Angiogenesis in a Patient with Ischemic Limb: Induced by Intramuscular Injection of Vascular Endothelial Growth Factor and Fibrin Platform

Abstract

Direct revascularization of critical ischemia of the limb is often unsuccessful due to the anatomic extent and distribution of arterial occlusive disease, and no pharmacologic treatment has proved effective in treating this condition. Patients with ischemic limb may eventually require amputation and may develop serious morbidity and mortality. The goal of limb salvage in these patients has stimulated research into alternative treatment methods, including angiogenesis.

Attempts have been made to apply growth factors directly or to encode DNA for such factors, but it is unknown whether these factors remain at the target site long enough to be effective. We report our strategy of using vascular endothelial growth factor in a fibrin network, which enables the sustained release of biologic material at the target site.

Epidemiologic studies report that critical ischemia of the limb develops in approximately 500 to 1000 people per million per year. ¹ Direct revascularization may be unsuccessful in most of these cases due to the anatomic extent and distribution of arterial occlusive disease. ^2,3 No pharmacologic treatment has been shown to alter the natural history of critical ischemia of the leg. ⁴ Patients with this condition may eventually require amputation and may develop serious morbidity and mortality. ^5,6 The goal of limb salvage in this group of patients has stimulated research into alternative treatment methods, including angiogenesis. ^7–9

In angiogenesis, vascular cells proliferate and form new capillary structures. ¹⁰ Ischemic tissue injury causes the release of endogenous biochemical agents, which include growth factors that stimulate angiogenesis and growth of the collateral vessels. ^11–13 It is also known that under pathologic conditions in which blood flow is limited by stenosis in the vascular system, the heart or any other organ is capable of growing new arteries, provided that the vascular narrowing or occlusion does not proceed too quickly. However, this physiologic response to pathophysiologic processes is often inadequate to prevent clinical manifestations of ischemia.

Attempts have been made to apply growth factors directly or to encode DNA for such factors, ^14,15 but it is unknown whether these factors remain at the target site long enough to be effective.

A fibrin network is critical for effective wound healing, and it is biodegradable through routine tissue fibrinolysis. Since fibrin sealant is lysed slowly, it can serve as a vehicle to deliver various agents that may act to help heal wounds, ^16–24 to promote new vessel growth, ^17,25 or to store and slowly release antibiotics and other therapeutic agents. ¹⁹ Following balloon injury in an experimental animal, we demonstrated the possibility of reconstructing the arterial wall using fibrin sealant to deliver endothelial cells. ²⁶

In this case report, we present an angiogenic strategy that uses vascular endothelial growth factor (VEGF) in a fibrin network, which enables the sustained release of biologic material at the target site.

Case History

In July 1998, a 66-year-old man was referred to us because he was experiencing pain in both legs on minimal exertion (walking 15 feet) and sometimes at rest. Treatment with analgesics and pentoxifylline provided incomplete relief. He was also being treated for hypertension and coronary artery disease. He had no history of diabetes, smoking, or cerebrovascular disease.

Physical examination revealed no carotid bruit, and cardiovascular examination was normal. Femoral pulses were palpable, but the popliteal pulse was feeble, and the dorsalis pedis pulses were absent. Noninvasive testing showed moderate reduction (0.60) of the ankle-brachial index (ABI) on the right leg at rest, moderate digital ischemia of the right 1st, 2nd, 3rd, and 4th toes, and severe ischemia of the right 5th toe. Peripheral angiography revealed bilateral occlusion of the superficial femoral artery (SFA) and diffuse distal disease of the arteries of the right lower extremity (Fig. 1A). Under the compassionate-use protocol of the Human Research Review Board of the Medical College of Wisconsin, the patient was deemed appropriate for angiogenic treatment.

Fig. 1 Angiogram of a patient with critical limb ischemia before treatment (A) and 3 months following treatment (B). Note vessel growth and better visualization of peripheral arteries.

The procedure was performed on an outpatient basis. Intramuscular application of VEGF and modified fibrin glue was carried out with a transdermal needle attached to a FibriJet™ dual syringe system (Micro-medics Inc.; Eagan, Minn). The patient was given diazepam, and 5 mL of lidocaine was administered topically. We then administered the following fibrin glue composition: 5,000 U of thrombin (Parke Davis; Morris Plains, NJ); 10 mL of cryoprecipitate (prepared at the Blood Center of Southeastern Wisconsin, Milwaukee, Wis); 100 μg of deferoxamine (Ciba; Summit, NJ); and 100 μg of VEGF (Intergen; New York, NY). This cocktail was injected into the right popliteal region, close to the popliteal artery.

Two weeks after the procedure, the patient was feeling better and could walk approximately 500 feet without experiencing significant pain, a more than thirty-fold increase of walking distance. Clinical examination remained unchanged, and the patient's need for analgesics decreased at this time. The patient was followed up once every 4 weeks thereafter. His ABI at 4 weeks was 0.69, a slight increase over the prior examination; he had residual arterial ischemia of the right 1st digit but not of the other 4.

Twelve weeks later, the patient no longer required any analgesics for his claudication pain, and his exercise tolerance had improved remarkably. Four months after treatment, angiography revealed substantial growth of new vessels in the infra-popliteal segment at the site where growth factor and fibrin had been injected (Fig. 1B). Noninvasive ABI measurements performed 20 weeks after the procedure showed significant increase (0.75) in comparison with the preprocedural ABI.

Discussion

Several studies have shown that angiogenic growth factors are effective in experimental models of myo-cardial ischemia ²⁷ and hind-limb ischemia. ^28,29 Moreover, VEGF was used in clinic to treat critical limb ischemia ^30,31 and advanced coronary artery disease. Providing further support for the concept, Isner and colleagues ³⁰ reported improvement of peripheral arterial ischemia after they used a hydrogel-coated balloon catheter to transfect the peripheral arteries with cDNA encoding VEGF. Subsequently, the same group initiated intramuscular injection of VEGF₁₆₅-encoding naked human plasmid cDNA in 9 patients (10 ischemic limbs). ³¹ In this initial series of intramuscular (rather than intra-arterial) injections of an angiogenic gene, blood flow improved in 8 of 10 limbs, as demonstrated by magnetic resonance angiography, and there was an overall increase of 30% in ABI, a key measure of improved limb blood supply. Patients were excluded if they had conditions that might be associated with untoward sequelae of “inappropriate angiogenesis,” such as cancer or diabetic retinopathy.

Isner's group ³² also injected naked plasmid DNA directly into the myocardium through a minimally invasive incision in the chest wall. They concluded that the procedure is safe and may lead to reduced symptoms and improved myocardial perfusion in selected patients with chronic myocardial ischemia.

Despite this very encouraging early experience, it is not known whether new collaterals will have hemodynamic significance. Recent trials using VEGF, recombinant fibroblast growth factor-2 (FGF-2), and basic fibroblast growth factor (bFGF) demonstrate minimal or no improvement of myocardial perfusion following angiogenic procedures. ^33–36

Recently, Terai and colleagues ³⁷ presented data suggesting that VEGF may play a role in the pathogenesis of acute Kawasaki syndrome. Indeed, the 1st peak of VEGF levels is observed in the early stage of the illness, when skin rash, edema of the extremities, or both, are present. Studies of skin histopathology in acute Kawasaki syndrome demonstrate vascular dilatation with endothelial gap formation and sub-endothelial edema. ^38,39 Pathologic examination has also demonstrated that coronary artery vasculitis is present in all patients with Kawasaki syndrome. ³⁸ Other studies have demonstrated that there is endothelial injury and adventitial edema in the coronary arteries of patients with Kawasaki syndrome. ³⁹ Moreover, recent studies have documented abnormal endothelial responses in patients who have Kawasaki syndrome and normal coronary anatomy. ⁴⁰

In view of current clinical studies involving the use of VEGF and related growth factors in the treatment of diffuse coronary artery disease, these data raise questions regarding the safety of the therapeutic angiogenesis procedure: could VEGF therapy possibly induce further damage to coronary circulation? Additional studies are needed to investigate the safety of introducing VEGF into ischemic tissue. It is possible that concomitant anti-inflammatory therapy or use of delivery vehicles that localize the effect of VEGF ^18,41,42 could make this therapy safer. Last, the possibility that VEGF accelerates atherosclerosis should be investigated. ⁴³

We believe that fibrin glue enhances the effect of VEGF. Even without the addition of growth factors, fibrin glue applied subcutaneously has been shown to stimulate angiogenesis. ^18,44 Experiments demonstrate that the glue becomes vascularized (Fig. 2), indicating that plasma proteins alone can perform some of the functions of the extracellular matrix involved in anchoring endothelial cells to the vascular wall. As described above in regard to ischemic tissue, the fibrin glue serves as a temporary matrix for the gradual development of granulation tissue that is characterized by a high degree of vascularity. Intravascular administration of growth factors without fibrin glue can stimulate angiogenesis in some animal models, ^15,45–47 but not in others, ⁴⁸ which further supports the value of adding the glue.

Fig. 2 Angiogenesis in an experimental model of dynamic cardiomyoplasty in a sheep. Fibrin glue was used to form a provisional reservoir between the myocardial wall and transplanted skeletal muscle. A) Transmission electron micrograph. Note neocapillaries and arterioles formed in a fibrin network. B) Immunohistochemical micrographs of latissimus dorsi muscle show evidence of neovascularization.

Fasol and colleagues ¹⁷ demonstrated that implantation of modified fibrin glue containing the angiogenic growth factor bFGF induced significant site-directed formation of new blood vessel structures in a rat model.

Laube and colleagues^* applied human recombinant basic fibroblast growth factor in a fibrin matrix directly to the epicardium during conventional bypass grafting in 8 patients with diffuse coronary artery disease. Follow-up angiography and thallium scans demonstrated induction of a collateral network of capillaries.

Thrombin and fibrinogen obtained from animal sources can lead to antibody production. ^49–51 This problem would be eliminated by the use of human fibrinogen. Although use of human fibrinogen carries a theoretical risk of disease transmission, a summary of several European studies has shown no evidence of human immunodeficiency virus or hep-atitis transmission in over 1,000,000 patients. ⁵² Alternatively, use of autologous cryoprecipitate ⁵³ or recombinant DNA ⁵⁴ would provide safe sources of fibrin meshwork.

We have previously demonstrated that VEGF and fibrin accelerated the wound healing process in a patient who had bilateral ischemic gangrene of the feet. Unfortunately, the patient still required bilateral below-knee amputation; however, this was less extensive and severe than anticipated before the protocol. ⁴²

In the case reported here, it is clear that there was substantial improvement in symptoms and, consequently, in the patient's quality of life. We have objectively demonstrated by angiography the growth of new blood vessels after treatment with VEGF and fibrin. The fibrin glue aids in the slow release of the growth factor and thus prolongs its availability, sustaining angiogenesis and improving oxygen supply. We propose that this strategy can be used as a possible therapeutic intervention in the management of limb ischemia by inducing growth of new blood vessels. Further clinical study appears warranted.