Basic Fibroblast Growth Factor Increases Collateral Blood Flow in Rats With Femoral Arterial Ligation
The potential for exogenous infusion of basic fibroblast growth factor (bFGF) to increase collateral blood flow to dependent tissue was quantified in adult male rats with peripheral arterial insufficiency. Occlusion of the femoral artery at a proximal site did not infringe on resting blood flow to the distal hindlimb muscle, but did remove the blood flow reserve. Blood flow to the hindlimb muscles was measured with radiolabeled microspheres using an isolated hindlimb preparation perfused in the descending aorta (Krebs-Henseleit bicarbonate, 5% albumin medium containing red blood cells [40% hematocrit]) at 100 mm Hg. Calf muscle blood flow changed modestly (≈50%) with infusion of only the carrier (heparin/saline), increased markedly over the first 2 weeks of bFGF infusion (1 μg/d into the femoral artery), but did not change further with infusion for 4 weeks. Waiting 2 weeks after 1 week of bFGF infusion did not further increase the intermediate improvement in blood flow. The improved collateral blood flow and increased muscle capillary density likely contributed to the enhanced muscle performance observed during nerve stimulation in situ. X-ray films of arterial casts identified an expansion of upper thigh vessels that likely served as collaterals. In animals with peripheral arterial insufficiency, short-term exogenous infusion of bFGF is effective at inducing vascular expansion that is sufficient to improve the flow reserve of dependent distal tissue and enhance muscle function. This raises the expectation that a similar response in patients with peripheral arterial insufficiency would significantly improve morbidity, including the symptoms of intermittent claudication.
Recent evidence indicates that the heparin-binding growth factors (aFGF, bFGF, and VEGF) exert angiogenic effects in vivo after experimental vascular occlusion. Administration of each of these growth factors appears to be effective in prompting angiogenesis after occlusion of the primary supply vessel(s) to the hindlimbs of rabbits and rats. Angiographic evaluation of vessel enlargement and expansion,1 2 3 4 recovery of transcutaneous Po2,1 improved indexes of blood flow,2 3 4 5 6 and enhanced capillarity1 3 4 provide evidence that vascular adaptations can be induced in response to growth factor administration after ischemia. Based on this evidence, the extent of ischemia is expected to be lessened; however, there has been little quantitative assessment of the actual blood flow changes associated with this vascular remodeling. An exception is the work of Bauters and coworkers,2 4 in which direct measurement of blood flow through the internal iliac artery nicely demonstrated an increase after occlusion; however, measurement of collateral flow was not ensured nor was the flow to reach the distal ischemic tissue determined. Thus, at present, the nature and extent to which growth factor–induced vascular remodeling improves blood flow to the distal limb tissue most at risk for ischemia is not known. Although frank ischemia leading to muscle atrophy and tissue pathology appears to be lessened by growth factor administration,1 7 it is unclear whether growth factor–induced vascular remodeling can be sufficient to establish a flow reserve useful to enhance the function of involved limb muscles. Any increase in flow reserve could meaningfully improve mobility, especially in conditions exhibiting intermittent claudication.
The purpose of the present study was to quantify the improvement in collateral-dependent blood flow induced by bFGF infusion in an experimental model of peripheral arterial insufficiency in which hindlimb blood flow capacity is markedly diminished without limiting flow below resting tissue needs. Using an isolated perfused hindlimb preparation, blood flow was then determined with radiolabeled microspheres while resistance of the distal limb muscles was minimal, and their contraction performance was measured during stimulation of the motor nerve. The nearly threefold increase in collateral-dependent blood flow to the calf muscles during short-term infusion of bFGF significantly improved muscle performance during contractions.
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-h/12-h light/dark cycle and fed Purina rat chow and tap water ad libitum. This research project was approved by the Committee for the Humane Use of Animals of the State University of New York Health Science Center at Syracuse.
The effect of intra-arterial infusion of bFGF on collateral-dependent blood flow was assessed in rats after bilateral ligation of the femoral artery. Femoral artery ligation markedly limits flow reserve to the distal hindlimb musculature of adult rats.8 9 10 However, flow capacity remains greater (approximately two to four times) than that observed during resting conditions, depending on muscle fiber type.11 Thus, the animals serve as a model of intermittent claudication and not the more severe peripheral arterial insufficiency that leads to ischemia at rest and complications resulting in pathological changes, tissue necrosis, or gangrene observed with more proximal vascular obstructions.6
At the time of surgical ligation of the left femoral artery, a catheter was inserted upstream through the ligature for delivery into the distal iliac artery, the source of collateral flow (see below for exception). The catheter was connected to an Alzet osmotic pump (2-week capacity) for infusion of bFGF or carrier in animals of the following groups: (1) control heparin-infused group (100 IU/d; for 2 weeks, n=8; for 4 weeks, n=4), (2) 1-week bFGF-infused group (1 μg/d containing 100 IU/d heparin, n=6), (3) 2-week bFGF-infused group (1 μg/d containing 100 IU/d heparin, n=8), (4) 4-week bFGF-infused group (1 μg/d containing 100 IU/d heparin, n=5), and (5) group infused for 1 week with bFGF with a 2-week time delay before blood flow determination (1 μg/d containing 100 IU/d heparin, n=8). All animals were evaluated over a short time period (2 to 5 days) after the experimental times given above for the groups. The 4-week duration of infusion of bFGF was achieved by replacing the first osmotic pump at day 14. An additional group of animals (n=7) was evaluated 3 days after ligation of the femoral arteries without any infusion to characterize the initial condition established by femoral artery ligation. Heparin was infused with bFGF to optimize growth factor effectiveness, since heparin binding extends the circulating time and increases the plasma concentration during exogenous administration.12 All rats were limited to cage activity.
Femoral Artery Ligation and Isosmotic Pump Installation
The surgical procedure for ligation of the femoral artery has been described in detail in our previous work.9 In brief, under ketamine-acepromazine anesthesia (100 mg/0.5 mg per kilogram body weight), the right femoral artery was ligated just distal to the inguinal ligament with a 3-0 surgical suture; topical antibiotic powder (Neo-Predef, Upjohn) was placed on the wound before closure with skin clips. The left femoral artery was similarly ligated, except that a catheter (PE-50) was inserted upstream through the ligature; however, in some of the early animals (ie, 3 [in the 2-week group] of 27 receiving bFGF and 3 [in the 2-week group] of 12 receiving heparin), the catheter was placed into the femoral artery for infusion downstream. The responses of these animals were not different from the responses of upstream-infused animals and have been combined in their respective groups. The catheter was connected to an osmotic pump containing either saline, heparin, or heparin plus bFGF. The catheter was primed with the same solution as in its pump. A subcutaneous tunnel was made under the abdominal skin for placement of the pump. The incision was closed with skin clips after application of topical antibiotic powder. Rats in the acute group received bilateral femoral artery ligation without installation of an osmotic pump.
Miniosmotic pumps delivering 0.50±0.02 μL/h for 14 days (Alzet model 2002, Alza Corp) were used after preparation according to the manufacturer's instructions. In preliminary work, we verified that the delivery of the pump was essentially complete (95±1% of its contents [saline containing 51Cr-EDTA and 5% BSA] at 19 days, n=3). Further, at the time of autopsy, we routinely evaluated pump contents for any residual volume. Pumps were filled to achieve one of the following: heparin infusion at 340 IU/kg body weight, as used previously9 or bFGF infusion at 4.0 μg/kg body weight plus heparin infusion at 340 IU/kg body weight. Each pump also contained 5% glycerol to stabilize protein and 0.02% sodium azide as a bacteriostat, as previously described by others.13
A second bFGF-containing pump was inserted after 14 days to achieve the 4-week infusion of bFGF. With the animals under ketamine-acepromazine anesthesia, the expended pump was removed, and the patency of the femoral artery catheter was checked, refilled with the bFGF-heparin solution, and then connected to a new pump. Each animal made an uneventful recovery and was returned to its cage.
Each rat was prepared for hindquarter perfusion as described in detail previously.14 In brief, the animal was anesthetized with sodium pentobarbital (60 mg/kg), and a midline abdominal incision was performed to allow access to and removal of the testes, bladder, seminal vesicles, prostate gland, and small and large intestines in order to expose the descending aorta and inferior vena cava. An ≈1.5-cm length of the aorta and vena cava just below the renal artery branch was dissected free and prepared for cannulation. The skin was then removed from both hindlimbs. The left hindlimb was prepared for muscle stimulation as follows: the left sciatic nerve trunk was exposed, high in its course near gluteus muscle, ligated, cut, and made ready for placement on electrodes. Distal branches were severed to limit stimulation to the distal limb muscles. After perfusion began, the free end of the nerve was placed over a platinum bipolar electrode connected to a Grass model S48 stimulator. Isometric tension of the GPS muscle group was measured by attaching the Achilles tendon to a Cambridge level system (series 300 B, Cambridge Technology Inc) for measurement of isometric force development. The distal hindlimb was fixed by a pin though the distal femur.
After surgical preparation, the animal was transferred to a temperature-regulated (37°C) Plexiglas chamber. After an intra-arterial (carotid) injection of heparin (2000 IU), the abdominal aorta or vena cava was cannulated with a 16-gauge (aorta) or 14-gauge (vena cava) polytetrafluoroethylene catheter (IV catheter, Becton Dickinson), respectively. Once the perfusion catheters had been inserted and secured in place, perfusion was begun immediately, and the animals were killed with 0.5 mL pentobarbital sodium injected via the catheter in the carotid artery. The period of ischemia before initiating perfusate flow was generally <15 seconds. The perfusion medium (≈300 mL) was recirculated after the initial volume (20 to 30 mL) of venous effluent had been discarded. Total inflow rate was measured by using timed collections of the venous effluent with a graduated cylinder. The flow rate and perfusion pressure were gradually increased until the net perfusion pressure (aortic pressure) reached 100 mm Hg. The perfusion pressure in the aorta was determined by subtracting the pressure drop across the 16-gauge catheter from the pressure measured by an on-line transducer. When perfusion pressure and perfusion rate were stable, muscle contractions were started. At the end of muscle contractions, tissue-specific blood flows were determined by using radiolabeled microspheres. Ligatures were put at the base of the rat tail and on the right and left feet to block flow to these areas.
The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing washed bovine erythrocytes, 4 g/100 mL BSA, 100 μU/mL bovine insulin, amino acids typical for rat blood, and 5 mmol/L glucose (maintained constant with periodic additions of glucose during perfusion), with a 39% to 40% hematocrit, as used routinely.9 15 16 17
Tetanic contractions were elicited with supramaximal square-wave pulses (6 to 8 V, 0.1-millisecond duration) delivered in 100-millisecond trains at 100 Hz to the sciatic nerve. Resting muscle length was adjusted to produce maximal active tension. Sequential 10-minute contraction periods were performed at train frequencies of 4, 8, 15, 30, and 45 tetani per minute. Tension output of the GPS muscle group was continuously recorded with a Gould 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.
Blood Flow Distribution
The distribution of blood flow in the hindlimb was determined with radiolabeled 85Sr microspheres after the 10 minutes of stimulation at the end of contraction, as described previously.14 Tissues of both hindlimbs (see Tables), except the right GPS muscles taken for histochemistry (see below), and the trunk between kidney and the base of the tail were dissected as previously described.9 15 The fast-twitch white and red quadriceps muscle sections were obtained from the superficial and deep vastus lateralis muscle, respectively. Microsphere radioactivities of all tissues were determined at a 2% error with a gamma counter (Beckman Gamma 8000). The fraction of radioactivity found in a tissue, relative to the total activity infused, is determined by its relative blood flow. Absolute blood flow was calculated as follows:where CPMtissue is counts per minute in tissue and CPMtotal injected is total counts per minute injected (ie, the sum of all tissues perfused and counted). Muscle blood flow is expressed per 100 g of tissue weight.
Muscle Sampling and Histochemical Analyses
Before stimulation of the left distal limb muscles, the GPS muscle group of the right limb was resected to obtain samples for histochemistry. A ligature was placed around the lower leg just distal to the knee to eliminate bleeding. This did not impact on blood flow to the more proximal tissue, as blood flows to the quadriceps muscles of the left and right limbs were the same (data not shown). The superficial fast-twitch white (predominately type IIb) and the deep fast-twitch red (predominately type IIa) sections of the gastrocnemius muscle (medial and lateral heads, respectively), slow-twitch red soleus muscle (predominately type I), and the plantaris muscle (mixed fiber type) of this right limb were then used to assess capillarity. An ≈5-mm-thick cross section was cut from each muscle sample, fixed to a cork with Tissue-Tek (Miles Inc), and frozen in isopentane chilled by liquid nitrogen. The muscle samples were stored at −80°C for histochemical analysis of capillarity.
Capillarity was determined from 10-μm frozen sections using a modification of the alkaline phosphatase method of Seligman et al,18 as performed previously.16 17 A periodic acid-Schiff reaction for glycogen and glycoprotein was performed after removal of glycogen with diastase. Finally, the section was subsequently counterstained with metanil yellow. This sequence of reactions and stains renders the capillaries blue-black, the fibers yellow, and the external lamina just outside the sarcolemma magenta. Muscle sections from control and bFGF-treated animals were paired on the same slide. Myocyte and capillary number and the number of capillary contacts surrounding fibers (capillary-to-fiber contacts) were determined from 20 nonoverlapping fields (0.06 mm2 each) from ×400 projections of each muscle. The capillaries around fibers were tabulated for at least 100 fibers for each section.
A qualitative assessment of vascular remodeling was made with x-ray films of arterial casts created by perfusing the descending aorta with liquid methacrylate containing lead (Polyscience, Inc). The liquid methacrylate was mixed with sufficient catalyst to harden in ≈5 minutes. Initially, the liquid plastic was infused at a constant rate with a syringe pump until flow stopped, ≈1.5 to 2.5 mL, depending on the treatment group. Similar filling conditions were achieved with slow, deliberate, manual delivery from the syringe with the same volume delivered at the cessation of flow. Standard planar x-ray films were taken of the skinned lower carcass.
Recombinant human bFGF was obtained from Scios Nova Inc and R&D Systems. Radioactive 85Sr-labeled microspheres (14.2±0.92 μm diameter) with a specific activity of 9.6 mCi/g were obtained from NEN in a suspension of 10% dextran containing 0.05% Tween 80 surfactant. Beef lung heparin sodium was obtained from Upjohn Co. BSA (fraction V) and the reagents used for biochemical analysis were obtained from Sigma Chemical Co. Fresh bovine erythrocytes were prepared by extensive washing (>20 vol) of acid-citrate-dextrose-collected blood obtained at a local meat packer.
Analysis of the Data
Results are presented as mean±SE. The data were subjected to ANOVA. Significant differences for main treatment effects and individual mean comparisons, made by Tukey's procedure,19 were recognized at P<.05.
Even though the arterial perfusion pressures in the descending aorta were similar across groups (≈100 mm Hg), total inflows to the hindquarters were highest (P<.025) in animals that received bFGF infusion for 2 and 4 weeks (Table 1⇓). Thus, the calculated total vascular resistance of the hindquarter was lowest in the groups treated with bFGF for the longest duration. As discussed below, this was due to lower collateral resistance.
Hindlimb Blood Flow
As shown in Table 2⇓, bFGF infusion significantly increased blood flow (P<.001) to the entire hindlimb. Although there was some change in blood flow to the proximal hindlimb tissue (4-week bFGF-infused group), the primary region of improved blood flow was the distal hindlimb. This is best illustrated by flows to the GPS muscle group (Fig 1⇓). Compared with the initial blood flow observed after ligation, blood flow tended to increase with only 1 week of bFGF infusion, increased significantly (P<.001) with 2 weeks of bFGF infusion, but did not increase further with 4 weeks of bFGF infusion. Interestingly, blood flows of animals receiving only 1 week of bFGF infusion, but evaluated after 2 weeks, did not increase further. This implies that the increase in blood flows with 2 weeks of bFGF infusion did not occur simply as a result of the longer duration for vascular development.
Changes in blood flows to individual muscles and/or muscle fiber type sections were consistent with their location within the proximal or distal region of the hindlimb (Table 3⇓). Specifically, all of the individual muscle sections of the distal hindlimb exhibited improved blood flow with bFGF infusion. Note that blood flows were not uniform within the tissue sections but varied in a predicted manner on the basis of muscle fiber type composition. Even with the heterogeneity of blood flows within the distal tissues of a group, the influence of bFGF infusion appears to be uniform, with significant increases in all muscle fiber sections.
In contrast to the response of the distal hindlimb tissue, the only improvement in blood flow to any tissues of the proximal hindlimb was observed in the 4-wk bFGF-infused group (Table 3⇑). Furthermore, this improvement was not consistent across all tissues but was limited in its distribution; the increase in blood flow in the quadriceps (P<.005) of the 4-week bFGF-infused group is the most noteworthy.
A functional benefit in muscle performance was likely imparted by the increase in blood flows to the GPS muscle group with bFGF infusion. As illustrated in Fig 2⇓, the ability of this muscle group to sustain tetanic tension development was markedly better in the bFGF-treated animals. This is evident during the easy contraction conditions (lower frequency), where force is high primarily because of the contributions of the fatiguable fast-twitch white fibers, and during the intense contraction conditions (higher frequency), where the relatively high blood flow high-oxidative fibers remain and contribute to tension development. The responses of the 2- and 4-week bFGF-infused groups were similar and have been combined for simplicity; furthermore, the response of the 1-wk bFGF-infused animals was intermediate to that shown but was not included for clarity of illustration.
As illustrated in Fig 3⇓, fill of the liquid plastic to define the larger conduit vessels of the proximal hindlimb was modest in the heparin control group. The iliac artery leading to the occlusion of the femoral artery is evident. Note the hypogastric trunk, which originates deep off the distal iliac and gives rise to supply vessels that are filled distally. The arterial cast of the bFGF-infused animals was both more abundant and filled further distally. In fact, the femoral artery, distal to the femoral occlusion, is filled with material.
Capillary Contact Per Fiber
Muscle capillarity of normal animals (acute ligated group, n=4) was not different from that obtained for the heparin-infused control group (n=6); therefore, the values were combined in Table 4⇓ for comparison with the bFGF-infused groups. Muscle capillarity was increased by bFGF infusion, but only after a 4-week duration. Furthermore, the enhanced muscle capillarity was found in the two high-oxidative fiber type sections, but not in the low-oxidative white gastrocnemius section.
The primary finding of the present study is that administration of the angiogenic growth factor, bFGF, led to quantitative improvement in blood flow to the tissue most at risk of ischemia with obstruction of its primary arterial supply. After the initial insult of femoral artery occlusion, which eliminated the flow reserve to the calf muscles, administration of bFGF improved blood flow ≈200% to 300% during muscle contractions, whereas without the angiogenic growth factor, blood flow increased only marginally (≈50%; see Fig 1⇑). It is unlikely that the known vasodilatory effect of bFGF20 contributed to this increase in flow observed in the rats after the infusion of bFGF. First, our dose of bFGF infusion (1 μg/d) is well below that necessary to elicit a hypotensive response on bolus administration (0.3 μg20 ). Second, our blood flows were determined during in situ perfusion using a large blood volume (≈300 mL) in the absence of any added bFGF. Rather, we interpret this improvement in blood flow with bFGF infusion as an increase in flow capacity of the collateral circuit circumventing the proximal obstruction. The vascular resistance of the distal limb tissue was effectively minimized by muscle contractions, a powerful stimulus for vasodilatation.11 21 Normal flow capacity of the calf muscles of a nonligated rat has been found in previous studies15 21 to be in excess of 250 mL/min per 100 g (expressed at the same perfusion pressure [100 mm Hg] as in the present study). By comparison, the measured blood flows to the calf muscles in our femoral-ligated groups are far below their normal flow capacity (eg, only 5% to 20% of normal). Thus, the primary vascular resistance defining flow to the calf muscles has become the series collateral resistance. For example, using the 12 mL/min per 100 g blood flow to the calf muscles of the acute group (see Fig 1),⇑ it can be calculated that the upstream collateral resistance is approximately 20-fold greater than the calf muscle minimal resistance established by our contraction conditions (per Mackie and Terjung11 ).
Similar to the observations of others,3 4 7 we have found an expansion of the vessels that circumvent the vascular obstruction, in this case apparent from the x-ray films of the arterial casts (see Fig 3⇑). In contrast to the modestly defined arterial tree in the control animals, the bFGF-treated animals exhibited a greatly expanded vascular tree that was more abundant and extended further distally. These vessels likely served as collateral vessels to meaningfully increase the downstream perfusion pressure. This would provide the net benefit of an improved blood flow to the distal tissue and would contribute to the retrograde filling of the distal femoral artery evident in Fig 3⇑.
Recent work has demonstrated that the angiogenic growth factor–induced gain in distal perfusion is sufficient to improve the deficit in pressure index,2 3 7 eliminate muscle atrophy,7 alleviate tissue hypoxia,1 and minimize tissue necrosis.1 7 It may be that the recovery of flow in these studies was even greater than that evident in their qualitative assessments. The present findings raise the potential that sufficient vascular expansion can be induced to recover flow reserve well beyond resting needs. Blood flows to the various fiber sections of the calf muscles of the bFGF-infused rats increased to ≈4- to 10-fold greater than those obtained previously for resting muscle.11 The functional benefit is apparent by the improved muscle performance observed during contractions (see Fig 2⇑). Although the attendant greater oxygen delivery is likely the primary contributor to the improved muscle performance, the enhanced capillary density demonstrated here (see Table 4⇑) and elsewhere1 4 7 also could have been important. Coincident with an increased muscle capillary density is an enhanced capillary/tissue oxygen exchange capacity.17 In fact, this adaptation can be evident in claudicants,22 especially when physically active, and should improve muscle function even if blood flow is not improved.17 Thus, both actions of bFGF (ie, those expanding the conduit vasculature that circumvent the obstruction and those expanding the microvasculature within muscle) should prove beneficial in peripheral arterial insufficiency.
A second important finding of the present study is evident in the time course of response to bFGF infusion (see Fig 1⇑). Collateral blood flow increased progressively over the first 2 weeks of bFGF infusion. Although we do not have definitive evidence, this rapid response implicates a process involving the expansion of existing vessels, an inference apparent previously.2 3 4 The initiating stimulus could be related to hemodynamic changes established by the vascular obstruction (eg, flow, pressure, and shear stress), which are thought to be important in vascular remodeling.23 Interestingly, the partial improvement in blood flow to the calf muscles observed at only 1 week of bFGF infusion did not increase further when a longer duration for vascular development was permitted, in the absence of continued bFGF infusion. Thus, it appears that our limited 1-week application of bFGF did not optimize vascular remodeling. Whether a higher dose would have been more effective7 is not known. Importantly, the partial recovery of flow capacity in the first week did not preempt the vasculature from continued responsiveness to bFGF-induced changes evident during the second week of bFGF infusion. The absence of any further increase with 4 weeks of bFGF infusion, however, implies that vascular remodeling in the upper hindlimb was complete. On one hand, it may be that the stimuli that led to vascular expansion abated as the flow capacity partially recovered. This could be related to modulation of the bFGF receptor, as the continued infusion of bFGF during the second week was effective at inducing flow improvement, whereas it was not during the subsequent 2 weeks. It is difficult to argue convincingly that an alteration in local ischemia within the upper hindlimb is the dominant variable, since the primary ischemic tissue is far downstream distally (see Table 2⇑); rather, enlargement of the conduit vessels would subsequently reduce flow velocity and shear and thereby arguably diminish the prompting stimulus. On the other hand, there is a suggestion that neovascular changes are still progressing throughout the 4 weeks of bFGF infusion. Although blood flows to the distal hindlimb muscles were virtually identical in the 2- and 4-week bFGF-infused groups, blood flow to the quadriceps muscle of the proximal hindlimb was greater (see Table 3⇑). This muscle group is near the ligation site and could have benefited from neovascular sprouting from the vessel stump, similar to the elongation observed on occasion by Bauters et al.2 Our arterial vessel imaging does not permit sufficient resolution to provide any insight. Therefore, future morphological evaluation of the proximal region surrounding the obstruction could demonstrate two actions of bFGF that provide collateral flow: (1) a rapid-onset enlargement of existing conduit vessels to improve bulk flow distally and (2) sprouting angiogenesis to improve flow capacity locally nearer the site of the obstruction.
It is well recognized that a flow deficit is a powerful stimulus for vascular remodeling, even without the intervention of exogenous growth factor(s).23 Further, Bauters et al2 and Takeshita et al3 found an direct relationship between an improvement in apparent blood flow and angiographic score prompted by exogenous VEGF and the initial vascular deficit caused by the occlusion. This implies that the greater the initial flow deficit, the greater is the improvement in vascular remodeling, and argues that ischemia is required for optimal effectiveness of growth factor–induced angiogenesis. However, when the flow deficit was too great, establishing the risk of tissue necrosis, growth factor administration was counterproductive.1 Thus, control and knowledge of the extent of ischemia produced by a vascular obstruction is an important experimental requisite. Peripheral ischemia was introduced in our experiments with bilateral ligation of the femoral artery just distal to the inguinal ligament. This single proximal obstruction deprived the distal hindlimb muscle of its flow reserve, thereby introducing the symptoms of intermittent claudication, but did not infringe on resting blood flow needs (see Mackie and Terjung11 ). It should be recognized that this model does not represent the broad spectrum of vascular obstructions found clinically in patients with peripheral arterial insufficiency. Furthermore, the large improvement in collateral-dependent blood flow observed in the present study with bFGF infusion may not be characteristic, as the single proximal obstruction site may have simplified vascular remodeling. However, as with previous work,1 2 4 7 24 25 it is apparent that significant and beneficial vascular remodeling can be induced with angiogenic growth factor administration after the introduction of ischemia.
We included heparin with the infusion of bFGF in an attempt to optimize vascular remodeling. This may not have been necessary, as others have recently shown that the angiogenic response to growth factors is found regardless of the presence of added heparin.1 2 4 7 24 25 Furthermore, our delivery of bFGF into the affected limb appears unnecessary, as the angiogenic growth factors are effective via systemic administration.4 25 This was even suggested in the present experiment by similar improvement in collateral blood flow to the tissue of the contralateral hindlimb; unfortunately, we were not able to make systematic evaluation, since most of the contralateral calf muscles were taken for histological evaluation before blood flow determination. Vascular remodeling in the contralateral limb would occur in response to bFGF delivered via arterial inflow after the initial venous return after infusion into the left hindlimb. Thus, the route of administration may not specifically require arterial infusion of bFGF near the site of vascular occlusion (authors' unpublished data, 1995).
In summary, the present study demonstrated that bFGF infusion produced a marked improvement in collateral blood flow to the calf muscles of rats with femoral artery ligation. This increase in blood flow was greatest after 2 weeks of bFGF infusion and significantly improved muscle performance during contractions. Similar vascular remodeling in patients could meaningfully improve collateral blood flow and greatly impact patient morbidity.
Selected Abbreviations and Acronyms
|aFGF, bFGF||=||acidic FGF, basic FGF|
|FGF||=||fibroblast growth factor|
|VEGF||=||vascular endothelial growth factor|
This study was supported by National Institutes of Health grant HL-37387. The excellent technical assistance of J. King and K. Furukoski and a generous gift of bFGF from Dr Judith Abraham (Scios Nova Inc) are gratefully acknowledged.
- Received December 6, 1995.
- Accepted March 26, 1996.
Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg.. 1992;16:181-191.
Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol.. 1994;267:H1263-H1271.
Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest.. 1994;93:662-670.
Takeshita S, Pu L-Q, Stein LA, Sniderman AD, Bunting S, Ferrara N, Isner JM, Symes JF. Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation.. 1994;90:228-234.
Yang HT, Terjung RL. Angiotensin-converting enzyme inhibition increases collateral-dependent muscle blood flow. J Appl Physiol.. 1993;75:452-457.
Yang HT, Ogilvie RW, Terjung RL. Heparin increases exercise-induced collateral blood flow in rats with femoral artery ligation. Circ Res.. 1995;76:448-456.
Yang HT, Ogilvie RW, Terjung RL. Training increases collateral-dependent muscle blood flow in aged rats. Am J Physiol.. 1995;268:H1174-H1180.
Mackie BG, Terjung RL. Blood flow to different skeletal muscle fiber types during contraction. Am J Physiol.. 1983;245:H265-H275.
Gorski J, Hood DA, Terjung RL. Blood flow distribution in tissues of perfused rat hindlimb preparations. Am J Physiol.. 1986;250:E441-E448.
Yang HT, Dinn RF, Terjung RL. Training increases muscle blood flow in rats with peripheral arterial insufficiency. J Appl Physiol.. 1990;69:1353-1359.
Yang HT, Ogilvie RW, Terjung RL. Low-intensity training produces muscle adaptations in rats with femoral artery stenosis. J Appl Physiol.. 1991;71:1822-1829.
Yang HT, Ogilvie RW, Terjung RL. Peripheral adaptations in trained aged rats with femoral artery stenosis. Circ Res.. 1994;74:235-243.
Seligman AM, Heymann H, Barrnett RJ. The histochemical demonstration of alkaline phosphatase activity with indoxyl phosphate. J Histochem Cytochem.. 1954;2:441-442.
Steel RGD, Torrie JH. Principles and Procedures of Statistics. New York, NY: McGraw-Hill Publishing Co; 1960.
Cuevas P, Carceller F, Ortega S, Zazo M, Nieto I, Gimenez-Gallego G. Hypotensive activity of fibroblast growth factor. Science.. 1991;254:1208-1210.
Hudlicka O, Brown M, Egginton S. Angiogenesis in skeletal and cardiac muscle. Physiol Rev.. 1992;72:369-417.
Banai S, Jaklitsch MT, Shou M, Lazarous DF, Scheinowitz M, Biro S, Epstein SE, Unger EF. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation.. 1994;89:2183-2189.
Lazarous DF, Scheinowitz M, Shou M, Hodge E, Rajanayagam MAS, Hunsberger S, Robison WG, Stiber JA, Correa R, Epstein SE, Unger EF. Effects of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart. Circulation.. 1995;91:145-153.