Heparin Increases Exercise-Induced Collateral Blood Flow in Rats With Femoral Artery Ligation
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.
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.
Materials and Methods
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.
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.
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
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.
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.
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.
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).
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 2.5×105 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: where CPMtissue is counts per minute in tissue, and CPMtotal injected is total counts per minute injected. The total CPM injected was obtained by summing the CPM of all tissues, since the hind-limb vasculature effectively traps nearly all (97%) 15-μm microspheres.28 Muscle blood flow is then calculated per gram of tissue wet weight.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
- © 1995 American Heart Association, Inc.
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