ISCHEMIC LIMB SALVAGE USING NEOANGIOGENICGENE THERAPY

INTRODUCTION:

Angiogenic gene therapy is a new experimental

treatment approach in peripheral arterial occlusive

disease (PAOD), which stimulates new blood vessels

formation in ischemic territories by local administration

of growth factors genes that are able to induce the

synthesis of angiogenic proteins, such as VEGF (vascular

endothelial growth factor), or VEGF synthesis modulating

proteins (1) like HGF (hepatocyte growth factor) (2).

Studies on experimental (3) and clinical models (4,5)

showed that HGF stimulates endothelial proliferation and

angiogenesis, one of the main pathways being VEGF

production stimulation.

We provided this new therapy for several patients

with critical limb ischemia (CLI) and no option for surgical

and/or percutaneous revascularization in order to avoid

lower limb major amputations.

CASE:

We present the case of a 48-year-old male patient

diagnosed in 2007 with peripheral arterial occlusive

disease, with left superficial femoral artery occlusion and

impaired filling of distal vessels. The patient was a heavy

smoker (30-40 cigarettes per day), had short distance

intermittent claudication, at approximately 50 meters,

and an ulcerative lesion on the external side of the left

69

Journal of Experimental Medical & Surgical Research

Cercetãri Experimentale & Medico-Chirurgicale

Year XVIII · Nr.2/2011 · Pag. 69 - 72 E x p e r i m e n t a l

M e d i c a l S u r g i c a l

R E S E A R C H

J O U R N A L o f

SUMMARY: In the present paper, we describe the case of a 50-year-old patient, heavysmoker, diagnosed at the age of 48 with peripheral occlusive arterial disease (occlusion ofthe left superficial femoral artery). A revascularization procedure was attempted but failedbecause of extensive thrombosis in the popliteal artery and poor distal arterial bed. In thenext period the patient developed trophic lesions of the calf; skin graft was performed butwith no result. Considering the current symptoms (rest pain), the peripheral angiographicaspect and the trophic lesions in the calf, in September 2008, VEGF and HGF gene therapywas performed, followed by a second dose administration at one month from the firstadministration. The patient was monitored for the next 18 month and the clinical outcomewas good, with rest pain relief and healing of trophic lesions.Keywords: angiogenic gene therapy, VEGF, HGF, critical limb ischemia

ISCHEMIE DE MEMEBRU INFERIOR TRATATÃ CU TERAPIE ANGIOGENICÃRezumat: În acest articol prezentãm cazul unui pacient în vârstã de 50 de ani, mare fumãtor, diagnosticat la vârsta de 48 de ani cu boalã arterialã ocluzivã perifericã (ocluzie arterafemuralã superficialã stângã). Iniþial s-a tentat o intervenþie de revascularizare, eºuatã dincauza trombozei extensive a arterei poplitee ºi a calitãþii precare a patului vascular distal. Înperioada imediat urmãtoare pacientul dezvoltã leziuni trofice gamba stângã; se realizeaza oplastie cu piele libera despicata dara fara rezultat. Având în vedere simptomatologiaprezentã (dureri de repaus continue) ºi aspectul patului vascular distal în septembrie 2008se practicã administrarea de terapie genicã cu VEGF si HGF, urmatã de o a douaadministrare o lunã mai târziu. Pacientul a fost monitorizat pentru urmãtoarele 18 luni iarevoluâia clinicã a fost bunã, cu dispariþia durerilor de repaus ºi vindecarea leziunilor trofice.Cuvinte cheie: terapie angiogenicã, VEGF, HGF, ischemie criticã

G. Taranu1, Andreea Rata1, L. Tamas2, Anca Tursie1, A. Anghel2,M. Ionac1

Received for publication: 07.01..2011

Revised: 21.02.2011

1. - Vascular Surgery Department, Emergency County Hospital Timisoara, 10 I. Bulbuca, 300736 Timisoara, Romania2. - Biochemistry Department, University of Medicine and Pharmacy "Victor Babes" Timisoara, 2 Eftimie Murgu, 300041Timisoara, Romania

Correspondence to: Dr. G. Taranu (gptgeorgel@yahoo.com), Emergency County Hospital Timisoara, 10 I. Bulbuca, 300736 Timisoara, Romania

calf, but no other associated conditions. In March 2007

surgery was performed, with an attempt of

revascularization, but abandoned because of precarious

distal vessels.

The evolution was poor and the patient's symptoms

became more severe, with the appearance of rest pain

and trophic lesions on the medial as well as the

anterior-external aspect of the left calf. In July 2008 the

patient received a skin graft in order to cover the tissue

defects of the medial aspect of the left calf. The

procedure unfortunately failed, leaving tissue defects on

the medial and anterior-external aspects of the left calf.

In these circumstances, in September 2008 the

patient was admitted in our department and the

assessment by peripheral angiography showed the same

level of left superficial femoral artery occlusion with

precarious distal vessels in the calf - occluded peroneal

and posterior tibial arteries, gracile anterior tibial artery

with distal occlusion. At that moment we decided to

apply gene therapy in order to avoid amputation. The first

administration of gene therapy product was well

tolerated, without side effects. Another dose was

administered one month later, our experience showing

that such an approach could be more effective than single

dose.

The VEGF and HGF genes were transfected by

plasmidial constructs produced according to the Good

Manufacturing Practice Regulations (GMP). The

pBLAST-VEGF and pBLAST-HGF plasmids were prepared

by cloning the appropriate full-length human cDNA into

pBLAST (InvivoGen) plasmids. A volume of 50 ml saline

solution, containing 1013 copies of both purified vectors,

was administered by intramuscular injection into the

lower thigh (4-6 injections) and the calf muscles at 3-4

cm intervals (10-12 injections), on a trajectory with

potential angiogenic benefit according to the most recent

angiography of the ischemic leg.6-9

After the injection of the gene therapy product, the

patient was monitored for 24 hours, in case of possible

immediate adverse reactions (fever, shivering and

allergic reactions). One month later we administered a

second dose following the same procedure.

For clinical outcome we assessed objective

parameters (ankle-brachial index, treadmill testing,

vascular ultrasonography) and subjective parameters

(duration of pain, analgesics intake, quality of life). The

final evaluation was focused on two main objectives: the

prevention of lower limb major amputations (at calf or

thigh level) and/or the healing of trophic lesions

(ulcerative lesions).

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Figure 1. Ischemic ulcers before gene therapy (A and B) and healed lesions 18 months later (C and D).

Patient evolution was slowly favorable, with a slow

regression of symptoms (decrease in rest pain intensity

to almost no pain at all, marked decrease in analgesics

consumption and walking distance increase) and trophic

lesions which tended to granulate. Under these

circumstances, at six months after the first

administration, a new skin graft was successfully

applied. Therefore, at one year from the first

administration of gene therapy, the patient's trophic

lesions were completely healed (Figure 1), having the

same aspect after one year and six months (last

check-up). The control angiography (at 14 months)

showed the same aspect of the peripheral lesions, and

neoformation vessels could not be detected at a CT

scanner minimal resolution of 2 mm. The evolution of

monitored parameters is shown in Table 1.

DISCUSSION:

Neo-angiogenic therapy offers new perspectives,

making it possible to avoid lower limb amputation in

patients with advanced peripheral vascular diseases of

various etiologies (atherosclerotic, inflammatory, and

diabetic).(1,6)

Current data in literature showed positive results using

gene therapy in patients with critical limb ischemia.

Rajagopalan reported good results using another type of

angiogenic protein - HIF (hypoxia-inducible

factor-1?).(10)

Recently, Shigematsu published the results of a

clinical trial which used HGF in patients with critical limb

ischemia, with a significant rate of ulcers healing and rest

pain improvement compared with placebo therapy in

control group.(11)

A controversial situation was recorded in the case of

patients with chronic limb ischemia, where the results

are still unclear. Some authors reported good results after

gene therapy for this kind of patients(7,8) but other

papers showed no significant improvement .(12)

This case report represents a good example for the

potential of angiogenic gene therapy for patients with

critical limb ischemia and supports the positive results

mentioned above. The patient's prognosis was poor, and

a lower limb amputation was taken under consideration

(procedure refused by the patient). For this particular

case one of the possible mechanisms involved in the

favorable clinical evolution was the positive feed-back

associated with the improvement of walking ability -

when the rest pain diminishes and trophic lesions are

cured the walking distance will increase constantly (as

demonstrated by the walking test, see table 1) - which

was proved to stimulate also the development of

collateral circulation.(13)

Also, the second gene therapy dose administration

after one moth from the first dose administration proved

to be benefic to the patient, the results showing a better

evolution which allowed us to avoid amputation.

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Time scale ABI* Walking test Rest pain Analgesics**

Pain onsetStop

exercise

Firstinjection

0.36 45s 2m 16sYes, severe

Daily, opiates drugs andusual analgesics

One month (reinjection)

0.39 2m 15s 3m 23sLess severe, especially at

night3-4 times/week, opiates

drugs and usual analgesics

Threemonths

0.40 2m 48 s 4m 15s Only from time to time1-2 times/week, mostly

usual analgesics

Six months 0.43 2m 55s 4 m 40sNo rest pain, claudication

at 100-200 metersOnce in 1-2 weeks time,

only usual analgesics

Twelvemonths

0.51 4m 10s 5m 45sNo rest pain, claudication

at ~ 300 m

Occasionally, 1-2times/month, only usual

analgesics

Eighteenmonths

0.50 4m 36s 5m 49s No rest pain,

Claudication at ~ 300 mNo need for analgesics

Table 1. Evolution of monitored parameters

*ABI - Ankle brachial index

**Usual analgesic medication: acetaminophen and NSAIDs, administered in therapeutic dosage. Opiate drugs: tramadolum,in therapeutic dosage.

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