completed angiogenesis clinical trials in patients with myocardial or peripheral ischemia did not evidence therapeutic efficacy, except for the accomplishment of soft end-points (Losordo and Dimmeler, 2004a,b; Yla-Herttuala et al., 2004). None of these agents tested so far has been approved for clinical use. Trials using autologous progenitor cells are too preliminary and lack of adequate controls to allow drawing strong conclusions regarding efficacy (Losordo and Dimmeler, 2004a,b). The present position paper aims at challenging the rationale of therapeutic angiogenesis. It also suggests possible solutions
Fig. 2. The cartoon graph illustrates the methods currently used for revascularization of obstructed arteries and the alternatives offered by biological revascularization using the delivery of vascular growth factors (GF), endothelial progenitor cells, or anti-apoptotic agents that impede the too rapid destabilization of neovessels.
to the problems that impede the medical translation of a potentially formidable concept. 1.1. Angiogenesis clinical trials Therapeutic angiogenesis has been initially validated in experimental animal models and then rapidly tested in man. Although the initial series of non-blinded, uncontrolled studies reported spectacular results, the conclusions of subsequent double-blinded, randomized, controlled trials generate, at the most, a prudent optimism. These trials used VEGFs or fibroblast growth factors (FGFs), which were delivered as recombinant proteins or by gene transfer (Henry et al., 2003; Simons et al., 2002; Lederman et al., 2002; Grines et al., 2002; Hedman et al., 2003; Makinen et al., 2002; Stewart et al., 2002; Rajagopalan et al., 2003). In the Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis (VIVA) trial, intracoronary or intravenous infusions of recombinant VEGF did not meet primary endpoints based on exercise tolerance test (ETT). Similarly, the FGF Initiating RevaScularization Trial (FIRST), using recombinant FGF-2, resulted in improved ETT at 90 days, but showed no efficacy at 180 days. At variance, the same factor showed efficacy in a secondary intention-to-treat analysis (the TRAFFIC, Therapeutic Angiogenesis with Recombinant Fibroblast growth Factor-2 for Intermittent Claudication). The Angiogenic GENe Therapy (AGENT) trial, which consisted in a single intracoronary administration of FGF4-encoding adenovirus (Ad-FGF4), demonstrated the greatest improvement in patients with more severe symptoms or with lower titres of antiadenovirus antibodies. In the KAT (Kuopip angiogenesis), intracoronary VEGF165 gene transfer at the occasion of coronary angioplasty improved myocardial perfusion as evaluated by angiography. Similarly, catheter-mediated VEGF gene therapy increased limb muscle vascularity in patients with limb ischemia. Tissue specific effects were evidenced when comparing the REVASC and RAVE trials (Regional Angiogenesis with Vascular Endothelial growth factor in peripheral arterial disease), both using a VEGF121 adenovirus, with the former showing improved electrocardiogram during ETT, whereas the latter showing no amelioration of clinical symptoms.
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1.2. Prospects for improvement The pioneering idea was that atherosclerosis might be associated with specific deficit in growth factor statement. This credence is contradicted by the huge heterogeneity in the expression levels among patients with critical arterial obstructions (Slevin et al., 2000; Porcu et al., 2002, 2004 Heeschen et al., 2003). For instance, we found that there is no correlation between the severity of carotid artery stenosis and the circulating levels of angiogenic factors (Fig. 3A). Approximately, one third of these patients showed VEGF-A levels below the levels of normal distribution, but the low VEGF-A subgroup was equally distributed among all the categories of stenosis (Porcu et al.,
2004). Furthermore, the low VEGF-A phenotype was associated with normal or upregulated b-FGF levels. Only circulating tissue kallikrein (TK) was found to be increasingly augmented with the degree of arterial obstruction and correlated with the number of angiographically visible collaterals (Fig. 3B). TK is synthesized in the kidney and arteries and exerts a potent angiogenic action (Emanueli et al., 2000, 2001), through a PI3 kinase–Akt–eNOS pathway, but independent of VEGF-A (Emanueli et al., 2004). Individual difference in expression levels of kallikrein might be inherited or environmentally determined. Recently, a missense polymorphism in exon 3 of the TK gene was recognized, which changes an active site arginine at position 53 for a histidine (R53H), resulting in a major loss of kallikrein activity in vitro and
Fig. 3. (A) Circulating levels of VEGF-A, bFGF, CPR, and TK in patients with various degrees of carotid stenosis. The green shadow represents the range of normal distribution in healthy volunteers. Each dot represents one single patient. Full dots represent patients with bilateral carotid stenosis. (B) Correlation between TK levels and angiographically visible collaterals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in vivo (Azizi et al., 2005a,b). The 53H allele is found with a frequency of 0.03 in Caucasians, and approximately 5% to 7% of the population is 53H heterozygous. The partial genetic deficiency in TK activity in R53H heterozygous subjects is associated with an inward remodeling of large muscular arteries which is not adapted to a chronic increase in wall shear stress, indicating a new form of arterial dysfunction (Azizi et al., 2005a,b). These data underline the possible role of TK in arterial function and remodeling. Whether atherosclerotic 53H heterozygous patients could be the best candidates for gene therapy with pro-angiogenic TK remains to be elucidated. Similarly, polymorphisms of VEGFs and VEGF receptors might be associated with vascular pathologies and abnormal responsiveness to angiogenic stimulation. Altogether, these results suggest the possible utility of applying a pharmacogenomic approach to the angiogenesis field. 1.3. Introducing new in vitro assays and imaging systems of human angiogenesis Translation of therapeutic angiogenesis into valid medical applications has been hindered by the lack of assays suitable for studying human angiogenesis. We are presently testing the validity of an adaptation of the rat arterial ring bioassay to human vascular explants. This ex vivo culture system permits to evaluate the effect of added angiogenic or anti-angiogenic factors on the capillary sprouting from the adventitial side of the artery. The model has additional features that make it very useful for screening of therapeutic agents: (a) Because cells of the artery outgrowth have not been modified by repeated passages in culture, microvessels developed in this system are indistinguishable from microvessels formed during angiogenesis in vivo; (b) The effect of angiogenesis agonists can be measured in the absence of serum which may otherwise bind, inactivate or simulate the action of the substances being tested; (c) The model can be used to study not only angiogenesis but also vascular regression because neovessels are eventually reabsorbed during the third week of culture. Validation of assays for tracking the fate of injected angiogenic factors may lead to significant improvements in the logistics of angiogenesis gene therapy. We used an immunoassay for tracking gene transfer of human TK in mice. Following intramuscular or intramyocardial injection of TK, the expression of the human transgene in myocytes was evaluated by immunohistochemistry, while an ELISA was used to evaluate the spill-over from infected tissue into the circulation (Emanueli et al., 2004). Myofibers expressed transgenic protein in variable amounts that in some cells appeared as small dots, whereas in others it was associated with the cell membrane. No immunoreactivity was observed in muscles injected with saline or when anti-human TK antibody was pre-absorbed with the purified human enzyme or replaced by non-immune serum. Furthermore, immunoreactive TK was recognized in the circulation for a limited time as compared with the duration of transgene expression in muscles. Alternatively, the pharmacokinetics of gene therapy can be followed by detecting luciferase expression with a bioluminescence camera.
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Angiography is used to visualize collateral vessels but complexity and multiple potential sources of the collaterals and the lack of sufficient radiographic spatial resolution limit this method's ability to accurately measure collateral blood flow. One special problem of X-ray angiography consists of its invasiveness. In addition, traditional angiography provides 2-D projection images and exposes to the risk for allergic reaction or kidney toxicity due to the use of iodinated contrast agents. CT scan is a promising approach for 3D anatomic study of neovascularization development after ischemia, albeit spatial resolution is still a limiting parameter. Magnetic resonance imaging (MRI) is a rapidly gaining importance for regional blood flow assessments, as well as assessments of blood vessel function and integrity. High spatial resolution, contrast-enhanced MRI, with first-pass gadolinium-based contrast agent, can be used to visualize arteries and to obtain regional and muscle-specific perfusion measures. Both methods could provide, in a near future, a number of data points to help researchers understand and quantitatively measure lower extremity perfusion. 1.4. Drug-related factors Major bottlenecks of current angiogenesis therapy are related to insufficient or undetermined dosing and unresponsiveness to specific growth factors. It is highly improbable that monotherapy can resolve the complex situation of chronic atherosclerosis, especially if the strategy is applied empirically without prior phenotyping. Furthermore, factors like VEGF-A are difficult to be used for therapeutic applications, owing to their short half-life and peculiar pharmacokinetics. Local bolus injection of VEGF-A, as recombinant polypeptide or encoding gene, results in transient elevation of the growth factor and formation of capillaries that rapidly regress. Likewise, in transgenic animals, conditionally switching on VEGF-A expression for a short time resulted in the formation of leaky and irregularly shaped vessels that rapidly disappear after cessation of VEGF-A stimulation (Dor et al., 2002). At first glance, the most obvious solution to secure the persistence of newly formed vasculature would require sequential/cyclic treatments. However, these treatments may not be amenable to internal organs, including the ischemic heart. Furthermore, technical hurdles in monitoring ongoing revascularization make problematic the assessment of optimal dosage. As mentioned above, some of these problems can be solved by taking advantage of pharmacogenomics or ex vivo angigenesis assays. Other possible solutions are illustrated in Fig. 4. Gene transfer is seemingly more suitable for ensuring long-term therapeutic effects after a single application. Furthermore, devises enabling controlled release could be considered for persistent expression of the therapeutic agent. One traditional approach in therapeutics consists of considering drug combination for nonresponders to monotherapy. The combination of 2 or more growth factors that act on different mechanisms of neoangiogenesis may represent a solution to clinical failure of single agents. Once decided for a combinatory approach, the next question is “with which ingredients”. A possible strategy contemplates the use of agents acting through complementary mechanisms. For instance, polymeric systems
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1999) or HIV-related neuropathy (Schifitto et al., 2001). A large-scale phase III clinical trail of 1019 patients randomized to receive either rhNGF or placebo for 48 weeks failed to confirm the earlier indications of efficacy (Apfel et al., 2000). Among the explanations offered for the discrepancy between the two sets of trials was a robust placebo effect, inadequate dosage, different study populations, and changes to the formulation of rhNGF for the phase III trail. As a result of the phase III outcome, patent holder decided to suspend further development of rhNGF. At the best of our knowledge, no clinical trial has been conducted using TK. 2. Conclusions Fig. 4. Drug-related factors that impede the success of angiogenesis therapy (light blue) and possible solutions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for dual delivery of VEGF-A and platelet-derived growth factor-BB (PDGF-BB) are reportedly more effective than monotherapy with either factor in promoting the formation of mature vessels (Richardson et al., 2001). Other synergic combinations comprise VEGF-A and angiopoietin-1 (Ang-1) or FGF-4 (Chae et al., 2000; Rissanen et al., 2003). Importantly, organ- and context-dependent effects should be considered as Ang-1 stimulates angiogenesis in skin and ischemic limbs (Visconti et al., 2002), while suppressing neovascularization in the heart (Visconti et al., 2002). Finally, it could be advantageous combining VEGF-A with angiogenesis activators that are VEGF-independent, such as tissue kallikrein (Emanueli et al., 2004) or proteins of the Wnt family (Doufourq et al., 2002). An alternative to combinations contemplates the use of transcription factors lying upstream to multiple mechanisms of the angiogenic cascade. For instance, hypoxia-inducible factor1alpha (HIF-1α) activates the transcription of VEGFA, VEGF receptor FLT-1, insulin-like growth factor-2 (IGF-2) and angiopoietin-2 (Ang-2) (Vincent et al., 2002; Elson et al., 2001). Ad-mediated HIF-1α gene therapy is currently under clinical testing for myocardial ischemia. Among other candidate therapeutic transcription factors, the early growth response-1 (Egr-1) stimulates angiogenesis and tissue healing through transcriptional activation of several growth factors including FGF-2 (Fahmy et al., 2003). The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. Therefore, Prox1 represents a potential target for lymphatic reprogramming. Pleiotropic factors, including nerve growth factor or TK, acting through multiple pathways offer unique opportunities owing to their ability to stimulate vascular, neural and muscular repairs (Emanueli et al., 2004, 2002). In clinical studies, NGF reportedly improves the healing of pressure and vasculitic ulcers. The healing effect was characterized by blood vessel formation and re-establishment of pain sensation, which were completely absent before treatment (Bernabei et al., 1999; Tuveri et al., 2000; Landi et al., 2003). Two sets of phase II clinical trails suggested that recombinant human NGF (rhNGF) may ameliorate the symptoms associated with diabetic (Apfel,
Once considered an alternative to traditional revascularization in no-option patients, angiogenesis therapy is opening unprecedented opportunities for life-threatening diseases. However, several technical hurdles need to be addressed for full exploitation of this promising strategy. This will require joining expertise in vascular biology, genomics, pharmacogenomics, and cardiovascular biology. The integration of these disciplines will open new avenues to regenerative medicine. Acknowledgments This research has been supported in part by a FIRB project grant from the Italian Minister of Scientific Research and by a Juvenile Diabetes Foundation grant. This paper is also dedicated to the memory of Paolo Madeddu's father, Dr. Andrea Madeddu, a practitioner who died of cancer in 2005. References Apfel, S.C., 1999. Neurotrophic factors in the therapy of diabetic neuropathy. Am. J. Med. 107, 34S–42S. Apfel, F.C., et al., rhNGF Clinical Investigator Group, 2000. Efficacy and safety of recombinant human nerve growth factor in patients with diabetic polyneuropathy: a randomized controlled trial. JAMA 284, 2215–2221. Azizi, M., et al., 2005a. Arterial and renal consequences of partial genetic deficiency in tissue kallikrein activity in humans. J. Clin. Invest. 115, 780–787. Azizi, M., et al., 2005b. Polymorphism of kallikrein gene and abnormalities of the endothelial function. Med. Sci. 21, 584–585. Bernabei, R., et al., 1999. Effect of topical application of nerve-growth factor on pressure ulcers. Lancet 354, 307. Chae, J.K., et al., 2000. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler. Thromb. Vasc. Biol. 20, 2573–2578. Dor, Y., et al., 2002. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J. 21, 1939–1947. Doufourq, P., et al., 2002. FrzA, a secreted frizzled related protein, induced angiogenic response. Circulation 106, 3097–3103. Elson, D.A., et al., 2001. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor1alpha. Genes Dev. 15, 2520–2532. Emanueli, C., et al., 2000. Adenovirus-mediated human tissue kallikrein gene delivery induces angiogenesis in normoperfused skeletal muscle. Arterioscler. Thromb. Vasc. Biol. 20, 2379–2385. Emanueli, C., et al., 2001. Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia. Circulation 103, 125–132. Emanueli, C., et al., 2002. Nerve growth factor promotes angiogenesis and arteriogenesis in ischemic hindlimbs. Circulation 106, 2257–2262.
C. Emanueli, P. Madeddu / Vascular Pharmacology 45 (2006) 334–339 Emanueli, C., et al., 2004. Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer. Circulation 110, 1638–1644. Fahmy, R.G., et al., 2003. Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth. Nat. Med. 9, 1026–1032. Grines, C.L., et al., 2002. Angiogenic GENe Therapy (AGENT) trial in patients with stable angina pectoris. Circulation 105, 1291–1297. Hedman, M., et al., 2003. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation 107, 2677–2683. Heeschen, C., et al., 2003. CAPTURE (c7E3 Anti-Platelet Therapy in Unstable REfractory angina) Investigators. Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes. Circulation 107, 524–530. Henry, T.D., et al., 2003. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation 107, 1359–1365. Husain, D., et al., 2005. Safety and efficacy of intravitreal injection of ranibizumab in combination with veterporfin PDT on experimental choroidal neovascularization in the monkey. Arch. Ophthalmol. 123, 509–515. Jain, R.K., Munn, L.L., 2000. Leaky vessels? Call Ang1! Nat. Med. 6, 131–132. Laflamme, M.A., Murry, C.E., 2005. Regenerating the heart. Nat. Biotechnol. 23, 845–856. Landi, F., et al., 2003. Topical treatment of pressure ulcers with nerve growth factor: a randomized clinical trial. Ann. Intern. Med. 139, 635–641. Losordo, D.W., Dimmeler, S., 2004a. Therapeutic angiogenesis and vasculogenesis for ischemic disease: Part I. Angiogenic cytokines. Circulation 109, 2487–2491. Losordo, D.W., Dimmeler, S., 2004b. Therapeutic angiogenesis and vasculogenesis for ischemic disease: Part II. Cell-based therapy. Circulation 109, 2692–2697. Lederman, R.J., et al., 2002. Therapeutic Angiogenesis with Recombinant Fibroblast growth Factor-2 for Intermittent Claudication (the TRAFFIC study): a randomised trial. Lancet 359, 2053–2058. Makinen, K., et al., 2002. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Molec. Ther. 6, 127–133. Porcu, P., et al., 2002. Reversal of angiogenic growth factor upregulation by revascularization of lower limb ischemia. Circulation 105, 67–72.
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Porcu, P., et al., 2004. Circulating tissue kallikrein levels correlate with severity of carotid atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 24, 1104–1110. Rajagopalan, S., et al., 2003. Regional Angiogenesis with Vascular Endothelial growth factor in peripheral arterial disease: a phase II randomized, doubleblind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 108, 1933–1938. Rakic, J.M., et al., 2005. Pegaptanib and age-related macular degeneration. N. Engl. J. Med. 352, 1720–1721. Richardson, T.P., et al., 2001. Polymeric system for dual growth factor delivery. Nat. Biotechnol. 19, 1029–1034. Rissanen, T.T., et al., 2003. Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model. FASEB J. 17, 100–102. Sandri, M., et al., 2005. Effects of exercise and ischemia on mobilization and functional activation of blood-derived progenitor cells in patients with ischemic syndromes results of three randomized studies. Circulation 111, 3391–3399. Schifitto, G., et al., 2001. Long-term treatment with recombinant nerve growth factor HIV-associated sensory neuropathy. Neurology 57, 1313–1316. Simons, M., et al., 2002. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor: 2. Double-blind, randomized, controlled clinical trial. Circulation 105, 788–793. Slevin, M., et al., 2000. Serial measurement of vascular endothelial growth factor and transforming growth factor-beta1 in serum of patients with acute ischemic stroke. Stroke 31, 1863–1870. Stewart, D.J., et al., 2002. A phase 2, randomized, multicenter, 26-week study to assess the efficacy and safety of BIOBYPASS (AdGVVEGF121) delivered through minimally invasive surgery versus maximum medical treatment in patients with severe angina, advanced coronary artery stenosis, and no options for revascularization. Circulation 106, 23–26. Tuveri, M., et al., 2000. NGF, a useful tool in the treatment of chronic vasculitic ulcers in rheumatoid arthritis. Lancet 356, 1739–1740. Vincent, K.A., et al., 2002. Harnessing the response to tissue hypoxia: HIF-1 alpha and therapeutic angiogenesis. Trends Cardiovasc. Med. 12, 362–367. Visconti, R.P., et al., 2002. Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF). Proc. Natl. Acad. Sci. U. S. A. 99, 8219–8224. Yla-Herttuala, S., et al., 2004. Gene therapy for ischemic cardiovascular diseases: some lessons learned from the first clinical trials. Trends Cardiovasc. Med. 14, 295–300.
Therapeutic angiogenesis: Translating experimental concepts to medically relevant goals Costanza Emanueli a,⁎, Paolo Madeddu a,b,⁎ a
Experimental Cardiovascular Medicine, Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW Bristol BS2 8HW, United Kingdom b Multimedica Research Institute, Via Fantoli, Milan, Italy Received 5 August 2006; accepted 5 August 2006
Abstract Angiogenesis is central to many physiological and pathological phenomena. In physiological angiogenesis, new vessels are well shaped and their growth is finely tuned to match the metabolic needs of tributary tissues. Accordingly, neovascularization is activated by physical exercise and destabilized by non-use. In contrast, pathological blood vessels that are observed in retinal neovascularization, cancer or in ischemic tissues are leaky, irregularly shaped, and tend to form arterial–venous fistulae. A great deal of attention is focused on new approaches for medical manipulation of vascular growth. These methods are aimed at facilitating the reperfusion of ischemic tissues or eradicating pathological vasculature. In this position paper, we challenge the rationale of therapeutic angiogenesis for the cure of myocardial and peripheral ischemia. Therapeutic angiogenesis aims at combating the insufficiency of, or insensitivity to angiogenic factors in the setting of atherosclerotic-induced arterial occlusion. However, clinical evidence indicates that such a defect is not common among patients with ischemic disease, as a whole. Genetic and environmental factors could account for the great heterogeneity in the expression of the master angiogenic factors. Future improvements in the strategy would require the introduction of in vitro assays and in vivo imaging systems for assessing human angiogenesis. Finally, the promise is to find individualized angiogenesis-based therapies for a genuine cure of ischemia and prevention of organ failure. © 2006 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Neovascularization; Myocardial and peripheral ischemia
1. Introduction Tissues depend on oxygen and nutrients for their metabolic needs. Because there is a threshold limit for the diffusion of these elements, vessels need to grow whenever cells increase in number or size. Accordingly, muscular hypertrophy is associated to physiologic neovascularization, while vessels tend to regress under conditions of non-use (Sandri et al., 2005). The healing of damaged tissues represents one prototypical situation in which the rapid formation of new vessels is urgently required. However, the process is not invariably working for health ⁎ Corresponding authors. Experimental Cardiovascular Medicine Bristol Heart Institute, University of Bristol Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW Bristol BS2 8HW, United Kingdom. Tel.: +44 117 928 3582; fax: +44 117 928 3581. E-mail addresses: [email protected] (C. Emanueli), [email protected] (P. Madeddu). 1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2006.08.012
benefit (Fig. 1). In fact, under pathological circumstances, excessive or disordered vascular growth can facilitate fluid extravasation and hemorrhage, which constitute major causes of retinal damage and blindness in diabetic patients. Furthermore, tumor cells use leaky neovessels to spread and metastasize (Jain and Munn, 2000). The other way round, defective neoangiogenesis may jeopardise the healing of ischemic tissues. The rationale behind the anti-angiogenic therapy for the cure of cancer or retinopathy is that if an angiogenic factor and/or its receptors are over-expressed, then suppressing pathological vessels could arrest tumoral growth in synergy with chemotherapy. This led to the construction of vascular endothelial growth factor (VEGF)-based new drugs such as Lucentis, a Fab humanized monoclonal anti-VEGF antibody (Husain et al., 2005) or Macugen, an anti-VEGF aptamer (Rakic et al., 2005). A novel corollary strategy envisions to transform the leaking cancerous vessels into stable and mature vessels.
C. Emanueli, P. Madeddu / Vascular Pharmacology 45 (2006) 334–339
335
Fig. 1. Graphic illustrates the central role of angiogenesis in physiological and pathological processes. Medical manipulation of angiogenesis enables us to control or modify the course of ischemic or cancer disease.
The other way round, in patients with limb ischemia, supply side of angiogenic factors is thought to benefit tissue repair by compensating a putative endogenous deficit in production/ release (Fig. 2). More recently, transplantation of progenitor vascular cells has emerged as a powerful method to provide the substrate for revascularization and regenerate the injured heart (Laflamme and Murry, 2005). These concepts were initially tested in experimental animal models and are now under scrutiny in the clinic. Completed angiogenesis clinical trials in patients with myocardial or peripheral ischemia did not evidence therapeutic efficacy, except for the accomplishment of soft end-points (Losordo and Dimmeler, 2004a,b; Yla-Herttuala et al., 2004). None of these agents tested so far has been approved for clinical use. Trials using autologous progenitor cells are too preliminary and lack of adequate controls to allow drawing strong conclusions regarding efficacy (Losordo and Dimmeler, 2004a,b). The present position paper aims at challenging the rationale of therapeutic angiogenesis. It also suggests possible solutions
Fig. 2. The cartoon graph illustrates the methods currently used for revascularization of obstructed arteries and the alternatives offered by biological revascularization using the delivery of vascular growth factors (GF), endothelial progenitor cells, or anti-apoptotic agents that impede the too rapid destabilization of neovessels.
to the problems that impede the medical translation of a potentially formidable concept. 1.1. Angiogenesis clinical trials Therapeutic angiogenesis has been initially validated in experimental animal models and then rapidly tested in man. Although the initial series of non-blinded, uncontrolled studies reported spectacular results, the conclusions of subsequent double-blinded, randomized, controlled trials generate, at the most, a prudent optimism. These trials used VEGFs or fibroblast growth factors (FGFs), which were delivered as recombinant proteins or by gene transfer (Henry et al., 2003; Simons et al., 2002; Lederman et al., 2002; Grines et al., 2002; Hedman et al., 2003; Makinen et al., 2002; Stewart et al., 2002; Rajagopalan et al., 2003). In the Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis (VIVA) trial, intracoronary or intravenous infusions of recombinant VEGF did not meet primary endpoints based on exercise tolerance test (ETT). Similarly, the FGF Initiating RevaScularization Trial (FIRST), using recombinant FGF-2, resulted in improved ETT at 90 days, but showed no efficacy at 180 days. At variance, the same factor showed efficacy in a secondary intention-to-treat analysis (the TRAFFIC, Therapeutic Angiogenesis with Recombinant Fibroblast growth Factor-2 for Intermittent Claudication). The Angiogenic GENe Therapy (AGENT) trial, which consisted in a single intracoronary administration of FGF4-encoding adenovirus (Ad-FGF4), demonstrated the greatest improvement in patients with more severe symptoms or with lower titres of antiadenovirus antibodies. In the KAT (Kuopip angiogenesis), intracoronary VEGF165 gene transfer at the occasion of coronary angioplasty improved myocardial perfusion as evaluated by angiography. Similarly, catheter-mediated VEGF gene therapy increased limb muscle vascularity in patients with limb ischemia. Tissue specific effects were evidenced when comparing the REVASC and RAVE trials (Regional Angiogenesis with Vascular Endothelial growth factor in peripheral arterial disease), both using a VEGF121 adenovirus, with the former showing improved electrocardiogram during ETT, whereas the latter showing no amelioration of clinical symptoms.
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1.2. Prospects for improvement The pioneering idea was that atherosclerosis might be associated with specific deficit in growth factor statement. This credence is contradicted by the huge heterogeneity in the expression levels among patients with critical arterial obstructions (Slevin et al., 2000; Porcu et al., 2002, 2004 Heeschen et al., 2003). For instance, we found that there is no correlation between the severity of carotid artery stenosis and the circulating levels of angiogenic factors (Fig. 3A). Approximately, one third of these patients showed VEGF-A levels below the levels of normal distribution, but the low VEGF-A subgroup was equally distributed among all the categories of stenosis (Porcu et al.,
2004). Furthermore, the low VEGF-A phenotype was associated with normal or upregulated b-FGF levels. Only circulating tissue kallikrein (TK) was found to be increasingly augmented with the degree of arterial obstruction and correlated with the number of angiographically visible collaterals (Fig. 3B). TK is synthesized in the kidney and arteries and exerts a potent angiogenic action (Emanueli et al., 2000, 2001), through a PI3 kinase–Akt–eNOS pathway, but independent of VEGF-A (Emanueli et al., 2004). Individual difference in expression levels of kallikrein might be inherited or environmentally determined. Recently, a missense polymorphism in exon 3 of the TK gene was recognized, which changes an active site arginine at position 53 for a histidine (R53H), resulting in a major loss of kallikrein activity in vitro and
Fig. 3. (A) Circulating levels of VEGF-A, bFGF, CPR, and TK in patients with various degrees of carotid stenosis. The green shadow represents the range of normal distribution in healthy volunteers. Each dot represents one single patient. Full dots represent patients with bilateral carotid stenosis. (B) Correlation between TK levels and angiographically visible collaterals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in vivo (Azizi et al., 2005a,b). The 53H allele is found with a frequency of 0.03 in Caucasians, and approximately 5% to 7% of the population is 53H heterozygous. The partial genetic deficiency in TK activity in R53H heterozygous subjects is associated with an inward remodeling of large muscular arteries which is not adapted to a chronic increase in wall shear stress, indicating a new form of arterial dysfunction (Azizi et al., 2005a,b). These data underline the possible role of TK in arterial function and remodeling. Whether atherosclerotic 53H heterozygous patients could be the best candidates for gene therapy with pro-angiogenic TK remains to be elucidated. Similarly, polymorphisms of VEGFs and VEGF receptors might be associated with vascular pathologies and abnormal responsiveness to angiogenic stimulation. Altogether, these results suggest the possible utility of applying a pharmacogenomic approach to the angiogenesis field. 1.3. Introducing new in vitro assays and imaging systems of human angiogenesis Translation of therapeutic angiogenesis into valid medical applications has been hindered by the lack of assays suitable for studying human angiogenesis. We are presently testing the validity of an adaptation of the rat arterial ring bioassay to human vascular explants. This ex vivo culture system permits to evaluate the effect of added angiogenic or anti-angiogenic factors on the capillary sprouting from the adventitial side of the artery. The model has additional features that make it very useful for screening of therapeutic agents: (a) Because cells of the artery outgrowth have not been modified by repeated passages in culture, microvessels developed in this system are indistinguishable from microvessels formed during angiogenesis in vivo; (b) The effect of angiogenesis agonists can be measured in the absence of serum which may otherwise bind, inactivate or simulate the action of the substances being tested; (c) The model can be used to study not only angiogenesis but also vascular regression because neovessels are eventually reabsorbed during the third week of culture. Validation of assays for tracking the fate of injected angiogenic factors may lead to significant improvements in the logistics of angiogenesis gene therapy. We used an immunoassay for tracking gene transfer of human TK in mice. Following intramuscular or intramyocardial injection of TK, the expression of the human transgene in myocytes was evaluated by immunohistochemistry, while an ELISA was used to evaluate the spill-over from infected tissue into the circulation (Emanueli et al., 2004). Myofibers expressed transgenic protein in variable amounts that in some cells appeared as small dots, whereas in others it was associated with the cell membrane. No immunoreactivity was observed in muscles injected with saline or when anti-human TK antibody was pre-absorbed with the purified human enzyme or replaced by non-immune serum. Furthermore, immunoreactive TK was recognized in the circulation for a limited time as compared with the duration of transgene expression in muscles. Alternatively, the pharmacokinetics of gene therapy can be followed by detecting luciferase expression with a bioluminescence camera.
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Angiography is used to visualize collateral vessels but complexity and multiple potential sources of the collaterals and the lack of sufficient radiographic spatial resolution limit this method's ability to accurately measure collateral blood flow. One special problem of X-ray angiography consists of its invasiveness. In addition, traditional angiography provides 2-D projection images and exposes to the risk for allergic reaction or kidney toxicity due to the use of iodinated contrast agents. CT scan is a promising approach for 3D anatomic study of neovascularization development after ischemia, albeit spatial resolution is still a limiting parameter. Magnetic resonance imaging (MRI) is a rapidly gaining importance for regional blood flow assessments, as well as assessments of blood vessel function and integrity. High spatial resolution, contrast-enhanced MRI, with first-pass gadolinium-based contrast agent, can be used to visualize arteries and to obtain regional and muscle-specific perfusion measures. Both methods could provide, in a near future, a number of data points to help researchers understand and quantitatively measure lower extremity perfusion. 1.4. Drug-related factors Major bottlenecks of current angiogenesis therapy are related to insufficient or undetermined dosing and unresponsiveness to specific growth factors. It is highly improbable that monotherapy can resolve the complex situation of chronic atherosclerosis, especially if the strategy is applied empirically without prior phenotyping. Furthermore, factors like VEGF-A are difficult to be used for therapeutic applications, owing to their short half-life and peculiar pharmacokinetics. Local bolus injection of VEGF-A, as recombinant polypeptide or encoding gene, results in transient elevation of the growth factor and formation of capillaries that rapidly regress. Likewise, in transgenic animals, conditionally switching on VEGF-A expression for a short time resulted in the formation of leaky and irregularly shaped vessels that rapidly disappear after cessation of VEGF-A stimulation (Dor et al., 2002). At first glance, the most obvious solution to secure the persistence of newly formed vasculature would require sequential/cyclic treatments. However, these treatments may not be amenable to internal organs, including the ischemic heart. Furthermore, technical hurdles in monitoring ongoing revascularization make problematic the assessment of optimal dosage. As mentioned above, some of these problems can be solved by taking advantage of pharmacogenomics or ex vivo angigenesis assays. Other possible solutions are illustrated in Fig. 4. Gene transfer is seemingly more suitable for ensuring long-term therapeutic effects after a single application. Furthermore, devises enabling controlled release could be considered for persistent expression of the therapeutic agent. One traditional approach in therapeutics consists of considering drug combination for nonresponders to monotherapy. The combination of 2 or more growth factors that act on different mechanisms of neoangiogenesis may represent a solution to clinical failure of single agents. Once decided for a combinatory approach, the next question is “with which ingredients”. A possible strategy contemplates the use of agents acting through complementary mechanisms. For instance, polymeric systems
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1999) or HIV-related neuropathy (Schifitto et al., 2001). A large-scale phase III clinical trail of 1019 patients randomized to receive either rhNGF or placebo for 48 weeks failed to confirm the earlier indications of efficacy (Apfel et al., 2000). Among the explanations offered for the discrepancy between the two sets of trials was a robust placebo effect, inadequate dosage, different study populations, and changes to the formulation of rhNGF for the phase III trail. As a result of the phase III outcome, patent holder decided to suspend further development of rhNGF. At the best of our knowledge, no clinical trial has been conducted using TK. 2. Conclusions Fig. 4. Drug-related factors that impede the success of angiogenesis therapy (light blue) and possible solutions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for dual delivery of VEGF-A and platelet-derived growth factor-BB (PDGF-BB) are reportedly more effective than monotherapy with either factor in promoting the formation of mature vessels (Richardson et al., 2001). Other synergic combinations comprise VEGF-A and angiopoietin-1 (Ang-1) or FGF-4 (Chae et al., 2000; Rissanen et al., 2003). Importantly, organ- and context-dependent effects should be considered as Ang-1 stimulates angiogenesis in skin and ischemic limbs (Visconti et al., 2002), while suppressing neovascularization in the heart (Visconti et al., 2002). Finally, it could be advantageous combining VEGF-A with angiogenesis activators that are VEGF-independent, such as tissue kallikrein (Emanueli et al., 2004) or proteins of the Wnt family (Doufourq et al., 2002). An alternative to combinations contemplates the use of transcription factors lying upstream to multiple mechanisms of the angiogenic cascade. For instance, hypoxia-inducible factor1alpha (HIF-1α) activates the transcription of VEGFA, VEGF receptor FLT-1, insulin-like growth factor-2 (IGF-2) and angiopoietin-2 (Ang-2) (Vincent et al., 2002; Elson et al., 2001). Ad-mediated HIF-1α gene therapy is currently under clinical testing for myocardial ischemia. Among other candidate therapeutic transcription factors, the early growth response-1 (Egr-1) stimulates angiogenesis and tissue healing through transcriptional activation of several growth factors including FGF-2 (Fahmy et al., 2003). The homeobox gene Prox1 is expressed in a subpopulation of endothelial cells that, after budding from veins, gives rise to the mammalian lymphatic system. Therefore, Prox1 represents a potential target for lymphatic reprogramming. Pleiotropic factors, including nerve growth factor or TK, acting through multiple pathways offer unique opportunities owing to their ability to stimulate vascular, neural and muscular repairs (Emanueli et al., 2004, 2002). In clinical studies, NGF reportedly improves the healing of pressure and vasculitic ulcers. The healing effect was characterized by blood vessel formation and re-establishment of pain sensation, which were completely absent before treatment (Bernabei et al., 1999; Tuveri et al., 2000; Landi et al., 2003). Two sets of phase II clinical trails suggested that recombinant human NGF (rhNGF) may ameliorate the symptoms associated with diabetic (Apfel,
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