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Improvement in Chronic Ischemic Neuropathy After Intramuscular phVEGF165 Gene Transfer in Patients With Critical Limb Ischemia
Drasko Simovic, MD;
Jeffrey M. Isner, MD;
Allan H. Ropper, MD;
Ann Pieczek, RN;
David H. Weinberg, MD
Arch Neurol. 2001;58:761-768.
ABSTRACT
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Objective To investigate the effects of vascular endothelial growth factor gene
therapy on ischemic neuropathy in patients with critical limb ischemia.
Design An open-label, dose-escalating trial. Patients with angiographically
proven critical leg ischemia received injections of phVEGF165 human
plasmid in the muscles of the ischemic limb. Testing before treatment and
at 3 and 6 months included (1) symptom severity score, (2) clinical examination
score, and (3) electrophysiologic studies. Clinical and electrophysiologic
examiners were masked to each other's findings.
Setting A tertiary care referral hospital and a major teaching affiliate of
Tufts University School of Medicine, Boston, Mass.
Results Of 29 consecutive patients enrolled, 17 (19 limbs) completed the 6 months
of study. Six patients had diabetes. Compared with baseline studies, treated
patients had significant clinical improvements in the symptom score (P<.01), sensory examination score (P<.01), total examination score (P = .01),
peroneal motor amplitude (P = .03), and quantitative
vibration threshold (P = .04). Improvement in the
vascular ankle-brachial index in treated legs (P<.01)
corresponded to improvement in neuropathy in the same limb. Neurologic improvement
was seen in 4 of 6 patients with diabetes who completed the study. No clinical,
electrophysiologic, or vascular improvements were observed in untreated legs.
Conclusions Ischemic neuropathy might be a reversible condition, and therapeutic
angiogenesis might be an effective treatment. The presence of diabetes does
not preclude a response to this therapy.
INTRODUCTION
CHRONIC AND severe arterial occlusive disease damages peripheral nerves
and results in a regional neuropathy in the ischemic limb.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
Clinical and electrophysiologic features of this chronic ischemic neuropathy
have been reported previously.1, 3, 5, 6, 9, 10, 11
The symptoms are predominantly sensory and might be easily obscured by ischemic
limb pain.
Animal models of hind limb ischemia have established that certain angiogenic
growth factors can be used to elicit angiogenesis in vivo.12, 13, 14, 15
This is consistent with the paradigm of angiogenesis established by Folkman16 that endothelial cells can migrate, proliferate,
and remodel in response to certain growth factors and in doing so form new
sprouts from parent vessels that constitute angiogenesis. Vascular endothelial
growth factor (VEGF) was identified as an endothelial cell–specific
mitogen that plays a critical role in regulating endogenous neovascularization
in response to tissue ischemia.12, 13, 14, 15, 17, 18, 19, 20, 21, 22
Preclinical studies have documented that VEGF promotes angiogenesis in vivo,
and recent preliminary clinical trials of gene therapy have established that
the results of these animal studies can be extrapolated to humans with ischemic
limbs.23, 24, 25, 26
The present prospective study of 29 consecutive patients undergoing
phVEGF165 gene transfer was undertaken to investigate the impact
of therapeutic angiogenesis on peripheral neuropathy caused by critical limb
ischemia.
PATIENTS AND METHODS
PATIENT COHORT
Patients qualified for a phase 1, open-label, dose-escalating trial
of direct intramuscular gene transfer of naked DNA encoding VEGF165 (phVEGF165) if they (1) had chronic critical limb ischemia, including
rest pain or nonhealing ulcers, for a minimum of 4 weeks; (2) had angiographically
documented severe vascular occlusive disease in the affected extremity; and
(3) were not candidates for surgical or percutaneous revascularization based
on usual practice standards.25, 27, 28
The cohort for this study consisted of sequential patients meeting the selection
criteria. This study was a component of the clinical trial entitled Intramuscular
Gene Therapy for Therapeutic Angiogenesis (FDA IND5777) as approved by the
Recombinant DNA Advisory Committee of the National Institutes of Health, the
Human Institutional Review Board and Institutional Biosafety Review Board
of St Elizabeth's Medical Center (Boston, Mass), and the US Food and Drug
Administration.
INTRAMUSCULAR phVEGF165 TRANSFER
Preparation and purification of the plasmid from cultures of phVEGF165-transformed Escherichia coli were performed
in the Human Gene Therapy Laboratory at St Elizabeth's Medical Center as described
previously.29 The plasmid into which the VEGF
complementary DNA has been inserted, phVEGF165, is a simple eukaryotic
expression plasmid that uses the 763–base pair (bp) cytomegalovirus
promoter/enhancer to drive VEGF expression. This promoter/enhancer has been
used to express reporter genes in a variety of cell types and can be considered
to be constitutive. Downstream from the VEGF complementary DNA is the SV40
polyadenylation sequence. Also included in this plasmid is a fragment containing
the SV40 origin of replication that includes the 72-bp repeat, but this sequence
is not functionally relevant (for autonomous replication) in the absence of
SV40 T antigen. These fragments occur in the pUC118 vector, which includes
an E coli origin of replication and the ß-lactamase
gene for ampicillin resistance.
Each treated limb was injected with a total of 3000 to 9000 µg
of phVEGF165 in 2 or 3 intramuscular injection sessions 4 weeks
apart using a 27-gauge needle. At each treatment session, 8 injections were
made into readily palpable muscles below the knee.25
In most patients, 1 of the 8 injections was performed into a foot muscle.
NEUROLOGIC ASSESSMENT
Patients were prospectively evaluated by 2 neurologists, one performing
the clinical assessment (D.H.W.) and the other performing the electrophysiologic
testing (D.S.). The examiners were masked to each other's results and to the
findings of patients' vascular examinations. Both lower extremities were studied,
except in patients whose contralateral limb had been amputated. Because this
was an open-label study with no sham injections, there were no true control
limbs. We tested the contralateral leg to assess the natural course of the
disease. Each patient was evaluated before treatment and 3 and 6 months after
phVEGF165 gene transfer.
Neurologic Scales
Semiquantitative scales (modified from Notermans et al30)
were used to assess changes in neurologic function before and after treatment
and to compare the treated and untreated legs.
A symptom score (SS) encompassed 5 neuropathy-related symptoms: (1)
distal leg weakness, (2) proximal leg weakness, (3) numbness, (4) paresthesias,
and (5) pain (excluding pain at the site of skin ulceration or from vascular
claudication). Each symptom was graded on a scale from 0 to 3 (0 indicates
none; 1, mild; 2, moderate; and 3, severe). The maximum possible SS (indicating
the most severe symptoms) was 15.
Lower extremity sensory testing was graded by a sensory examination
score (SES), which evaluated (1) intensity of sensory deficit for pinprick
and light touch compared with a proximal, normal region using a scale from
0 to 4 (0 indicates normal; 1, >75% of normal; 2, 50%-74% of normal; 3, 25%-49%
of normal; and 4, <25% of normal); (2) distribution of sensory symptoms
for light touch and pinprick graded on a scale from 0 to 4 (0 indicates normal;
1, abnormal to toes; 2, abnormal to ankle; 3, abnormal to middle of calf;
and 4, abnormal above middle of calf); (3) vibration sense at the toes and
ankle using a scale from 0 to 4 (0 indicates normal; 1, mild loss; 2, moderate
loss; 3, severe loss; and 4, absent), and (4) proprioception at the great
toe (6 trials) graded on a scale from 0 to 4 (0 indicates 6 correct; 1, 4-5
correct; 2, 3 correct; 3, 1-2 correct; and 4, none correct). The maximum possible
SES was 32.
The motor examination score evaluated strength in proximal and distal
leg muscles using a scale from 0 to 4 (a reverse of the Medical Research Council
[MRC] scale as higher score indicates more weakness: 0 indicates MRC 5; 1,
MRC 4; 2, MRC 3; 3, MRC 2; and 4, MRC 1 or 0). The proximal muscle group included
the iliopsoas, quadriceps, and hamstrings, and the distal muscle group included
the tibialis anterior, gastrocnemius, extensor hallucis longus, and the combined
action of the extensor digitorum brevis and longus. The extensor digitorum
brevis was selected for clinical testing of the foot muscles because of the
ease of its strength testing and the convenience of standard electrophysiologic
testing. The reflex score assessed deep tendon reflexes at the knees and ankles
using a scale from 0 to 4 (0 indicates normal; 2, reduced; and 4, absent).
A total examination score (TES) was calculated as the sum of the SES,
motor examination score, and reflex score. The maximum possible TES (indicating
the most severe disability) was 64. For all the clinical measures, therefore,
improvement was reflected by a reduced score.
Electrophysiologic Testing
Nerve conduction studies were performed using standard techniques.31 Tibial and peroneal compound muscle action potentials
(CMAPs) were recorded with surface disk electrodes from the abductor hallucis
and extensor digitorum brevis muscles, respectively. No recordings were obtained
from the tibialis anterior. Sural sensory nerve action potentials were also
studied. Both limbs were tested, when possible, and repeated studies were
performed by the same examiner using the identical anatomical landmarks and
distances. Quantitative sensory testing was performed using a CASE IV (Computer
Aided Sensory Evaluator) device (WR Medical Electronics Co, Stillwater, Minn)
to determine the thresholds for vibration and cold sensation in the legs.32, 33, 34, 35, 36
One trial for vibration and cold threshold was performed using the 4-2-1 testing
algorithm.35 This test was omitted if a skin
ulcer was present at the testing site or if a great toe was previously amputated.
Results were recorded in a standardized measurement unit (ie, just noticeable
difference).34
VASCULAR TESTING
Each patient underwent vascular testing that included measurement of
the ankle-brachial index (ABI) to document the severity of the vaso-occlusive
disease and response to therapeutic angiogenesis.25
Change in the ABI for individual limbs was assessed to evaluate the relation
between vascular and neurologic outcomes. Improvement in the vascular outcome
measure for each individual limb was defined as an ABI increase of at least
0.1, the magnitude of improvement that has been previously suggested to constitute
a successful outcome of surgical or catheter-based revascularization.37 Because the principal inclusion criterion for this
study was clinical and angiographic evidence of critical limb ischemia, patients
with noncompressible arteries were not excluded, although the ABI was not
meaningful.
SERUM VEGF LEVELS
Venous blood samples were analyzed using an enzyme-linked immunosorbent
assay at baseline and weekly after the initial gene transfer. Samples were
immediately centrifuged for 20 minutes at 3600 rpm at 4°C, and the serum
was stored at -20°C until analysis. Serum VEGF concentration was
determined with an immunoassay according to the manufacturer's instructions
(R&D Systems, Minneapolis, Minn). Results were compared with the standard
curve of human VEGF concentrations, with a lowered detection limit of 5 pg/mL.
Samples were checked using serial dilution at least in duplicate.
STATISTICAL ANALYSIS
Changes among baseline, 3-month, and 6-month assessments of SS, SES,
TES, and electrophysiologic measures were analyzed. To compare clinical and
electrodiagnostic data with the ABI in individual patients, we defined overall
neurologic improvement at 6 months as fulfilling at least 2 of the 3 following
measures: (1) 2-point or greater decrease in SS, (2) 4-point or greater decrease
in TES, and (3) 30% or greater improvement in peroneal or tibial CMAP amplitude
or summed motor amplitude. A 30% amplitude increase was selected to reduce
the possibility of improvement reflecting test-to-test variability. The t test, Wilcoxon rank sum test, and Fisher exact test were
used for all comparisons. P<.05 was considered
statistically significant.
RESULTS
Twenty-nine consecutive patients undergoing phVEGF165 gene
therapy were enrolled for neurologic study; 7 did not complete the 3-month
follow-up, yielding 22 patients for analysis at that point (50% women; mean
age, 54 years; age range, 23-77 years). Of the 7 excluded patients, 4 had
limb amputations, 1 was lost to follow-up, 1 refused testing, and 1 had lower
limb bypass surgery. Because of rapid progression of vascular insufficiency
in the contralateral leg, 2 patients had both legs treated with phVEGF165; thus, the total number of treated limbs analyzed at 3 months was
24. Five additional patients did not complete the 6-month follow-up (2 refused
testing, 1 had lower limb bypass surgery, 1 had a limb amputation, and 1 accidental
death occurred), leaving a total of 19 limbs (17 patients) for analysis at
that point.
TRANSGENE EXPRESSION
Enzyme-linked immunosorbent assay analysis of serially obtained venous
blood samples provided evidence that gene transfer was successful in achieving
constitutive overexpression. In patients who completed 6 months of study,
mean ± SEM values for VEGF increased from 18 ± 5 pg/mL at baseline
to peak levels of 68 ± 20 pg/mL and then returned to 22 ± 6
pg/mL by 5 weeks. The VEGF levels before and after gene therapy were significantly
different from peak values (P<.05). There was
no relation between the magnitude of clinical improvement and circulating
VEGF levels.
CLINICAL SCORES
Mean SSs and SESs in treated limbs improved significantly at 3 months
(Table 1), and these changes persisted
at 6 months. Mean TESs significantly improved at 6 months as well (Table 2 and Figure 1 and Figure 2).
At 6 months, the SS was improved in 12 (63%) of 19 limbs and the TES was improved
in 9 (47%) of 19 limbs. Neither the SS (P<.001,
treated vs untreated) nor the TES (P = .004, treated
vs untreated) improved in any of the untreated limbs .
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Table 1. Outcome Measures at 3-Month Follow-up*
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Table 2. Outcome Measures at 6-Month Follow-up*
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Figure 1. Changes in symptom scores for
treated legs between baseline and 6-month follow-up. A indicates mean score;
vertical line, SD. P<.01, baseline vs 6 months.
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Figure 2. Changes in total examination scores
for treated legs between baseline and 6-month follow-up. A indicates mean
score; vertical line, SD. P = .01, baseline vs 6 months.
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ELECTROPHYSIOLOGIC MEASURES
At 6 months, mean peroneal CMAP amplitudes (P
= .03) and vibration thresholds (P = .04) significantly
improved in treated limbs (Table 2).
Eleven (58%) of 19 limbs had absent sural responses. The mean (SD) sural sensory
nerve action potential amplitude in treated legs was 4.48 (6.05) µV
at baseline and 5.87 (7.30) µV at 6 months, which was not a statistically
significant change (P = .06). Ten (53%) of 19 limbs
showed improvement in electrophysiologic measures at 6 months (7 limbs had
increased peroneal and summed CMAP amplitudes, 2 had an increased peroneal
CMAP amplitude, and 1 had an increased summed CMAP amplitude), whereas no
improvements were seen in any of the contralateral, untreated limbs (P = .002, treated vs untreated). Table 3 summarizes the comparison of amplitude and conduction velocity
changes between treated and untreated legs during the 6 months of study. The
mean percentage increase for the peroneal CMAP amplitude was 50% in the treated
legs, whereas in the untreated legs it decreased by 11% (P = .02). Similarly, the mean summed CMAP amplitude increased 16% in
treated legs vs a decrease of 9% in untreated legs (P
= .04). The percentage change in the peroneal CMAP and summed CMAP amplitudes
for each individual limb is presented in Figure 3 and Figure 4.
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Table 3. Comparison of Mean Percentage Changes at 6-Month Follow-up*
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Figure 3. Changes in peroneal motor amplitude
at 6 months. A indicates mean percentage change; vertical line, SD; and solid
circle, improvement from 0 to 160 µV (unable to calculate percentage).
P = .02, treated vs untreated legs.
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Figure 4. Changes in summed motor amplitudes
at 6 months. A indicates mean percentage change; vertical line, SD.
P = .04, treated vs untreated legs.
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There were no differences in the neurologic improvement rates among
patients injected with different doses (3000-9000 µg) of phVEGF165.
ANKLE-BRACHIAL INDEX
The mean ABI improved in treated limbs at 3 and 6 months (Table 1 and Table 2).
The ABI improved in 8 of 10 limbs that satisfied at least 2 of 3 neurologic
improvement criteria for individual limbs. Of the 2 remaining limbs, 1 had
noncompressible arteries and 1 had no improvement. The ABI significantly improved
in only 1 of 9 limbs that did not satisfy the criteria for overall neurologic
improvement (P = .005). All other limbs had an ABI
change of less than 0.1 (4 limbs had noncompressible arteries, so that no
meaningful change in the ABI could be demonstrated).
Eight patients with diabetes were enrolled in this study: 4 had neurologic
improvement in the treated limbs, 2 did not complete the study (1 had bypass
surgery and 1 had an amputation), and 2 had no improvement. Three of the 4
improved limbs in patients with diabetes also had a significant improvement
in the ABI (Table 4).
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Table 4. Outcome Measures in Patients With Diabetes*
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COMMENT
Chronic occlusive arterial disease has a recognized adverse effect on
peripheral nerves, although there have been few studies detailing the clinical,
electrophysiologic, and histologic features of this chronic ischemic neuropathy.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
The symptoms generally include numbness, painful paresthesias, aching, and
burning, and the signs are reduced appreciation of pinprick, light touch,
and vibration sensation; mild distal weakness; and depressed or absent deep
tendon reflexes.5, 11 An asymmetric
neuropathy develops in many patients, particularly in those without diabetes.11 The main electrophysiologic features of ischemic
neuropathy are consistent with a sensorimotor polyneuropathy with axonal features.11 Two studies38, 39
examined electrophysiologic changes after surgical revascularization, one
demonstrating improvement only in the peroneal motor conduction velocity and
the other with no improvement in multiple electrophysiologic outcome measures.
Hunter and colleagues39 concluded that ischemic
neuropathy is an irreversible condition on the basis of these data.
Use of phVEGF165 gene transfer for therapeutic angiogenesis
was established in rabbit and murine models of hind limb ischemia and ultimately
led to the first clinical trial of cardiovascular gene therapy in patients
with critical limb ischemia.12, 13, 14, 15, 23, 24, 25, 40, 41, 42, 43, 44, 45
Preliminary studies23, 24, 25
have suggested that increased limb vascularity and tissue perfusion after
phVEGF165 gene therapy might improve the clinical features of limb
ischemia (ie, restoration of tissue integrity and consequent limb salvage).
In our study of the effect of intramuscular phVEGF165 gene
transfer on chronic ischemic neuropathy, patients had reduced symptoms, decreased
neurologic disability, and improved electrophysiologic measures in treated
limbs. In contrast, none of the clinical or electrophysiologic measures improved
in contralateral, untreated limbs.
The clinical improvement in treated limbs was evident at the 3-month
examination as decreased neuropathic symptoms and improved SES. At 6 months,
the TES also improved. When individual patients were followed up longitudinally,
more than half of the treated limbs demonstrated significant improvement in
contrast to none of the untreated limbs.
Two electrophysiologic measures also improved in treated limbs after
6 months: mean peroneal CMAP amplitude and the quantified vibratory threshold.
There was no change in the motor or sensory conduction velocities, distal
latencies, or sural sensory nerve action potential amplitudes. This latter
observation is not surprising because 58% of limbs had absent sural nerve
sensory potentials at entry into the study, indicating severe sural nerve
damage that was unlikely to be reversible. Furthermore, mean percentage improvements
in the peroneal and summed motor amplitudes at 6 months were significantly
greater in treated vs untreated limbs.
The vascular outcome measure (ABI) also improved at 3 months and to
a greater extent at 6 months in treated limbs only. The ABI was improved in
8 of 10 limbs that had shown neurologic improvement. Both of the remaining
limbs had noncompressible arteries, precluding demonstration of improvement.
Thus, the neurologic and vascular improvements were linked.
Four of 6 patients with diabetes who reached the 6-month end point had
also improved in most outcome measures. Although few in number, this suggests
that the coexistence of diabetes does not preclude improvement in the associated
ischemic neuropathy. It is intriguing to consider how often the development
of a predominantly sensory neuropathy in patients with diabetes and coexisting
vascular obstruction can be incorrectly attributed to diabetes rather than
to ischemia.
The neurophysiologic mechanism of improvement after phVEGF165 treatment
is uncertain. Recovery is attributable to enhanced perfusion of ischemic nerves
through global revascularization of the limb, including via the vasa nervorum,
the nutrient arteries that constitute the microcirculation of the peripheral
nerves. In vivo and postmortem studies46, 47, 48, 49, 50
have suggested that neovascularization from angiogenic cytokines principally
involves small vessels of a dimension that would include the vasa nervorum
(<180 µm). Alternatively, our findings might reflect a direct effect
of phVEGF165 on neural elements. Soker and colleagues,51 for example, reported that the neuropilin-1 receptor,
a cell surface glycoprotein associated with axonal guidance in the developing
nervous system, binds phVEGF165.
The effects of ischemia on muscle could be a confounding variable in
our evaluation of muscle strength and motor neurophysiologic measures. Severe
acute ischemia has been reported to have a profound effect on muscle in animal
studies,52, 53 inducing early muscle
necrosis. In fact, the changes can precede and be more severe in muscle than
in peripheral nerve.52 In chronic ischemia
in humans, however, the situation seems different. In a comprehensive assessment
of muscle histopathologic specimens in 40 patients with severe peripheral
vascular disease, Farinon and colleagues9 concluded
that the major muscle alterations were the result of peripheral nerve damage.
Other studies have reached similar conclusions.54
Furthermore, the acute ischemic monomelic neuropathy induced by surgical vascular
shunting or acute large-vessel occlusions has been demonstrated to represent
multiple distal mononeuropathies with no evidence of a significant primary
muscle component.55, 56 We therefore
believe that the clinical and electrophysiologic improvements in this study
reflect effects on motor nerves and not on muscle.
In summary, our findings establish that ischemic neuropathy might be
a reversible condition and that therapeutic angiogenesis might be a novel
and effective treatment for ischemic neuropathy. We are aware of the limitations
of an open-label study with no placebo control group. However, the substantial
clinical improvement in treated limbs and the absence of improvement in untreated
contralateral limbs constitute strong data regarding the biological efficacy
of phVEGF165 gene therapy. Finally, our experience with the subset
of patients with diabetes, although small, demonstrates a degree of reversibility
that may, on further study, have significant clinical and pathophysiologic
implications. Our findings need to be confirmed in a larger controlled clinical
trial.
AUTHOR INFORMATION
Accepted for publication August 31, 2000.
From the Division of Neurology (Drs Simovic, Ropper, and Weinberg)
and the Department of Medicine (Cardiology) (Dr Isner and Ms Pieczek), St
Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass.
Corresponding author and reprints: David H. Weinberg, MD, St Elizabeth's
Medical Center, 736 Cambridge St, Boston, MA 02135.
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