angiogenic gene therapy in patients with … · georgel taranu et al 225 original articles...

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Georgel Taranu et al 225

ORIGINAL ARTICLES

ANGIOGENIC GENE THERAPY IN PATIENTS WITH CRITICAL LIMB ISCHEMIA – INITIAL CLINICAL EXPERIENCE

Georgel P. Taranu1, Mihai E. Ionac1, Lucian P. Jiga1, Andreea L. Rata1, Claudia F. Rus1, Edward P. Seclaman2, Andrei Anghel2

REZUMATObiective: Terapia neoangiogenetică reprezintă la ora actuală o metodă intens studiată în privinţa posibilităţii de a salva de la amputaţie membrele inferioare în cazul pacienţilor cu afectare vasculară periferică depăşiţi ca indicaţie de revascularizare endovasculară sau chirurgicală. Actualul studiu îşi propune să analizeze potenţialul terapeutic privind salvarea membrului inferior în această categorie de pacienţi a unei singure injectări versus injectare plus rapel la o lună precum şi existenţa unor posibile reacţii adverse sau efecte secundare ale metodei. Material şi metodă: Lotul de studiu a cuprins 7 pacienţi, împărţiţi în două subloturi: sublotul A – patru pacienţi cu o singură injectare, respectiv sublotul B – 3 pacienţi cu injectare iniţială plus rapel la o lună. Doza administrată intramuscular, la o injectare, a cuprins 1013 copii, atât a factorului de creştere endotelială (vascular endothelial growth factor – VEGF) cât şi a factorului de crestere hepatocitar (hepatocyte growth factor – HGF). Au fost urmărite la intervale de 1, 2, 3, 6 si 12 luni elemente clinice (durata şi caracterul durerii, necesarul de analgezice, evoluţia leziunilor trofice) cât şi elemente paraclinice (indice gleznă/braţ - IGB, test de mers pe covor rulant, Doppler vascular). Rezultate: În sublotul A trei din cei patru pacienţi au necesitat amputaţie majoră a membrului inferior afectat la un interval cuprins între 2-4 luni iar în sublotul B nu s-a consemnat nici o amputaţie pe durata urmăririi pacienţilor. Nu s-au consemnat reacţii adverse sau efecte secundare. Concluzii: Terapia neoangiogenetică este o metodă sigură de tratament, efectuarea rapelului la o lună creşte semnificativ rata de reuşită, elemente de prognostic nefavorabil au fost: IGB < 0,2 şi durerea de repaus > 12 săptămâni de la debut în momentul admiterii în studiu.Cuvinte cheie: neoangiogeneză, VEGF, boală vasculară periferică

ABSTRACTObjectives: Neoangiogenetic therapy represents nowadays a very intensive explored therapeutical approach regarding the opportunity to avoid lower limb amputation in patients with peripheral occlusive disease with no endovascular or surgical revascularization options. The aim of our study was to determine the therapeutic potential of this method (single administration versus one administration followed by another one four weeks later), focusing on limb salvage, as well as to determine possible adverse reactions or side effects and possible interference with associated conditions (the increase of morbidity or mortality). Material and methods: The total number was 7 patients, divided in two groups: group A – four patients with single injection and group B – three patients with two injections (the initial one was followed by a second injection four weeks later). The dose administration, via intramuscular injection, included 1013 copies of both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). All patients were supervised by periodic controls at 2 weeks, 4 weeks, 3, 6 and 12 months. Patients check up involved clinical evaluation of the general health status, local status, the need of pain killers as well as ankle-brachial index, echo-Doppler and walking test. Results: In group A three patients needed major amputation (thigh amputation) between 2-4 month while in group B no amputation was recorded. They were not side effects or adverse reactions. Conclusions:Neonagiogenetic therapy is a safe method with no side effects; the second injection significantly increases the success rates; ankle-brachial index ≤ 0.2 on admission and rest pain for more than 12 weeks are bad prognosis elements.Key Words: neoangiogenesis, VEGF, peripheral vascular disease

Received for publication: Sep. 23, 2009. Revised: Dec. 14, 2009.

1 Vascular Surgery Department, Clinical Emergency County Hospital Timisoara, 2 Department of Biochemistry, Victor Babes University of Medicine and Pharmacy Timisoara

Correspondence to:Georgel P. Taranu, Vascular Surgery Department, Clinical Emergency County Hospital Timisoara, I. Bulbuca 10, Timisoara, Tel: +40-748-331475, E-mail: [email protected]

INTRODUCTION

Critical lower limb ischemia is the most severe form of peripheral arterial occlusive disease or of other peripheral vascular conditions (such as thromboangiitis obliterans). Patients in this stage of the disease have a negative prognosis; in the first year 25% undergo major amputation, 25% die (usually because of a cardiovascular or cerebrovascular condition), and only 50% are alive and without amputation.1 The most important aim of the treatment is to decrease mortality

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rate, with a second target to reduce the rate of major amputations in these group of patients. The latter is achieved through a wide variety of revascularization procedures (interventional or surgical). Unfortunately, a significant percentage of patients with critical ischemia can not undergo revascularization because of extended lesions (especially in diabetes, thromboangiitis and chronic renal failure).

Therapeutic angiogenesis is a relatively recent treatment option, that is still in experimental stage, and is based on stimulating new blood vessels formation in ischemic areas by local injection of gene precursors that induce the synthesis of certain angiogenic proteins: VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), HIF-1 (hypoxia inducible factor-1). In order to be incorporated into the cellular structure of targeted tissues, the genetic material can be administered directly (naked DNA) requiring larger amounts of plasmids, or attached to a vector (usually an adenovirus) with a high risk of unwanted immune response.2

The peripheral ischemic disease, associated to diabetes mellitus or non-diabetic arteriopathy, affects especially the arteries of the lower limbs, and the final result of the blood flow obstruction through these arteries is represented by the necrosis of the ischemic tissues. The therapeutic possibilities are limited, the disease evolution having as a result the amputation of the affected limb. Lately, a special interest has been observed for the angiogenic gene therapy, destined to reestablish the blood flux at the level of the lower limbs. The transfer of genes that code factors implied in angiogenesis (VEGF, HGF, erythropoietin 2) has given preliminary encouraging results.

VEGF has both angiogenic and vasculo-protection effects through the stimulation of the endothelial production of nitric oxide and prostacyclins, with vasodilatatory and platelets antiagregating properties. The treatment with adenoviral vector has been proved more efficient from the point of view of the duration of the induced effects, 61% of the patients have presented an increased level of the antibodies anti-adenovirus and of the hepatic enzymes. That is why the administration of plasmidial vector remains the preferred method of angiogenic gene therapy in the case of the peripheral ischemic disease, even if the repetition of the therapy is probably needed every six months. The main advantages of nonviral vectors are represented by the possibility of including of up to 40 kb DNA fragments, the absence of the immune response, and a low risk of adverse effects. The relative low capacity of penetration through the cellular wall, and the transitory expression of the target

proteic product are the major disadvantages. Recently, the utilization of the HGF (hepatocyte growth factor) gene came into view, associated with VEGF in the treatment of cardiac and peripheral ischemic disease.3

HGF has been initially described as a growth factor that stimulates the regeneration of the hepatic tissue, consecutive to hepatectomy.4 Studies on experimental and clinical models have demonstrated that HGF stimulates the endothelial proliferation and angiogenesis.5-7 Unlike VEGF that has no receptors at the level of smooth muscle cells, HGF stimulates their migration.

The present paper describes the implementation of angiogenic gene therapy using plasmidial vectors that contain the genes VEGF165 and HGF in severe chronic ischemia of the inferior limbs. Although susceptible to be rapidly degraded by endonucleases, it has been observed that the intramuscular injected plasmid penetrates rapidly in the mytocytes nuclei, and after that it ensures the expression of the therapeutic gene, contained by these vectors.

MATERIAL AND METHOD

Our study included a total of 7 patients admitted during July 2008 and July 2009. The inclusion criteria are shown in Table 1.

Table 1. Inclusion criteria.

1 Age 21-80 years

2 Diagnosis Peripheral arterial occlusive disease (PAOD)

Thromboangiitis obliterans – Buerger’s Disease (TAO)

3 Clinical stage

Rest pain for at least one month ± distal ulceration or gangrene

4 Correct and complete conservative treatment, for at least one month

5 Patients without indication of interventional and/or surgical revascularization

The patients without indication of interventional revascularization are patients that have long chronic occlusions, with thin or invisible distal vessels; the patients without surgical indication have thin distal vessels, no calf vessels that can be grafted (with or without prior occluded grafts), and not long enough autologue veins, or are patients that have an associated pathology that increases too much the operative risk.

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The exclusion criteria for our study are shown in Table 2.

Table 2. Exclusion criteria.

Prostaglandin treatment less than 3 months prior to the study

Interventional or surgical revascularization procedures or attempts in the affected member during the last 3 months

Sympathectomy during the last 3 months

Active infection (gangrene) in the affected limb with indication of acute surgery (amputation)

Ostheomielitis in the affected limb (radiological)

Acute coronarian events or surgical and/or interventional myocardial revascularization procedures in the last month

Transient ischemic attack or cerebral stroke during the last 3 months

Deep vein thrombosis in the last 3 months

Associated condition with vital prognosis less than 3 years

Neoplasia

Liver dysfunction with ALT and AST higher than 3 times the upper normal values, albumin < 35 g/L, bilirubin > 2 mg/dL, medical history of hepatic encephalopathy or ascitis

Chronic renal failure with creatinine > 2.5 mg/dL

Diabetes mellitus complicated with proliferative retinopathy

Chronic inflammatory conditions

Chronic immunosuppressive treatment

Coagulation disorders

Inability to comply with the protocol or to follow the patient

Inclusion in another study that uses an experimental drug or procedure during the last month

Pregnancy, lactation period or inadequate treatment with birth control medication

All patients included in our study have been thoroughly examined in order to strictly determine the initial degree of ischemic condition, as well as to assess the general state of health and to find any associated conditions that would constitute contraindications (e.g. neoplasm, proliferative retinopathy, etc.). A full list of these investigations is shown in Table 3.

Table 3. Data, tests and other preliminary investigations.

Demographics

Medical history (including medication)

General and local clinical examination

Ankle-brachial index

Walking test on graded treadmill (for patients that can undergo the test); Gardner protocol was used (constant speed of 3.22 km/h, increasing with 2% degrees every 2 minutes noting the interval before pain appears and the total walking time)

Echo Doppler

Laboratory tests: hemoleucogram, VSH, fibrinogen, PCR, blood sugar level, urea, creatinine, AST, ALT, bilirubin, proteins, cholesterol, triglycerides, LDL, HDL, INR, aPTT, LDH, CPK, CPK-MB; pregnancy test (for women at procreation age)

Resting EKG

Chest X-Ray

Bone X-Ray for patients with trophic lesions

Eye fundus examination

Cardiologic examination

Neurologic examination

Neoplasm screening: urologic/gynecologic examination, gastroenterological examination

Peripheral angiography

The inclusion, exclusion criteria and patients testing was established according to existing data from the literature.8,9

1013 copies of both purified vectors (VEGF and HGF in pBLAST49mcs-InvivoGen) were brought to 50 ml in sterile Falcon tubes with 0.9% NaCl sterile perfusion solution. The obtained solution was then subjected to a standard sterility verification protocol, by cultivating a small amount on bullion medium for 5 days followed by subsequent cultivation on aerobe and anaerobe specific media, respectively. No bacterial growth was observed in any case. Administration was performed by intramuscular injection, as recommended by some authors, of a total of 50 ml saline solution containing 1013 copies of vectors into the lower thigh (4-6 injections) and calf muscles (10-12 injections) (at 3–4 cm intervals), on a trajectory with potential angiogenic benefit, as judged by the most recent angiography of the ischemic leg.8,10,11

The patients were divided into two groups: group A-4 patients, who were administered only one injection, and group B- 3 patients, who were administered two injections (initial injection followed by a second four weeks later).

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228 TMJ 2010, Vol. 59, No. 3-4

All patients were followed-up periodically at 2 weeks, 4 weeks, 3, 6 and 12 months. Patients check up involved clinical evaluation of the general health status, local status, the need of pain killers as well as ankle-brachial index, echo-Doppler, walking test and a control angiography at 12 months.

The aim of our study was to determine the therapeutic potential of this method, focusing on limb salvage, as well as to determine possible adverse reactions or side effects and possible interference with

associated conditions (the increase of morbidity or mortality).

RESULTS

Initial patient data can be found in Table 4 for group A and in Table 5 for group B.

In group A four injections were administered for four limbs. The patients were supervised for 1 to 3 months with an average of two months (the

Table 4. Group A initial data.

Pat. 1 Pat. 2 Pat. 3 Pat. 4

Age (years) 71 42 64 49

Gender M M F M

Smokers Yes Yes No Yes

Diagnosis PAOD TAO PAOD PAOD

Actual signs Rest pain Yes Yes Yes Yes

Trophic lesions No Yes No No

Pain duration (weeks) 12 14 20 18

Daily analgesic medication

Usual pain killers + opiates drugs (Tramal 3×50 mg/day)

Usual painkillers + opiate drugs (Tramal 3×100 mg/day)

Usual pain killers + opiates drugs (Tramal 3×50 mg/day)

Usual pain killers

Lesion SFA occlusion; calf arteries occlusion

Calf arteries occlusion

Ilio-femoral occlusion, SFA occlusion, calf arteries occlusion

SFA occlusion, calf arteries occlusion

History of revascularization of the affected limb

No No AFB (thrombosed) Left functional FPB

ABI Injected limb 0.18 0.15 0.12 0.22

The other limb 0.73 0.7 amputated amputated

Walking test The beginning of pain 2 min 30 sec 30 sec Could not be performed (thigh amputation)

Could not be performed (thigh amputation)Test interruption 4 min 10 sec 1 min 30 sec

Associated condition Coronarian disease No No No Yes

Stroke No No No No

DM No No No No

AHT No No Yes Yes

Others No Motor and sensitive peripheral neuropathy

3rd degree mitral valve regurgitation

Aortic abdominal aneurysm (no surgical indication)

Side effects upon first injection Pyrexia No No Allergic reaction

PAOD – peripheral arterial occlusive disease; TAO – thromboangiitis obliterans; DM- diabetes mellitus; AHT – arterial hypertension; SFA – superficial femoral artery; AFB – aorto-femoral by-pass; FPB – femuro-popliteal by-pass.

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explanation for the short period of supervision is that three of the four patients had to be amputated after 2 to 4 months from injection).

In group B a total of six injections were administered for 3 limbs (3 injections at first presentation and 3 after 1 month). The patients were supervised for 3 to 11 months, with an average of 8 months.

Adverse reactions, side effectsIn group A, only one of the patients had a brief

episode of pyrexia (37.9°C) treated with antipyretic drugs and another patients had an allergic reaction (shivering, pyrexia, rash) treated with hydrocortisone hemisuccinate, calcium, antipyretic and antialergic drugs.

In group B, only one patient had pyrexia (37.8°C) after the first injection, treated with antipyretics. There were no allergic reactions at the second injection.

Existing conditions were not aggravated, nor did

new conditions appear after the treatment. The evolution of studied parameters and the rate

of major lower limb amputations are shown in Table 6 for group A and Table 7 for group B.

Usual analgesic medication was algocalmin, piafen and NSAIDs, administered in therapeutic dosage.

In group A, three of the four patients had to be amputated (thigh amputation) after 2 to 4 months from the injection.

Concerning ankle-brachial index, two patients showed no significant increase and two patients showed an increase of 0.23, respectively 0.12 (the latter had to be amputated because of the progression towards gangrene).

The walking test could not be performed for two of the patients (as they were already amputated of one leg) and the other two patients showed no significant improvement.

Table 5. Group B initial data.

Pat. 1 Pat. 2 Pat. 3

Age (years) 50 64 50

Gender M M M

Smoker Yes Yes Yes

Diagnosis TAO PAOD TAO

Actual signs Rest pain Yes Yes Yes

Trophic lesions No No Yes

Pain duration (weeks) 6 12 12

Daily medical analgesic Usual pain killers Usual pain killers Usual pain killers + opiates drugs (Tramal 3×50 mg/day)

Lesions Calf arteries occlusion SFA occlusion; calf arteries occlusions

SFA occlusion; calf arteries occlusions

History of revascularization of the affected limb

No Left thrombosed FPB No

ABI Injected limb 0.5 0.15 0.46

Other limb 0.7 Thigh amputation 0.6

Walking test The beginning of pain 1 min 20 sec Could not be performed (thigh amputation)

25 sec

Test interruption 2 min 20 sec 2 min and 16 sec

Associated conditions Coronarian disease No No No

Stroke No No No

DM No No No

AHT No AHT No

Other No No No

Side effects Pyrexia No No

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230 TMJ 2010, Vol. 59, No. 3-4

Table 6. The evolution of studied parameters for group A.

Initial 1 month 3 months 6 months

Pat. 1 Rest pain Yes Yes, slightly decreased in intensity

Not done(thigh amputation at 2 months for infected stab wound with evolution towards gangrene)

Not done (amputation at 2 months after injection)

Trophic lesion No No

Daily analgesic medication Usual pain killers + opiates drugs (Tramal 3×50 mg/day

Usual pain killers + opiates drugs (Tramal 3×50 mg/day

ABI injected limb 0.18 0.2

Walking test The beginning of pain

2 min 33 sec 2 min 41 sec

Test interruption 4 min 15 sec 4 min 39 sec

Pat. 2 Rest pain Yes Yes (same) Not done (amputation after 1 month and 3 weeks after injection for the progression from dry to wet gangrene)

Not done (amputation)

Trophic lesion Yes (dry gangrene I-II toes)

Yes (gangrene projection to 3rd toe)

Daily analgesic medication Usual pain killers + opiates drugs (Tramal 3x100 mg/day)

Usual pain killers + opiates drugs (Tramal 3x100 mg/day)


Walking test Beginning of pain 30 sec 43 sec


Pat. 3 Rest pain Yes Yes – slightly diminished

Yes – stronger than before

Not done (4 months amputation for trophic lesions and rest pain increase)Trophic lesion No No Yes (dry gangrene in

toes II and III)

Daily pain medication Usual pain killers + opiate drugs (Tramal 3×50 mg/day)

Usual pain killers + opiate drugs (Tramal 2×50 mg/day)

Usual pain killers + opiate drugs (Tramal 4×50 mg/day)

ABI injected limb 0,12 0,15 0,12


Not done (thigh amputation)

Not done Not done

Test interruption

Pac 4 Rest pain Yes Yes – slightly improved

Yes – improved (episodic nocturnal pain)

Not done (patient included in the studied for a period less than six months)Trophic lesion No No No

Analgesic medication Usual pain killers daily

Usual pain killer every 2 or 3 days

Usual pain killers rarely (one a week)

ABI injected limb 0.22 0.37 0.45


Not done Not done Not done

Test interruption

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Vascular echo-Doppler showed no improvement of blood flow in the large calf arteries (posterior tibial artery and anterior tibial artery), and the data was omitted.

The patients that didn’t need amputation showed a significant decrease of analgesic medication and a significant decrease in rest pain.

In group B, no amputation was required, all three patients having a favorable evolution.

Ankle-brachial index showed a significant increase (> 0.1) in all patients in group B, with values between 0.15 – 0.34 with a median value of 0.22.

Vascular echo-Doppler showed no significant improvement of blood flow in main calf arteries (anterior tibial and posterior tibial artery).

The walking test could not be performed for one patient (due to previous thigh amputation) and for the other two patients showed an increase in pain interval

Table 7. The evolution of studied parameters for group B.

Initial 1 month + second injection

3 months 6 months 12 months

Pat. 1 Rest pain Yes Yes – diminished a lot

Not done (patient admitted less than 3 months)Trophic lesion No No


Usual pain killers every 3-4 days



1 min 20 sec 3 min 27 sec


Pat. 2 Rest pain Yes Yes – diminished a lot

Yes – rarely (nocturnal cramps)

No Not done (patient admitted less than 12 months)Trophic lesions No No No No


Usual pain killers 1-2 times / week

Usual pain killers 2-3 times / month

Very rarely

ABI injected limb 0.15 0.30 0.41 0.49


Not done (thigh amputation)

Not done Not done Not done

Test interruption

Pat. 3 Rest pain Yes Yes – slightly diminished

Yes – diminished a lot (nocturnal cramps)

No Not done (patient admitted less than 12 months

Trophic lesions Yes (arterial ulceration antero-lateral and medial calf side)

30-40% healed 75% healed Fully healed

Analgesic medication Usual pain killers + opiate drugs (Tramal 3×50 mg/day)

Tramal 2×50 mg/day + usual pain killers every 2-3 days

Tramal 50 mg/day

None

ABI index injected limb 0.46 0.51 0.55 0.62


25 sec 2 min 3 sec 2 min 38 sec 2 min 55 sec

Test interruption 2 min 16 sec 3 min 33 sec 4 min 15 sec 5 min 10 sec

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232 TMJ 2010, Vol. 59, No. 3-4

of 2 minutes 30 seconds, respectively 2 minutes 7 seconds, and an increase of total walking time of 2 minutes 54 seconds, respectively 3 minutes 52 seconds.

Favorable evolution is also demonstrated by the decrease of pain medication and a decrease of rest pain.

For a better illustration we present the angiographic aspect of one patient from group B (patient number 3) at the moment of the first presentation. (Figs. 1,2) This patient was the only one in his group who showed trophic lesions on admissions. (Figs. 3, 4). These trophic lesions completely healed after six months. (Figs. 5, 6)

Figure 1. Occlusion of the left superficial femoral artery – anterior view (patient 3, group B).

Figure 2. Occlusion of the left calf arteries – posterior view (patient 3, group B).

Figure 3. Ischemic ulceration, medial aspect of the left calf – first presentation (patient 3, group B).

Figure 4. Ischemic ulceration, antero-lateral aspect of the left calf – first presentation (patient 3, group B).

Figure 5. Complete healing of the lesion, medial aspect of the left calf, six months later.

Figure 6. Complete healing of the lesion, antero-lateral aspect of the left calf, six months later.

Morbidity and mortalityNo aggravation of associated conditions was

reported during the follow-up of group A patients, nor new conditions developed and no deaths occurred. The same findings are also valid for group B.

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DISCUSSIONS

The use of genic products administration in order to induce neoangiogenesis in patients with peripheral vascular lesions is still a disputed subject; both positive and negative data exist in literature.8-12

Our findings suggest that the administration of these products is safe without adverse reactions or important side effects and without interference with existing conditions. Concerning limb salvage, we noted that the second injection after one month led to an important improvement. Failure was noted in patients with advanced disease (one case of severe trophic lesions – dry gangrene that progressed to wet gangrene that required amputation), in patients with multi-level lesions (one case with aorto-iliac and infrainguinal lesions). Also, we noticed that pain lasted longer in group A patients (three patients with rest pain more than 12 weeks) while in group B only one patient showed pain for more than 12 weeks. Four patients showed femuro-popliteal lesions associated with calf artery lesions, of which only one needed to be amputated. Patients who had an ankle-brachial index less than 0.2 on admission had a negative evolution (three out of four patients needed amputation). There were no significant differences in evolution related to the underlying condition (limb salvage in two of the four patients that had peripheral arterial occlusive disease, respectively in two out of three patients that had thromboangiitis obliterans). The administration of neoangiogenetic products can be extended to patients with diabetic neuropathy in the absence of peripheral vascular lesions.13

The most effective methods for proving the favorable evolution were the measuring of ankle-brachial index and the walking test. Doppler Duplex examination didn’t prove to be useful probably due to its limitation to explore small calf arteries.

In order to increase the efficacy of this method certain measures must be taken, focused on the increase of the intracellular admission of the injected products, as well as by associating certain maneuvers, such as vascular stenting and physical therapy.14-15 Also, the moment of the treatment seems to be very important; patients that experienced rest pain for more than 12 weeks had a worse prognosis (three patients had to be amputated).

CONCLUSIONS

Although the number of patients included in our study was relatively small, we were able to draw the following conclusions:

- Neonagiogenetic gene therapy is a safe method with no side effects;

- The second injection, four weeks after the first one, significantly increases the success rates of the therapy;

- The values of admission criteria parameter: ankle-brachial index ≤ 0.2 and rest pain for more than 12 weeks are bad prognosis elements for the neoangiogenetic gene therapy efficiency.

The final conclusion shows that an earlier therapy has considerably more successful rates as pointed out in a clinical study with VEGF only.16 This observation creates new questions, that of the optimal moment in starting gene therapy; the conditions are favorable for us to study the effect of gene therapy in patients with a less severe condition (e.g. intermittent claudication).

The statistical validation of these data requires more patients to be included in this study, as well as a diversification of the methods of post-therapy evaluation (such as musculo-cutaneous biopsies before and after injection as a way to reveal the neo-formation vessels).

ACKNOWLEDGMENTS

This work was supported financially by the Romanian Ministry of Education and Research, grant PNII number 41-052/2007.

REFERENCES

1. Inter-Society Consensus for the Management of Peripheral Artery Disease – TASC II – European Journal of Vascular and Endovascular Surgery, Volumes 33 and 34, 2007.

2. Craig Kent K. Therapeutic angiogenesis. In “Haimovici’s Vascular Surgery”, 5th Edition, Blackwell Publishing, 2004, p 176-82.

3. Gerritsen ME. HGF and VEGF: a dynamic duo. Circ Res 2005;96:272-3.4. Nakamura T, Nawa K, Ichihara A. Partial purification and characterization

of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 1984;122:1450-9.

5. Morishita R, Nakamura S, Hayashi S, et al. Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy. Hypertension 1999;33:1379-84.

6. Powella RJ, Dormandyb J, Simonsc M, et al. Therapeutic angiogenesis for critical limb ischemia: design of the hepatocyte growth factor therapeutic angiogenesis clinical trial. Vascular Medicine 2004;9:193-8.

7. Morishita R, Aoki M, Hashiya N, et al. Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease.Hypertension 2004;44:203-9.

8. Isner JM, Baumgartner I, Rauh G, et al. Treatment of thrombangeitis obliterans (Buerger’s disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg 1998;28(6):964-73.

9. Rajagopalan S, Olin J, Deitcher S, et al. Use of a constitutively active hypoxia-inducible factor-1α transgene as a therapeutic strategy in no-option critical limb ischemia patients – phase I dose-escalation experience. Circulation 2007;115:1234-43.

10. Shyu KG, Chang H, Wang BW, et al. Intramuscular vascular endothelial

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growth factor gene therapy in patients with cronic leg ischemia. Am J Med 2003;114(2):85-92.

11. Kim HG, Jang SY, Park JI, et al. Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease. Exp Mol Med 2004; 36(4):336-44.

12. Gavin K. VEGF gene transfer fails to help peripheral arterial disease patients. Bio-Medicine 2003, http://cmbi.bjmu.edu.cn/news/0304/16.htm.

13. Isner JM, Ropper A, Hirst K. VEGF gene transfer for diabetic neuropathy. Hum Gen Ther 2001;12:1593-4.

14. Mäkinen K, Manninen H, Hedman M, et al. Increased vascularity detected by digital substraction angiography after gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002;6(1):127-33.

15. Mac Gabhann F, Ji JW, Popel AS. Multi-scale computational models of pro-angiogenic treatments in peripheral artery disease. Ann Biomed Eng 2007;35(6):982-94.

16. Anghel A, Mut-Vitcu B, Savu L, et al. Clinical improvement after treatment with VEGF(165) in patients with severe chronic lower limb ischaemia. Genomic Med 2007;1(1-2):47-55.

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