ischemic limb salvage using neoangiogenic gene therapyjmed.ro/articole/240.pdf · ischemic limb...
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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
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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 ([email protected]), 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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