© 1995 American Heart Association, Inc.
Articles |
Heparin Increases Exercise-Induced Collateral Blood Flow in Rats With Femoral Artery Ligation
From the Department of Physiology, State University of New York, Health Science Center at Syracuse and the Department of Cell Biology and Anatomy (R.W.O.), Medical University of South Carolina, Charleston.
Correspondence to Dr Ronald L. Terjung, Department of Physiology, SUNY-HSC at Syracuse, 766 Irving Ave, Syracuse, NY 13210.
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Abstract |
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Abstract The potential for heparin to enhance the training-induced increase in collateral-dependent blood flow to the distal hind-limb muscles was evaluated after bilateral femoral artery ligation in adult male rats (
350 g). Rats received either saline
(n=34) or heparin (n=36) injections and were kept sedentary (limited to
cage activity) or exercised on a treadmill 5 days per week up a 15%
incline by one of two protocols: (1) exercise at a constant moderate
speed (20 m/min) for 6 wks or (2) exercise at a progressively
increased speed for 7 to 8 weeks (started at 20 m/min, increased at 15
minutes to 25 m/min, and then increased at 30 minutes to 30 m/min).
Heparin- and saline-treated rats, exercised by the moderate-speed
protocol, were run for the same time each day. Collateral-dependent
blood flow to the distal limb tissue was determined by using 15-µm
85Sr-labeled microspheres in an isolated hindquarter
preparation perfused in the descending aorta at 100 mm Hg. For
comparison with the above groups, sedentary animals with acute femoral
artery ligation and without femoral obstruction were included. Exercise
tolerance increased from 7 minutes initially to 30 to 40 minutes per
bout; tolerance was greater in the heparin-injected rats than in the
saline-injected rats (P<.05). Muscle performance of the
gastrocnemius-plantaris-soleus muscle group (GPS) during isometric
contractions in situ improved with training, was further increased by
heparin administration (P<.001), and generally scaled with
recovery of blood flow. Collateral-dependent blood flow in the GPS of
the heparin-injected exercised groups (40±4 and 56±3 mL/min per 100
g) was greater than that in the saline-injected exercised groups (19±3
and 30±5 mL/min per 100 g), and blood flow in both of these groups was
greater than that in the sedentary groups (14±4 and 12±2 mL/min per
100 g) (P<.005). Heparin injections did not alter
collateral-dependent blood flow in the absence of exercise training.
Thus, exercise appears to impart an essential stimulus for collateral
vessel development that is enhanced by heparin administration.
Key Words: angiogenesis • intermittent claudication • muscle contraction • microspheres • muscle fiber type
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now well established that heparin is involved in the regulation of angiogenesis. This probably occurs through interactions with a family of polypeptide growth factor mitogens that stimulate endothelial cell proliferation.1 2 Heparin, through its high-affinity binding,3 is thought to aid in the storage of angiogenic growth factors (such as basic fibroblast growth factor [bFGF]) in the extracellular matrix,4 5 protect it against inactivation, and serve to chaperon bFGF through different cellular compartments.2 Heparin potentiates angiogenesis initiated by other angiogenic mechanisms,6 7 possibly by mobilizing bFGF from the extracellular matrix and interacting with low-affinity receptors on the surface of endothelial cells.2
Recently, the role of heparin has been implicated in augmenting collateral circulation. Fujita et al8 demonstrated that daily heparin injections accelerated coronary collateral development in response to repeated brief occlusions (nine per day) of the left circumflex coronary artery in conscious dogs. Recovery from deficits in cardiac function and regional blood flow during ischemia was accelerated by heparin administration. More recently, Carroll et al9 measured a heparin-induced improvement in the rate and magnitude of collateral-dependent blood flow recovery in a porcine model of gradual coronary artery occlusion. In another experimental study in dogs, Unger et al10 demonstrated that revascularization of a collateral-dependent area of the heart from an implanted internal mammary artery was enhanced by heparin. This effect of heparin may be relevant to humans. Evidence of an improved collateral vessel function was found for a patient group with stable effort angina.11 In that study, heparin was administered before supervised treadmill exercise, which was performed twice daily. Exercise tolerance, the maximal double product, and the double product at the onset of angina and at the onset of ST segment depression were significantly improved in the heparin-treated group within 10 days but not in patients who exercised without receiving heparin. Repeat angiographic evaluation of the heparin-treated patients identified an increased collateral network in the myocardial area at risk. Interestingly, this influence of heparin apparently requires other factor(s) to prompt angiogenesis. Heparin administration, in the absence of daily exercise, in a similar group of patients with effort angina was without effect.12 Thus, heparin served to enhance the effect of physical activity.
The potential of heparin to enhance angiogenesis prompted by physical activity could also be beneficial in the presence of vascular obstructions causing peripheral arterial insufficiency. The onset of pain and a characteristic exercise intolerance are symptoms of intermittent claudication caused by flow inadequacies, which become evident in affected patients with exertion. Enhanced physical activity is a useful treatment for patients with intermittent claudication and uniformly leads to improvements in exercise tolerance.13 14 15 16 Peripheral adaptations within the active muscles can contribute to the improvement in exercise tolerance, even in the absence of an increase in oxygen delivery.17 18 The most significant recovery of function, however, would occur by an increase in total blood flow to the limb via expansion of collateral vessel function. The potential for exercise to improve collateral-dependent blood flow has been demonstrated with experimental arterial obstruction in rats19 and in some studies with patients.15 20 21 22 Although the factors controlling angiogenesis of the vasculature supplying myocytes are not well understood, events associated with exercise and ischemia appear to be important.23 The potential for heparin to amplify the angiogenic response to exercise raises the possibility of significantly alleviating the symptoms of intermittent claudication. In the present study, we hypothesize that treadmill exercise combined with heparin pretreatment improves collateral-dependent blood flow in rats with bilateral femoral artery ligation. A greater blood flow would serve to improve muscle function during contractions. Collateral-dependent blood flow was monitored by flow changes to the distal limb muscles of an isolated rat hindquarter preparation perfused at a constant aortic pressure.
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Materials and Methods |
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Animal Care
Adult male Sprague-Dawley rats (
325 g, Taconic
Farms, Germantown, NY) were housed in a temperature-controlled room
(20±1°C) with a 12-hour light/dark cycle and were fed Purina rat
chow and tap water ad libitum. All animals were initially given a brief
(5- to 10-minute) run on the treadmill (20 m/min at 15% grade;
model 4215, Quinton Instruments) daily for 10 days to become
familiarized with treadmill exercise. This experimental protocol was
approved by the Committee for the Humane Use of Animals of the State
University of New York, Health Science Center at Syracuse.
Experimental Design
Surgical ligation of both femoral arteries reduces blood flow
reserve enough to limit active hyperemia24 to an
approximately twofold to fourfold increase but does not reduce blood
flow below the rate observed for resting muscle.25 Rats
were divided into two main treatment groups: heparin and saline. Within
each division, animals were further divided into a sedentary group,
which was limited to cage activity, or either of two groups subjected
to a training protocol that involved exercise on a treadmill 5 days a
week. The first training protocol involved exercising at a constant
treadmill walking speed (20 m/min), which could be easily achieved by
femoral-ligated rats.24 Such a moderate-intensity exercise
protocol induces significant training adaptations in the absence of
heparin administration.17 Care was taken to exercise the
heparin-treated trained animals the same amount of time as their
saline-treated trained controls to impart the same training stimulus. A
second more demanding training protocol was used to evaluate the
interaction between exercise intensity and heparin. Animals were
trained by a fairly intense treadmill program that involved running at
higher speeds as exercise tolerance improved. Instead of matching the
performance of the heparin-injected animals with the performance of
their saline-injected controls, animals of these groups were trained to
their exercise tolerance. Daily run times were recorded for all
exercised animals. To evaluate if the higher running speed preempted
the ability to discern a difference in exercise tolerance, we
administered an exercise test that involved running at a lower constant
speed of 20 m/min. This exercise tolerance test was administered in a
blinded manner: the exercise manager did not know the identification of
the animal's treatment group.
Two additional groups were included: (1) Acute-ligated animals had
bilateral femoral artery ligation 3 days before the experiment and
provided initial baseline information for femoral artery ligation,
presumably in the absence of collateral vessel expansion. (2) Another
sedentary nonexercised group was included. In this group, each hind
limb was perfused in the femoral artery without obstruction at an
arterial pressure similar to the above groups (100 mm Hg) for
comparison with a high blood flow response. Although this group of
animals was part of another study,17 most of the specific
results presented here were not published previously. We simply
present this information as a comparison for blood flow to and the
functional response of lower limb muscle when blood flow is not
collateral dependent. Thus, a total of eight groups are identified in
the data tables.
Jugular Vein Catheterization and Femoral Artery Ligation
Under ketamine/acepromazine (100 mg/0.5 mg per kilogram body
weight) anesthesia, bilateral ligation of the femoral arteries was
performed aseptically just distal to the inguinal ligament with 3-0
surgical suture. Topical antibiotic powder (Neo-Predef, Upjohn) was
placed on the wound before closure with skin clips. Another incision
(1.5 cm in length) was made at the midline of the neck for
catheterization of the right jugular vein. A Micro-renathane catheter
(type MRE-040 [outer diameter, 0.040; inner diameter, 0.025],
Braintree Scientific, Inc) was precoated with tridodecylmethylammonium
chloride/heparin (TDMAC-heparin) complex (Polysciences,
Inc)26 at both outer and inner surfaces and sterilized by
gas. The catheter was inserted 1 cm into the jugular vein at the
bifurcation of the internal and external vein. After the catheter was
secured and exteriorized at the back of rat neck, the incision was
closed with 3-0 silk suture. Catheters were filled with sterilized
saline and flushed every day.
Heparin Treatment
Initial experiments established that intravenous injections of
100 U heparin per rat (250 to 300 U/kg) was sufficient to double the
clotting time, the biological reference shown by Fujita at
el8 for a dose that effectively improved collateral
function in the ischemic myocardium in dogs.
Heparin (100 U per rat, Sigma Chemical Co) was delivered intravenously
15 minutes before daily exercise. The catheters treated with
TDMAC-heparin usually remained patent for 3 to 4 weeks in the
heparin-treated group and 1 to 2 weeks in the saline-treated group.
Upon stasis in the catheter, heparin or saline was administered by
intraperitoneal injections 30 minutes before exercise, which has
been shown to be effective in delivering heparin to vascular
endothelium.27
Treadmill Exercise
Rats began exercising on the treadmill 48 hours after surgery.
Two training protocols were used. The first training protocol (moderate
intensity) involved heparin- and saline-treated animals walking at a
constant speed of 20 m/min at a 15% grade. The performance of the
saline-treated group established the run time for the heparin-treated
group. As fatigue, evidenced by a characteristic change in gait
followed by exaggerated hops, became apparent, the saline-injected
animals were taken off the treadmill. This determined the walk time for
the heparin-injected group. The rats were trained once a day, 5 days a
week, for 6 weeks.
The second training protocol required more intense exercise and
involved running at a higher speed as exercise tolerance improved.
After the first 15 minutes of walking at 20 m/min, the treadmill speed
was increased to 25 m/min. After 30 minutes of running, the treadmill
speed was increased to 30 m/min. The elevation of the treadmill
remained at a 15% grade throughout. Each animal ran until it was
fatigued. The rats in the second training protocol were trained once a
day, 5 days a week, for 8 weeks.
Hindquarter Preparation
After 6 to 8 weeks of training, rats were randomly taken from
each group for hindquarter perfusion preparation, as described in
detail previously.28 In brief, after anesthesia with
pentobarbital sodium (60 mg/kg), a midline incision on the abdomen
allowed access to and removal of the testes, bladder, seminal vesicles,
prostate gland, and small and large intestines to expose the descending
aorta and vena cava. The descending aorta and vena cava just below the
renal artery branch were dissected free and prepared for cannulation.
The skin was then removed from both hind limbs. The left hind limb was
prepared for muscle stimulation, as described in detail
previously.28 Briefly, the left sciatic nerve trunk
serving the distal limb was ligated and cut near the gluteus muscle.
The left distal hind limb was immobilized by a pin drilled though the
bone at the distal end of the femur.
Perfusion Procedure
After surgical preparation, the animal was transferred to a
temperature-regulated (37°C) Plexiglas chamber and given an
intra-arterial (carotid) injection of heparin (2000 U). After 5 to 10
minutes, the abdominal aorta or vena cava was cannulated with a
16-gauge (aorta) or 14-gauge (vena cava) polytetrafluoroethylene
(Teflon) catheter (intravenous catheter, Becton Dickinson),
respectively. Arterial flow began immediately, with only a brief period
of ischemia (<15 seconds). The animals were then killed with an
overdose of pentobarbital sodium injected into carotid artery. The
perfusion medium (300 mL) was recirculated after the initial volume
(20 to 30 mL) of venous effluent had been discarded. The flow rate was
measured by timed collections of the venous effluent. Flow rate and
perfusion pressure were gradually increased over 15 to 20 minutes until
the aortic pressure reached 100 mm Hg. This pressure was determined by
subtracting the pressure drop across the 16-gauge catheter for the
existing flow rate by using an on-line transducer at the height of the
hind limb. The free end of the nerve was placed over a platinum bipolar
electrode connected to a model S48 stimulator (Grass Instruments).
Isometric tension of the gastrocnemius-plantaris-soleus (GPS) muscle
group was measured by attaching the Achilles tendon to a lever system
(series 300 B, Cambridge Technology Inc). At the end of contractions,
blood flows among and within muscles were determined by using
radiolabeled microspheres.
Perfusion Medium
The perfusion medium consisted of Krebs-Henseleit bicarbonate
buffer28 containing washed bovine erythrocytes
(hematocrit, 40%), 4 g/100 mL bovine serum albumin, 100 µU/mL
bovine insulin, amino acids typical for rat blood, and 5 mmol/L glucose
(maintained with periodic additions of glucose during perfusion).
Muscle Stimulation
Resting tension was adjusted to establish maximal active force
development. Tetanic contractions were elicited with supramaximal
square-wave pulses (6 V, 0.1-millisecond duration) delivered in
100-millisecond trains at 100 Hz. Muscle performance was evaluated by
using a sequence of 10-minute contraction periods of increasing energy
demands, from 4, 8, 15, 30, and 45 tetani per minute (TPM). Tension
development of the GPS muscle group was recorded with a five-channel
polygraph (Gould Inc). Average tension for each contraction frequency
was calculated from the tension measured at the 5th and 10th minute of
contractions.
Tissue Blood Flow
Blood flows to the hind-limb tissues were determined with
radiolabeled 15-µm microspheres after the most demanding contraction
frequency (30 or 45 TPM), as described previously.28
Approximately 2.5x105 microspheres were carefully
dispersed and thoroughly mixed in 0.7 mL of perfusion medium and slowly
(20 seconds) infused into a port of the manifold supplying the
arterial catheter. All perfused tissues were then carefully dissected,
counted to a 1% error with appropriate correction for spillover, and
used for calculation of blood flow, as detailed
previously.28 Absolute blood flow was calculated as
follows:
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Materials
Radioactive 85Sr-labeled microspheres (15±1.0 µm)
with a specific activity of 10 mCi/g were obtained from NEN in a
suspension of 10% dextran containing 0.05% Tween 80 surfactant.
Heparin sodium (10 000 U per vial), extracted from beef lung, was
obtained from Upjohn Co. Bovine serum albumin (fraction V) and the
reagents used for biochemical analysis were obtained from Sigma.
Fresh bovine erythrocytes were prepared by extensive washing (>20 vol)
of acid/citrate/dextrose–titrated blood collected at a local meat
packer.
Analysis of the Data
The results are presented as mean±SEM. Statistical analyses
included repeated-measures ANOVA with individual mean comparisons made
by Tukey's procedure.29 The primary ANOVA evaluated the
main effects of heparin versus saline and the level of training. The
acute-ligated and normal groups were not included in this comparison.
Other ANOVA included repeated-measures evaluation of tissue blood flows
across limbs within each group. Significant differences for main
treatment effects and individual comparisons were recognized at
P<.05.
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Results |
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Exercise Responses
The exercise tolerance of animals that were trained by the constant-speed running protocol (identified as moderate intensity in Fig 1
) improved markedly after the first week of running
after femoral artery ligation. Running duration increased from 7
minutes initially to 40 minutes by week 6 (see Fig 1). We have shown
previously that sedentary animals, who have peripheral arterial
insufficiency and are limited to cage activity, do not appreciably
increase their exercise tolerance.18 It is important to
note that the heparin-treated trained animals run at 20 m/min were
exercised only as long as the saline-injected trained animals. Thus,
the exercise stimulus should be the same between groups.
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The exercise tolerance of animals that were trained by increased
treadmill speeds also improved. This more intense training protocol was
designed to test the possible action of heparin in expanding the
collateral network. As illustrated in Fig 1
, both saline- and
heparin-injected animals were able to run at 25 m/min, after the
initial 15-minute period at 20 m/min, without apparent difficulty.
Seven of 12 heparin-treated rats and 4 of 10 saline-treated rats were
able to run consistently at 30 m/min (P=NS). However, the
heparin-injected animals ran for a longer time (55±5 minutes,
P<.05) compared with the saline-injected group (39±5
minutes) during the exercise tolerance test at a constant speed of 20
m/min.
Perfusion Conditions
Table 1 shows that aortic pressure was initially
100 mm Hg, tended to increase during contractions
(P>.05), and was not different across groups. Thus,
perfusion pressure at the head of collateral channels supplying the
distal limb musculature should be similar across groups.
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Hind-limb muscle masses of the saline- and heparin-treated animals,
including the collateral-dependent tissue of the distal limb and the
GPS muscle group from which force development was measured, were not
different among groups. However, the muscle masses of these groups were
greater (P<.05) than corresponding sections of the
acute-ligated group (see Table 1). Initial force development of the GPS
muscle group was similar across groups, except for a difference between
the sedentary and intensely trained heparin-injected animals. The
reason for this difference is not known.
Muscle Performance
Fig 2 illustrates force development in the
different treatment groups during sequential contraction periods of
increasing energy demands. Muscle performance was the least in the
acute-ligated group and not appreciably increased by 6 to 8 weeks of
sedentary life (limited to cage activity); one free-hand drawn line is
shown for these groups in Fig 2 for simplicity. The development of
fatigue was not different between these saline- and heparin-injected
sedentary groups; therefore, the results have been combined to simplify
the illustration.
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Values for exercised
heparin-treated (trained-heparin) groups were significantly greater
(P<.001) than for trained-saline, sedentary-ligated, and
acute-ligated groups. Since there were no systematic differences
between saline- and heparin-treated sedentary groups, trained-saline
groups exercised at moderate (Mod) and high speed, or trained-heparin
groups exercised at Mod and high speed, for simplicity we have drawn
individual lines corresponding to each pair.On the other hand, the ability to sustain force development was markedly improved (P<.001) in the trained groups that received saline injections. Force development was not different between the moderate and intense training protocols; thus, the results for the saline-trained groups have been combined to simplify the illustration. Finally, force development was even better maintained in the trained animals given heparin injections. Again, there were no differences between the moderate and intense training protocols; thus, the results for the heparin-trained groups have been combined.
Training was necessary for improvement in muscle performance, with the
addition of heparin administration providing the greatest benefit.
Heparin administration alone did not alter muscle performance. The
performance of normally perfused muscle, with flow delivered through
the femoral artery without any obstruction, has been included from
previous work17 for comparison in Fig 2.
Hind-Limb Blood Flow
Ligation of the femoral artery significantly reduces blood flow
capacity to the hind limb. As illustrated in Table 2,
the distal limb tissue is most at risk of ischemia with flows only 20%
to 50% of that observed even in the proximal tissue of the same hind
limbs. Two to 4 days after ligation of the femoral arteries, blood flow
to the GPS muscle group was only 12±2 mL/min per 100 g (see
acute-ligated group; Table 2), 10% to 20% of flows observed
without vascular obstruction. For comparison, we have included normal
blood flows to the distal limb tissues, perfused through the femoral
artery without any vascular obstruction, that were obtained
previously.17 Six weeks of cage activity experienced by
the saline-injected sedentary group did not increase blood flow
significantly (20±4 mL/min per 100 g, .10>P>.05).
Furthermore, heparin administration during 7 to 8 weeks of limited cage
activity did not alter blood flow compared with flow in saline-injected
animals (Table 2). In the present study, blood flow to the bones
(femur and tibia/fibula) was found to be consistent with that
determined previously for the rat24 28 and changed among
groups in a pattern corresponding to limb flow.
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Participation in a training program increased blood flow to the entire
hind limb; however, the effect was dependent on the administration of
heparin. Total hind-limb blood flows (average for both limbs) for the
trained saline-treated groups were not different (P<.10)
from the value for the corresponding sedentary saline-treated control
group; however, total hind-limb blood flow of each heparin-treated
trained group was significantly greater (P<.05) than the
value for each corresponding trained saline-treated group. No training
effect was observed for the proximal limb tissue for either of the
training protocols. Rather, the significant increases
(P<.001) in blood flow were observed in the distal limb
tissues. This is the limb section most at risk of ischemia with femoral
artery ligation and most reliant on collateral-dependent blood flow. As
shown in Table 2, there appeared to be a general influence of exercise
intensity, with the highest blood flows to the GPS found with the
intense training protocol.
As shown in Table 2, the greatest impact of training was evident when
coupled with heparin injections. The improvements in blood flow were
quantitatively the greatest in the collateral-dependent region of the
distal limb tissue of the intensely trained heparin-treated group; for
example, GPS blood flow was approximately threefold higher compared
with the sedentary heparin-treated group.
Results presented in Table 2 identify significant differences in
tissue blood flows across limbs for some experimental groups. This
suggests that differences between limb preparation and/or subsequent
contractions of the distal muscles of one hind limb (left) may have
altered flow distribution. Although the contracting GPS of all groups,
except the two trained groups receiving daily heparin injections,
tended to have lower blood flows, significant differences were reached
in only three groups (Table 2). Subsequent analyses among the
individual muscle sections that constitute the distal hind-limb tissue
identified significant differences across limbs among only a few muscle
sections identified in Table 3. Each of these individual
muscle sections were among the GPS muscle group whose rest length was
stretched for optimal force determination. This suggests that muscle
stretch and/or contractions can reduce blood flow. Interestingly, the
experimental groups that exhibited the highest blood flows (eg, both
heparin-treated trained groups) gave no evidence of differences in
blood flows across limbs. Most important, significant main treatment
effects of heparin administration and training were observed for each
hind-limb analysis. Thus, the difference in flows among a few
individual tissues across limbs in some groups did not confound our
results. Blood flows for individual muscles given in Table 3 are the
average of values from corresponding sections from both limbs, since
only a few significant differences across limbs as identified were
evident.
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Discussion |
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The most significant finding of the present study demonstrates that heparin administration serves to increase blood flow to the hind-limb muscles of physically active animals with peripheral arterial insufficiency. Although the improvement in blood flow was evident in the entire hind limb (Table 2
), the most striking increases were
observed in the distal muscles. This is the primary tissue at risk of
ischemia with femoral artery occlusion and therefore the tissue most
reliant on collateral blood flow.24 30 We believe that
these flows to the distal limb tissue, measured in our perfusion
system, represent maximal values for collateral-dependent blood
flows. First, downstream resistance within the lower limb musculature
was minimized by muscle contractions. Second, blood flows to the GPS
muscles of the sedentary groups (Table 2) were similar to the maximal
collateral-dependent flows determined previously in femoral-ligated
animals during treadmill running.24 In that study,
evidence that maximal flows were achieved came from similar blood flows
at two demanding treadmill speeds.24 Most pertinent to the
present study, the influence of heparin on collateral-dependent
blood flow was contingent on physical activity. Blood flow to the GPS
muscle group was not altered by heparin injections if the animals were
kept sedentary and limited to cage activity (Fig 3). In
contrast, the exercise-induced improvements in collateral-dependent
blood flow were further expanded by heparin injections. This suggests
that the adaptive responses prompted by exercise and the actions of
heparin interact to expand collateral vessel function.
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In the first exercise protocol, care was taken to establish an
equivalent exercise stimulus between saline- and heparin-injected
trained animals. Daily exercise involved treadmill running at a
constant moderate speed, which could be reasonably accommodated by rats
with femoral obstruction.17 24 Duration of running was
increased substantially as exercise tolerance improved (Fig 1). The
heparin-injected animals were removed from the treadmill at the same
time as the saline-injected exercised control animals. Thus, the
difference in blood flow between these groups, illustrated in Fig 3,
represents a specific heparin effect that is not confounded by
differences in the exercise treatment. Our results also suggest that
the intensity of daily exercise is an important factor determining the
magnitude of collateral vessel expansion. Animals ran at higher speeds,
as their exercise tolerance permitted (Fig 1), and exhibited yet
greater increases in collateral-dependent blood flow to the GPS,
especially the heparin-injected group (Fig 3). Although the daily
exercise performances of these saline- and heparin-injected groups were
not matched, it is apparent that heparin administration was effective
in expanding the exercise-induced improvement in collateral-dependent
blood flow.
The increases in collateral-dependent blood flow to the GPS muscle
group of the trained groups were associated with improvements in
functional responses. Exercise tolerance increased progressively with
daily treadmill running (Fig 1). Although this response is not likely
confounded by the performance-enhancing metabolic effect of heparin in
increasing circulating nonesterified fatty acid
concentration,31 since lipid oxidation should not be
favored in relatively ischemic tissue, other actions of heparin cannot
be dismissed. Muscle performance during the in situ stimulation
protocol (Fig 2) was significantly improved by daily physical activity,
even in the trained groups receiving saline injections. Although
training-induced adaptions within the active muscle would serve to
improve oxygen exchange and increase muscle performance in the absence
of any blood flow increase,17 18 it is probable that the
increased blood flow was also important. This is especially true in the
heparin-treated trained groups, in which the improvements in muscle
performance and blood flow were remarkable (Fig 2), exhibiting the
greatest recovery from the deficits caused by obstruction of the
femoral artery. Plotting the relation between muscle performance and
measured blood flow among all of our treatment groups illustrates the
importance of blood flow (Fig 4). Even low energy
demands, created by a few tetanic contractions per minute, caused
deficits in performance in those groups in which collateral-dependent
blood flows were meager. As collateral-dependent blood flows increased,
muscle performance could be better sustained. Thus, as found in
patients,32 the greater the improvement in
collateral-dependent blood flow, the greater was the recovery of muscle
function.
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It is well known that exercise training prompts angiogenesis of the capillary network within active muscle.23 The basis for this response is unclear but may be related to factors such as wall shear established by blood flow, ischemia, metabolic factors, and/or release of angiogenic factors.23 Accepting that contractions establish a low vascular resistance in the distal musculature, our study adds support to previous work indicating that physical activity can prompt enrichment of the upstream vessels of the collateral network in the presence of peripheral arterial insufficiency.15 19 20 21 22 30 We now demonstrate that heparin serves to expand this exercise-induced response. This action of heparin is similar to that observed by the augmented collateral function in the heart in models with experimental ischemia8 and in patients with coronary insufficiency.12 Similar to the present study, heparin was effective in expanding collateral-dependent blood flow in patients with coronary insufficiency only when associated with physical activity.12 Thus, exercise appears to impart the essential stimulus for angiogenesis, which is then amplified by heparin administration.
On the basis of a summary of evidence,23 we hypothesize
that increased blood flow through existing collateral channels during
treadmill exercise24 prompts increased wall shear and
initiates endothelium-specific events that promote
angiogenesis.23 bFGF could be important in this process
because it is a potent endothelial cell mitogen found among the
extracellular matrix.4 5 Unger et al33
recently demonstrated that daily injections of bFGF into the coronary
circumflex artery, distal to the site of progressive vessel occlusion,
enhances angiogenesis and collateral-dependent blood flow to the
myocardium. Chleboun and Martins34 have provided evidence
that bFGF can impart similar effects with peripheral limb ischemia. In
the present study, it was determined that the administration of
heparin could serve to augment liberation of FGF from the extracellular
matrix,4 5 assist in FGF–endothelial cell
interaction,2 and thereby enrich the development of
angiogenesis. The involvement of bFGF is likely34 but not
assured, since another potentially more specific angiogenic growth
factor could be important. Takeshita et al35 showed that
vascular endothelial growth factor (VEGF) provides a potent stimulus to
angiogenesis in the ischemic limb of rabbits. It is apparent that
events related to exercise (eg, flow-established wall shear) provide
the foundation for adaptation and that heparin helps amplify the
response. Differences in collateral-dependent blood flow between the
moderate- and high-speed training protocols (Fig 3) imply differences
in the stimulus for angiogenesis. This may seem difficult to reconcile,
since running at even the lower treadmill speed is sufficient to
require peak collateral-dependent blood flow to the distal limb
tissue.24 However, a difference in stimulus for
angiogenesis may be imparted by increasing the hind-limb mass that is
relatively ischemic. Increased flow demands by some, more proximal,
musculature could increase flow through the collateral channels. This
would occur at higher running speeds, since the recruitment of
additional motor units is required to achieve a greater power
output.36 37 On the other hand, the lack of a
heparin-induced expansion of collateral-dependent blood flow in the
absence of exercise implies that femoral artery occlusion by itself
imparts an insufficient stimulus for angiogenesis, at least in the
relatively short time frame of the present study. This is not
surprising, since the flow capacity of the distal limb musculature in
the presence of femoral artery ligation exceeds the flow demands of
resting muscle. Thus, the extent of ischemia and the potency of
angiogenic intervention (eg, bFGF34 and
VEGF35 ) are likely to be important factors in the vascular
adaptation.
The actions of heparin in expanding exercise-induced collateral blood
flow could be most significant, if applicable to patients with
intermittent claudication. It should be recognized, however, that in
the present study peripheral arterial insufficiency was caused by
obstruction of each femoral artery at a proximal site. Although this
places the relatively large distal limb mass at risk of ischemia during
activity,24 the potential for collateral flow improvement
is appreciable. Thus, the magnitude of the flow increase observed in
our animals may not be applicable to that possible in patients.
Interestingly, the route for systemic heparin administration does not
appear to be critical. The vascular endothelium is the main site of
heparin distribution, whether administered by intravenous or
intraperitoneal routes.27 Thus, it is unlikely that the
initial 4-week period of intravenous, followed by 2 to 4 weeks of
intraperitoneal, administration of heparin confounded our results.
Although our dose of large molecular weight heparin was sufficient to
double clotting time, as also found with patients,8 11 12
no complications were found in our study.
Few studies have assessed the potential value of heparin in managing
patients with intermittent claudication. Mannarino et al38
did find that treatment with low molecular weight heparin for 6 months
lengthens pain-free walking time by 25%. Peak calf muscle blood
flow was not altered; however, this was not a training study. The
authors attributed the improved exercise tolerance to the
hemorrheological influence of heparin. We know of no studies that have
evaluated the actions of heparin in exercise-induced collateral
development in patients with intermittent claudication. Although a
number of training studies have provided evidence for an
exercise-induced increase in collateral blood flow in patients in the
absence of heparin,15 20 21 22 many clinical evaluations of
exercise responses have been unable to document improvements in
collateral blood flow.13 14 16 Whether this absence of
exercise benefit is confounded by complexities in the extent and nature
of the vascular obstructions or by inadequacies in the exercise
stimulus is not known. Evidence that physical activity can initiate
collateral vessel development15 19 20 21 22 30 and the
present findings that heparin serves to expand this response raise
the potential for more effective clinical management of claudicants not
encumbered by contraindications for exercise. If the mechanism of
action is found to be flow-initiated endothelial cell events within the
collateral channels, then routine simple walking for sufficient periods
of time should be effective in the presence of heparin
administration.
|
Acknowledgments |
|---|
This study was supported by National Heart, Lung, and Blood Institute grant HL-37387. The excellent technical assistance of Stacey Melnyk and Bruce Searles is gratefully acknowledged.
Received July 7, 1994; accepted November 8, 1994.
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References |
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