Therapeutic Angiogenesis Gains a Leg to Stand On
Authors: Andreas Rück, Christer Sylvén
Journal: Molecular Therapy (2008)

The field of angiogenic gene therapy has recently been somewhat dormant after an initial wave of enthusiasm fueled by positive animal studies and small, nonblinded clinical studies.1Yla-Herttuala S Alitalo K Gene transfer as a tool to induce therapeutic vascular growth.Nat Med. 2003; 9: 694-701Crossref PubMed Scopus (355) Google Scholar However, few adequately designed double-blind trials have been published thus far. A similar wave of enthusiasm attended the idea of regeneration of the myocardium, induced by homing and transdifferentiation of bone marrow derivatives that exhibited properties of myocardial progenitor cells.2Orlic D Kajstura J Chimenti S Limana F Jakuniuk I Quaini F et al.Mobilized bone marrow cells repair the infarcted heart, improving function and survivaly.Proc Natl Acad Sci USA. 2001; 98: 10344-10349Crossref PubMed Scopus (1951) Google Scholar Clinical studies evaluating the intracoronary or intramyocardial injection of such cells were rapidly initiated on the basis of the transdifferentiation paradigm even before three key reports were published that challenged the concept of transdifferentiation of progenitor cells into cardiomyocytes.3Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 607-608Crossref PubMed Scopus (1922) Google Scholar,4Balsam LB Wagers AJ Christensen JL Kofidis T Weissmann IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1554) Google Scholar,5Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow–derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (917) Google Scholar Consequently, treatment effects observed in such cell therapy trials are now referred to not as arising from cell transdifferentiation but instead as an outcome of “paracrine” effects, without further elaboration about the nature of such effects. In this issue of Molecular Therapy, however, Nikol et al. report the findings of the first phase II randomized placebo-controlled clinical trial to explore the efficacy of angiogenic gene therapy to counteract limb ischemia, a form of peripheral artery disease.6Nikol S Baumgartner I Van Belle E Diehm C Visoná A Capogrossi MC et al.Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia.Mol Ther. 2008; 16: 972-978Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar The Therapeutic Angiogenesis Leg Ischemia Study for the Management of Arteriopathy and Non-Healing Ulcer (TALISMAN) trial enrolled 125 patients with critical limb ischemia that was unsuitable for surgical revascularization and that was associated with nonhealing ischemic ulcers. This is a chronic and progressive disease characterized by severe chronic arterial insufficiency resulting in severe underperfusion of the leg that frequently leads to ischemic ulcers, tissue necrosis, and amputation. The objective of TALISMAN was to investigate the efficacy and safety of intramuscular delivery of the NV1FGF plasmid vector expressing fibroblast growth factor 1 (FGF-1)—used successfully to promote angiogenesis in animal studies7Muhlhauser J Pili R Merrill MJ Maeda H Passaniti A Crystal RG et al.In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor.Hum Gene Ther. 1995; 6: 1457-1465Crossref PubMed Scopus (70) Google Scholar,8Safi J Jr DiPaula AF Jr Riccioni T Kajstura J Ambrosio G Becker LC et al.Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart.Microvasc Res. 1999; 58: 238-249Crossref PubMed Scopus (57) Google Scholar,9Tabata H Silver M Isner JM Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA.Cardiovasc Res. 1997; 35: 470-479Crossref PubMed Scopus (75) Google Scholar—vs. placebo in subjects with critical limb ischemia who were at high risk of amputation. The study was carried out at sites in six European countries (Belgium, France, Germany, Italy, Switzerland, and the United Kingdom). NV1FGF (Centelion, Vitry sur Seine, France) is a pCOR DNA plasmid-based gene delivery system for local expression of FGF-1.10Soubrier F Cameron B Manse B Somarriba S Dubertret C Jaslin G et al.pCOR: a new design of plasmid vectors for nonviral gene therapy.Gene Ther. 1999; 6: 1482-1488Crossref PubMed Scopus (95) Google Scholar The pCOR plasmid backbone has several features that increase biosafety, including absence of an antibiotic resistance gene and lack of potential immunostimulatory sequence motifs found in ColE1 plasmids.11Witzenbichler B Mahfoudi A Soubrier F Le Roux A Branellec D Schultheiss HP et al.Intramuscular gene transfer of fibroblast growth factor-1 using improved pCOR plasmid design stimulates collateral formation in a rabbit ischemic hindlimb model.J Mol Med. 2006; 84: 491-502Crossref PubMed Scopus (16) Google Scholar The study revealed that the risk of major amputation was reduced by as much as half in patients who received the NV1FGF vector in four injections over a period of 6 weeks. Although the trial did not meet its primary end point—ulcer healing—this is an extremely important result in these patients, who lack other therapeutic options. Healing of ischemic wounds has turned out to be a complex process that is difficult to monitor under strict and robust core lab conditions. Limb salvage in this patient population, however, is a clear-cut end point and is critical to keep patients walking and reduce the overall high risk of cardiovascular morbidity and mortality in this patient group—in particular, the risk associated with limb amputation. Since its inception, the concept of therapeutic angiogenesis has continued to evolve at a slow but steady pace. Gene transfer systems have been improved by the availability of better vector technology. As for cardiac angiogenesis, there are three published phase II/III trials—the KAT,12Hedman M Hartikainen J Syvänne M Stjernvall J Hedman A Kivelä A et al.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. 2003; 107: 2677-2683Crossref PubMed Google Scholar AGENT,13Henry TD Grines CL Watkins MW Dib N Barbeau G Moreadith R et al.Effects of Ad5FGF-4 in patients with angina: an analysis of pooled data from the AGENT-3 and AGENT-4 trials.J Am Coll Cardiol. 2007; 50: 1038-1046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar and EUROINJECT ONE14Kastrup J Jørgensen E Rück A Tägil K Glogar D Ruzyllo W et al.Efficacy of direct intramyocardial plasmid VEGF-A165 gene therapy in patients with stable severe angina pectoris: a randomized double-blind placebo-controlled study—the Euroinject One Trial.J Am Coll Cardiol. 2005; 45: 982-988Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar,15Gyöngyösi M Khorsand A Zamini S Sperker W Streblow C Kastrup J et al.NOGA-guided analysis of regional myocardial perfusion abnormalities treated with intramyocardial injections of plasmid encoding VEGF A-165 in patients with chronic myocardial ischemia: subanalysis of the EUROINJECT-ONE Multicenter Double-Blind Randomized Study.Circulation. 2005; 112: I-157-I-165PubMed Google Scholar trials—all of which observed positive treatment effects. In the KAT trial, patients were treated with percutaneous coronary intervention (coronary artery dilatation) and thereafter with adenoviral or plasmid-based vascular endothelial growth factor 165 (VEGF-165) gene transfer. Myocardial perfusion as estimated by scintigraphy 6 months after the gene transfer was found to be significantly better in the group receiving adenoviral gene transfer as compared with baseline. In the AGENT trials, adFGF5 was given by intracoronary injection, and the primary end point was effects on treadmill exercise capacity as an index of improved coronary perfusion. Although no effect was observed in the treadmill test, in a post hoc analysis female patients performed better than those receiving placebo following adFGF5 treatment. These findings provided the basis for an ongoing prospective study, AWARE, which will recruit 300 women with documented coronary artery disease to be treated according to the AGENT protocol: adFGF5 or placebo given by intracoronary injection with follow-up after 6 months and analysis of the effect of treatment on treadmill exercise.16US National Institutes of Health Angiogenesis in Women With Angina Pectoris Who Are Not Candidates for Revascularization (AWARE) <http://www.clinicaltrials.gov/ct/show/NCT000438867>Google Scholar In the EUROINJECT ONE trial, patients were treated with intramyocardial injection of plasmid expressing VEGF-165 or with placebo plasmid. Although only 80 patients were enrolled, treatment effects were observed in the end-point core lab analyses: both myocardial perfusion (primary end point) and local wall motion (secondary end point and estimate of myocardial function) improved in the treatment area. The findings of Nikol et al.6Nikol S Baumgartner I Van Belle E Diehm C Visoná A Capogrossi MC et al.Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia.Mol Ther. 2008; 16: 972-978Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar provide a glimpse of hope for the treatment via gene therapy of patients with peripheral artery disease. This disease has increasing prevalence, high mortality, and no effective pharmacologic treatment in the more advanced stage, critical limb ischemia (TASC II 2007 guidelines17Norgren L Hiatt WR Dormandy JA Nehler MR Harris KA Fowkes FG et al.Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II).Eur J Vasc Endovasc Surg. 2007; 33: S1-S75Abstract Full Text Full Text PDF PubMed Scopus (2474) Google Scholar). It is the fourth completed phase II angiogenesis trial.18Kusumanto YH van Weel V Mulder NH Smit AJ van den Dungen JJ Hooymans JM et al.Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial.Hum Gene Ther. 2006; 17: 683-691Crossref PubMed Scopus (260) Google Scholar,19Makinen K Manninen H Hedman M Matsi P Mussalo H Alhava E et al.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.Mol Ther. 2002; 6: 127-133Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar,20Rajagopalan S Mohler ER 3rd Lederman RJ Mendelsohn FO Saucedo JF Goldman CK et al.Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication.Circulation. 2003; 108: 1933-1938Crossref PubMed Scopus (520) Google Scholar Although the demonstrated improved limb salvage is a milestone, the results should be confirmed in a larger trial. In this regard, we look forward to the results of the recently initiated phase III TAMARIS (Therapeutic Angiogenesis for the Management of Arteriopathy in a Randomized International Study) clinical trial, which involves the transfer of NV1FGF (the same gene and delivery agent used in the TALISMAN trial), which will enroll 490 patients worldwide. In the TAMARIS trial, the primary end point is combined amputation and death. The TAMARIS trial and other studies should answer some important remaining questions. What is the mechanism behind the reduced amputation rate in this patient population? Because the distal blood pressure and oxygen pressure were similar after delivery of both placebo and NV1FGF, one might speculate that the beneficial effect was due not to angiogenesis or arteriogenesis but instead to other FGF-related trophic effects. The possibility of systemic side effects, hinted at by the nonsignificant trends toward increased cardiac events and hematuria in the TALISMAN trial, suggests the need for further study, including follow-up of longer duration. Compared with standards for ordinary drug development—trials in which demonstration of treatment effects requires the recruitment of a large number of patients in phase III clinical trials—the results of therapeutic angiogenesis trials are very promising. At least in the cardiovascular area, the diseases have a high degree of complexity and the gene transfer technology is so demanding that only phase II/III trials with limited numbers of patients have been accomplished. At the present state of knowledge, an effect on secondary end points is also of relevance for the future design of proper trials with robust primary end points. The TALISMAN and TAMARIS trials exemplify this process. In parallel with phase III trials such as AWARE and TAMARIS, further preclinical developments in vector and administration technology will be important.