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Cardiovascular Research 2003 59(4):997-1005; doi:10.1016/S0008-6363(03)00522-4
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Copyright © 2003, European Society of Cardiology

Enhanced hindlimb collateralization induced by acidic fibroblast growth factor is dependent upon femoral artery extraction

James C Hersheya,*, Halea A Corcorana, Elizabeth P Baskina, David B Gilbertob, Xianzhi Maoc, Kenneth A Thomasc and Jacquelynn J Cooka

aDepartment of Pharmacology, Merck Research Laboratories, West Point, PA 19454, USA
bLaboratory Animal Resources, Merck Research Laboratories, West Point, PA 19454, USA
cCancer Research, Merck Research Laboratories, West Point, PA 19454, USA

james_hershey{at}merck.com

* Corresponding author. Tel.: +1-215-652-9943; fax: +1-610-652-3811.

Received 5 February 2003; revised 11 July 2003; accepted 14 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. Objective: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S117 in the absence of a peripheral blood flow deficit. Methods and results: Five days after femoral artery removal, aFGF-S117 (1, 3, or 30 µg/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 µg/kg of aFGF-S117, with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S117. When an efficacious dose of 3 µg/kg of aFGF-S117 was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. Conclusions: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF-S117 may have therapeutic potential for the treatment of arterial occlusive disorders.

KEYWORDS Angiogenesis; Growth factors; Collateral circulation; Arteries; Blood flow


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Peripheral vascular disease (PVD) is most commonly caused by arterial obstructions due to advanced atherosclerotic lesions and has been reported to affect ~12% of the elderly population [1]. The most common methods of treatment for patients with PVD are surgical revascularization techniques such as bypass procedures, endarterectomy and percutaneous balloon angioplasty. Unfortunately, many patients are excluded from these procedures due to the anatomical extent and distribution of the vascular obstructions. The ability to augment or accelerate neovascularization through the delivery of exogenous growth factors has received much attention since this type of therapy may be a useful adjunct or alternative to the conventional procedures used to treat patients suffering from PVD.

Early pre-clinical and clinical studies that utilized gene transfer of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), showed promise as an innovative approach to treat patients with critical limb ischemia. However, VEGF gene therapy was also associated with significant adverse effects such as peripheral edema (34% of patients in one study), a condition which is also observed in animal models [2,3]. Other unfavorable effects associated with VEGF overexpression include formation of fragile "angioma-like" capillaries and severe neointimal proliferation [4–6]. Moreover, there have recently been studies using various animal models of hindlimb ischemia and different delivery methods that have questioned the functional efficacy of VEGF [3,4,7]. These studies have demonstrated that although VEGF did stimulate angiogenesis, it did not induce arteriogenesis or improve collateral blood flow. Therefore, due to its uncertain efficacy and mechanism-based adverse effects, the clinical utility of VEGF for the treatment of arterial occlusive disorders has been questioned [8,9].

Clearly, it would be favorable to deliver a growth factor that could enhance the growth and development of pre-existing vessels (i.e. arteriogenesis), rather than solely stimulating the sprouting of capillaries (i.e. angiogenesis). Acidic fibroblast growth factor (aFGF or FGF-1) may have several therapeutic advantages over VEGF. For example, unlike VEGF, which is an endothelial cell specific growth factor, aFGF is a powerful mitogen which stimulates migration and proliferation of various cell types, such as smooth muscle cells and fibroblasts which may favor the development of thicker walled vessels. aFGF also stimulates endothelial cell production of matrix metalloproteinases required for vessel remodeling, and, finally, its high affinity to heparin sulfates on the surface of endothelial cells prolongs its half-life and pharmacodynamic action at the FGF-1 receptor. Indeed, aFGF has been shown to be active in animal models of dermal wound repair, large vessel re-endothelialization and myocardial ischemia [10–13].

The purpose of the present study was to determine if intramuscular administration of the previously described stabilized form of aFGF, denoted aFGF-S117 [12–15], could stimulate neovascularization and improve collateral blood flow in ischemic and non-ischemic rabbit hindlimbs. Compared to other members of the FGF family of proteins, a cysteine residue at position 117 is unique to aFGF and replacement of Cys117 to an isomorphorous serine (S117) does not diminish the intrinsic mitogenic activity of the protein but substantially increases the half-life of the active mitogen at physiologic temperature and pH both in the absence and presence of heparin [14,15].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal care
Fifty-four adult New Zealand White rabbits (male, 3–4 kg) were used for the completion of this study. The rabbits were individually housed in a temperature controlled (20±1°C) room with a 12 h/12 h light/dark cycle with food and water provided ad libitum. All animal related procedures were approved by the Institutional Animal Care and Use Committee at Merck Research Laboratories, West Point, PA and conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Experimental design
The effect of aFGF-S117 on collateral-dependent blood flow was assessed in rabbits after unilateral ligation and extraction of the femoral artery. We have previously shown that this method of femoral artery extraction has a transient (<5 days) effect on resting blood flow, but a marked and prolonged (<40 days) effect on reserve flow [16]. Thus, these animals serve as a model of intermittent claudication and not the more severe critical limb ischemia that is associated with resting pain.

For the initial surgery, the rabbits were pre-medicated with xylazine (2.5 mg/kg, i.m.) and anesthetized with ketamine (70 mg/kg, i.m.). Under sterile surgical conditions, a longitudinal incision was made on the medial thigh of one "experimental" hindlimb extending inferiorly from the inguinal ligament to a point just proximal to the patella. Through this incision, the external iliac artery was isolated, ligated twice and sectioned between the ligatures. The distal ligature was used to retract and dissect free the entire length of the common femoral artery to the point where it bifurcates into the saphenous and popliteal arteries. The ligation of the femoral artery results in retrograde thrombus propagation to the origin of the external iliac artery. Therefore, blood flow to the hindlimb after removal of the femoral artery is dependent upon flow from the internal iliac artery and collateral vessels. All rabbits were closely monitored by veterinary staff and received an analgesic (buprenorphine, 0.04 mg/kg s.c., b.i.d.) and antibiotic (enrofloxacin, 6.0 mg/kg, s.c.) for 3 days following surgery.

Five days after surgery, the rabbits were randomly assigned to one of four experimental groups and treated three times per week for two consecutive weeks with either the purified recombinant aFGF-S117 protein at 1 µg/kg (n=10), 3 µg/kg (n=12), 30 µg/kg (n=11), or the vehicle (n=12), which contained 0.1 mg/ml human serum albumin and 12 µg/ml heparin in PBS. The dose of aFGF-S117 indicates the amount administered during each of the six treatments. The aFGF-S117 protein was confirmed to be highly active in a standard Balb/c 3T3 cell mitogenesis assay (ED50=95 pg/ml). A separate group of naïve rabbits (n=9) underwent a sham operation (i.e. femoral artery remained intact) in order to study the effects of aFGF-S117 in the absence of a peripheral blood flow deficit. For all animals, the aFGF-S117 was administered as a purified protein via direct intramuscular injection into the hindlimb (0.5 ml/site over five muscle sites; adductor longus, adductor magnus, vastus medialis, semimembranosus and gastrocnemius). All hemodynamic and anatomic measurements were performed 20 days after surgery.

2.3 Measurement of hindlimb blood flow
Twenty days after the initial surgery, resting and reserve hindlimb blood flow were measured in each rabbit as previously described [16]. Briefly, anesthetized rabbits were placed in the supine position, intubated and mechanically ventilated with room air. A midline incision was made in the lower abdomen and the descending aorta was isolated at the point of bifurcation into the common iliac arteries. The common iliac arteries were then isolated and both epigastric arteries were ligated to prevent blood flow to the lateral compartments of the abdomen and pelvic cavity. Perivascular flow probes (1.5 RB, Transonic Systems, Inc.) were placed on the right and left common iliac arteries for continuous and simultaneous measurement of blood flow to both limbs. To obtain a functional measurement of collateral blood flow, the hyperemic response to a brief arterial occlusion (ao) was measured. An arterial clip was temporarily applied to the common iliac artery and four separate tests (10, 20, 40 and 60 s in duration) were performed in duplicate with 10 min recovery time allowed between tests. The results are presented as the increase in blood volume (ml), which equals the total area under the curve during the first 30 s after release of the arterial clip, minus the baseline blood flow during the time of integration.

2.4 Vascular imaging
Following hindlimb blood flow measurements, angiography was performed as previously described by us and others to obtain an anatomical index of the growth and development of the larger collateral vessels in the medial portion of the upper hindlimb [16,17]. For this, a polyethylene catheter was inserted into the common iliac artery of the experimental limb, with the tip of the catheter positioned just proximal to the origin of the internal iliac artery. The hindlimb was positioned 20 cm below the output beam of a fluoroscope (General Electric, Stenoscop), and the vascular bed was dilated with an intraarterial bolus injection of 300 µg sodium nitroprusside. Immediately thereafter, iodinated contrast media (Isovue-370) was infused intraarterially at a rate of 60 ml/min. Perfusion of the hindlimb was observed on a monitor in real time and an angiographic image was taken exactly 4 s after the start of contrast media infusion. As previously described, a circular grid overlay was used by one blinded investigator to count the number of vessels that transect a circle to obtain a quantitative measurement of the density of collateral vessels in the medial thigh region [16,17].

2.5 Histological analysis
Immediately after the animal was sacrificed (20 days post-surgery, 15 days after the start of dosing), both hindlimbs were dissected and transverse sections of the adductor and gastrocnemius muscles were removed, covered with OCT compound and frozen in liquid nitrogen. Tissue sections (10 µm thickness) were stained for alkaline phosphatase using the indoxyl-tetrazolium method to detect capillary endothelial cells and counterstained with eosin as previously described [16,18,19]. The number of capillaries was determined using an image analysis system (Phase 3 Imaging) that was programmed to distinguish between the dark blue alkaline phosphatase staining of the capillaries and the pink eosin staining of skeletal muscle cells. The system automatically identified and counted the number of capillaries in a chosen field (a total of 60 fields from six different sections were analyzed per muscle). To ensure that capillary density was not overestimated due to tissue edema or underestimated due to muscle atrophy, the total area of muscle was also determined to produce a measurement of capillaries/mm2 of muscle.

2.6 Statistical analysis
All data are presented as mean±S.E.M. Comparisons within the same animal (e.g. experimental vs. contralateral limb) were performed using a paired Student’s t-test. For intergroup comparisons, a one-way ANOVA followed by Fishers PLSD test for multiple comparisons was used. Statistical significance was accepted at the P≤0.05 level.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of aFGF-S117 on resting and reserve hindlimb blood flow
When measured 20 days after femoral artery removal, there was no significant difference in the resting hindlimb blood flow between the experimental and contralateral limbs in any treatment group (Fig. 1). This time course of reestablished resting flow is consistent with other reports using similar hindlimb models in rats and rabbits [20,21]. In contrast to the resting flow, reserve blood flow (i.e. collateral-dependent flow) was significantly increased in the experimental limbs of rabbits dosed with either 3 or 30 µg/kg of aFGF-S117 (Fig. 2). There was no difference in the efficacy observed between the two highest doses of aFGF-S117, and the 1 µg/kg dose was not different from vehicle-treated rabbits. The reserve blood flow measured in the contralateral limb, which did not undergo surgery or drug treatments, was similar among all groups. It is also noteworthy that the reserve blood flow capacity measured in the experimental limbs from the rabbits treated with either 3 or 30 µg/kg aFGF-S117 remained significantly below the values obtained in the contralateral limbs.


Figure 1
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  		Fig. 1 Resting hindlimb blood flow measured 20 days after femoral artery extraction was similar between limbs with no differences among treatment groups. The aFGF-S117 protein or the vehicle was delivered intramuscularly into the experimental limb (sqdiagf), while the contralateral limb ({blacksquare}) served as a internal control and did not undergo surgery or receive treatment.

 

Figure 2
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  		Fig. 2 Effect of aFGF-S117 on hindlimb reserve blood flow. The reserve flow capacities of the experimental (top panel) and contralateral (bottom panel) limbs were assessed by directly measuring the increase in blood flow (i.e. hyperemia) in response to a series of temporary arterial occlusions lasting 10, 20, 40 or 60 s. *P<0.05, compared to the vehicle group.

 
3.2 Collateral vessel growth and development
Representative angiographic images of the experimental limb from vehicle and aFGF-S117 treated rabbits are shown in Fig. 3A–D. In response to femoral artery removal, it can be noticed that the internal iliac artery gives rise to supply vessels that transverse the medial thigh region of the limb. Compared to the vehicle treated rabbits, the angiographic images from the rabbits that received 3 and 30 µg/kg of aFGF-S117 displayed a more dense network of vessels. The increased vascular density was evident by the increased angiographic score in rabbits that were treated with 3 and 30 µg/kg of aFGF-S117 (63.5±4.7 and 59.7±6.8, respectively) compared to rabbits that received 1 µg/kg (44.6±6.2) or the vehicle (40.5±2.1) (Fig. 4).


Figure 3
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  		Fig. 3 Representative angiographic images of the experimental limb of rabbits treated with either vehicle (A) or aFGF-S117 at doses of 1 µg/kg (B), 3 µg/kg (C) or 30 µg/kg (D). Note the increased density of collateral vessels in the medial thigh region of rabbits treated with 3 and 10 µg/kg.

 

Figure 4
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  		Fig. 4 Effect of aFGF-S117 on the growth and development of angiographically visible collateral vessels. The angiographic score represents the density of collateral vessels in the medial thigh region of the experimental limb. *P<0.05, compared to the vehicle group.

 
3.3 Skeletal muscle capillary density
Tissue sections from the adductor (composed of slow, type I fibers located in upper thigh) and gastrocnemius (composed of fast, type II fibers located in lower limb) muscles from both the experimental and contralateral limbs were stained for alkaline phosphatase (AP) to estimate capillary density. Analysis of the adductor muscle (Fig. 5, top panel) revealed no statistical differences in the capillary density between the experimental and contralateral limb muscles among any treatment group, however, as expected, the capillary density in the white adductor muscle was lower than that of the red gastrocnemius. As can be seen, the capillary density in the gastrocnemius muscles (Fig. 5, bottom panel) of the experimental limb in all groups was higher compared to the non-operated contralateral limb. The elevated capillary density in the gastrocnemius muscle of the experimental limb in the vehicle group is most likely due to the effect of ischemia alone since we observe a similar effect in operated, but untreated rabbits. More importantly, the capillary density in the gastrocnemius muscles obtained from the experimental limbs of the rabbits treated with 3 or 30 µg/kg aFGF-S117 (709±69 and 743±77 caps/mm2, respectively) was significantly increased compared to the corresponding experimental limb of vehicle treated rabbits (539±51 caps/mm2).


Figure 5
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  		Fig. 5 The effect of aFGF-S117 on skeletal muscle capillary density was determined for the adductor (top panel) and gastrocnemius (bottom panel) muscles from both the experimental (sqdiagf) contralateral ({blacksquare}) limbs. Top panel: In the adductor muscle, no differences in capillary density were observed either between limbs or among treatments groups. Bottom panel: There was a significant increase in the capillary density of gastrocnemius muscle from the experimental limb compared to the contralateral limb. This result was observed in all groups, however treatment with the two highest doses of aFGF-S117 resulted in a further increase in capillary density. *P<0.05 compared to the contralateral limb; {dagger}P<0.05 compared to the experimental limb of the vehicle group.

 
3.4 Effect of aFGF-S117 on non-ischemic hindlimbs
A separate group of rabbits (n=11) was utilized to determine the effects of aFGF-S117 on the existing hindlimb vasculature in the absence of femoral artery extraction. Normal rabbits underwent a sham operation (i.e. the femoral artery remained intact) and were dosed with the previously determined efficacious dose of 3 µg/kg aFGF-S117 using the identical dosing regimen as described above for rabbits that underwent femoral artery extraction. The same hemodynamic and anatomical measurements were performed 15 days after the start of dosing. Interestingly, as shown in Table 1, intramuscular administration of aFGF-S117 into a normal limb had no effect on any of the parameters measured (reserve blood flow, angiographic score or capillary density).


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  		Table 1 Effect of intramuscular administration of aFGF-S117 (3 µg/kg/dose) in sham-operated rabbits (n=9) with an intact femoral artery

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Following arterial occlusion or major stenosis, blood flow through collateral arteries is required to maintain tissue viability. Normally, these collateral vessels are either not present or do not function as major arterial conduits. Enhancing the growth and development of these collateral vessels by the administration of an exogenous growth factor represents an interesting approach for the treatment of arterial occlusive diseases. The primary finding of the present study was that intramuscular administration of a stabilized form of aFGF enhanced the development of collateral vessels and improved reserve blood flow in hindlimbs following femoral artery removal, but had no angiogenic or arteriogenic effect in normally perfused limbs.

The results from our study demonstrated that collateral-dependent blood flow in the experimental limbs of the rabbits administered 3 and 30 µg/kg of aFGF-S117 was ~70% higher compared to vehicle treated rabbits when measured 20 days after femoral artery removal. The increases in reserve blood flow and angiographic score were similar between the rabbits that received 3 and 30 µg/kg, while injections of 1 µg/kg aFGF-S117 had no effect and were no different from the vehicle group. Thus, maximally effective and no-effect doses of aFGF-S117 were defined. These results are similar to a previous study by Yang, using a rat model of hindlimb ischemia in which they showed intraarterial administration of bFGF into the iliac artery for 2 weeks at 5 or 50 µg/kg/day was equally effective in stimulating collateral-dependent blood flow, while 0.5 µg/kg/day was less effective [22]. Tabata et al. [23] also showed a similar dose–response profile using plasmid delivery of a secreted form of aFGF in a rabbit model of hindlimb ischemia. In their study, there was no improvement in any of the anatomic or functional parameters with low doses of plasmid (100–300 µg), however a statistically significant increase in these indices was demonstrated at a dose of 500 µg, with no further augmentation at higher doses (700–1000 µg). Furthermore, the doses found to be efficacious in our study are similar to the dose used in the phase II study to investigate the efficacy of recombinant FGF-2 in patients with intermittent claudication (the TRAFFIC study). In that study, a single intraarterial injection of 30 µg/kg of FGF-2 improved peak walking time during a graded treadmill-exercise test performed 90 days after treatment [24]. Interestingly, a second dose of FGF-2 administered 30 days after the first injection conferred no additional advantage. Although our study has shown that aFGF-S117 stimulated a significant increase in the reserve flow capacity of the experimental limb, we recognize that complete restoration did not occur, as the reserve values of the experimental limb were ~30% of the contralateral limb. This sub-maximal improvement is similar to the ~20% of normal response observed by others using bFGF [25], and it is possible that the partial improvements observed in these studies may be due the time frame of the experiment and/or sub-optimal protocols for the frequency and duration of growth factor administration.

There are several reports using various animal models of hindlimb ischemia or peripheral arterial insufficiency that have addressed the issues of duration and timing of growth factor treatment in relation to the time of end-point measurements. The blood flow measurements in our study were performed 20 days after surgery because there is an adequate window to observe efficacy beyond the well-known spontaneous collateralization induced by ischemia alone. The results obtained with our study design may not be observed with alternative dosing protocols and different days of end-point measurement (e.g. 30 or 40 days after surgery). However, others have found that bFGF-induced improvement in hindlimb collateral blood flow in rats was independent of the duration of bFGF administration [22]. In that report, intraarterial administrations lasting 1, 3, or 14 days yielded similar increases in calf muscle blood flow when measured 16 days after femoral artery ligation. Importantly, in those experiments, the bFGF treatment was initiated on the same day as the surgical ligation of the femoral artery. Those results suggested that the duration of treatment was not as important as the timing of the treatment as long as it coincided with the onset of ischemia. It has been shown that shortly after the experimental induction of a vascular occlusion, nearby vessels that likely serve as subsequent collateral conduits exhibit proliferative activity and upregulation of certain growth factor receptors that may be necessary for vascular remodeling [26]. Specifically, using a rabbit model similar to ours, Deindl and colleagues recently reported increased expression of FGF receptor-1 (FGFR-1) during the early phase of arteriogenesis [27]. Their elegant immunohistochemical studies performed 3 days after femoral artery ligation, showed specific localization of FGFR-1 in smooth muscle cells of the collateral arteries that crossed the medial thigh region of the limb. Therefore, administration of an exogenous growth factor, such as aFGF, may only need to be present during the "vascular activation" or early phase of arteriogenesis which temporally coincides with the development of tissue ischemia or the presence of a transcollateral pressure gradient. Thus, the results of our study complement the previous work of others by showing that responsiveness to aFGF-S117 was maintained when the initiation of treatment was temporally dissociated from the induction of the vascular obstruction by 5 days.

It is well recognized that a flow deficit is a powerful stimulus for vascular remodeling, even without the intervention of an exogenous growth factor(s) [16,26]. The remodeling of existing vessels to form functional arterial conduits has been termed "arteriogenesis", and is clearly distinct from de novo formation of new capillaries by the process of angiogenesis. In response to a vascular stenosis or occlusion, it is arteriogenesis which is believed to be the more important and compensatory form of vascular growth required to maintain tissue perfusion [7,16,28]. Although arteriogenesis was observed via angiography in all animals in the present study, in contrast to the modestly defined arterial tree seen in the vehicle group, the aFGF-S117 treated animals exhibited an expanded vascular tree that was more dense and extended further distally. These results are consistent with previous studies using rat and rabbit models of hindlimb ischemia that showed intraarterial administration of bFGF into the hindlimb led to increased proliferation of endothelial and smooth muscle cells [29], enlargement of existing conduit vessels and increased collateral-dependent blood flow [25,29,30]. It is also interesting to note that aFGF-S117 had an angiogenic effect (i.e. increased capillary sprouting) in a skeletal muscle located in the lower hindlimb. This result agrees with the work of Ito et al. who, using a similar rabbit model, showed that in the lower limb where the greatest perfusion deficits occur, endothelial cell proliferation takes place in the absence of smooth muscle cell proliferation, suggesting that angiogenesis predominates over collateral remodeling in this region [26].

Although the process of arteriogenesis is not completely understood, it is not likely related to the metabolic consequences of ischemia, since the tissue surrounding the collateral conduits has been shown by others to receive adequate blood flow [25,26,31]. It is more likely that the hemodynamic changes related to increased flow velocity, wall stress and shear stress, and/or monocyte involvement are important in the remodeling process [29]. Thus, it may be that the FGF receptors in the endothelium are up-regulated by these altered flow dynamics leading to an increased responsiveness to exogenous aFGF. However, it is unknown whether the beneficial effects of aFGF are a result of a primary arteriogenic affect or potentiation of this activity by facilitating or inducing the activity of other growth factors. We acknowledge that the beneficial effects of aFGF-S117 observed in the present study may not be characteristic of a clinical situation, since the method of arterial obstruction may have simplified the remodeling process. Also, prior knowledge of the area of vascular obstruction allowed site-specific delivery of the growth factor. Nonetheless, it is possible that the advantageous clinical situation may exist where following the identification of a peripheral vascular obstruction through imaging, site-specific delivery of aFGF-S117 into the tissue that is most likely to respond could minimize side-effects and enhance efficacy.

Our results clearly demonstrated that in the absence of femoral artery extraction, aFGF-S117 had no effect on any of the parameters of angiogenesis, arteriogenesis and hindlimb blood flow. When a previously determined effective dose of aFGF-S117 was administered to normal rabbit limbs with an intact femoral artery, we found no changes in limb blood flow (resting or reserve), angiographic score or capillary density. These results suggest that adequately perfused skeletal muscle is unresponsive to exogenous aFGF and, to our knowledge, this is the first evidence showing that existing vessels that ultimately serve as collateral conduits are unresponsive to aFGF, unless a vascular obstruction exists in the region. This finding supports the idea that creation or formation of a vascular obstruction produces site-specific alterations in nearby vessels which allow them to become responsive to exogenous aFGF.

Although the increased reserve blood flow measured in response to aFGF-S117 treatment appeared to be physiologically relevant, we did not evaluate its impact on exercise tolerance or muscle function. However, using a rat model of hindlimb ischemia, it has been shown that improvements in calf muscle function were proportional to the increases in hindlimb blood flow induced by either bFGF administration [25], increased physical activity [32], or a combination of bFGF administration and enhanced physical activity [33]. The goal of future experiments will be to determine whether the aFGF-S117-induced changes observed in this study would translate to enhanced exercise tolerance or performance. It should also be noted that our results with the S117 form of aFGF are consistent with and complement a previous study showing the efficacy of WT-aFGF in the same rabbit model [23]. However, it is difficult to directly compare our results with this study since the growth factor was delivered in different forms (plasmid vs. purified protein) and routes of administration (intra-arterial vs. intramuscular). Although both forms of aFGF (S117 and WT) function through the same receptor, it is unknown whether the prolonged stability and half-life of aFGF-S117 would translate into improved efficacy in vivo and/or increased side-effects compared to the WT form.

In summary, the present study demonstrated that intramuscular administration of a stabilized form of aFGF stimulated the growth and development of collateral vessels and produced a marked improvement in hindlimb reserve blood flow in a rabbit model of peripheral arterial insufficiency. The surgical removal of the femoral artery was a prerequisite for aFGF-S117 activity and suggests that alterations in blood flow or the induction of ischemia produces a local environment that is responsive to exogenous aFGF-S117. These results suggest aFGF-S117 may have therapeutic potential since similar vascular remodeling in patients with intermittent claudication could stimulate a meaningful improvement in collateral blood flow and enhance mobility.

Time for primary review 21 days.


   Acknowledgements
 
The authors gratefully acknowledge the surgical and animal care support from the Department of Laboratory Animal Resources, Merck Research Laboratories. We also thank William Huckle and Rosemary McFall for characterization of aFGF-S117 in cell-based assays.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Criqui M.H., Fronek A., Barrett-Connor E., et al. The prevalence of peripheral arterial disease in a defined population. Circulation (1985) 71:510–515.[Abstract/Free Full Text]
  2. Baumgartner I., Rauh G., Pieczek A., et al. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor. Ann Intern Med (2000) 6:880–884.
  3. Hershey JC, Baskin EP, Corcoran HA, et al. VEGF stimulates angiogenesis without improving collateral blood flow following hindlimb ischemia in rabbits. Heart and Vessels (in press).
  4. Lee R.J., Springer M.L., Blanco-Bose W.E., et al. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation (2000) 102:898–901.[Abstract/Free Full Text]
  5. Lazarous D.F., Shou M., Scheinowitz M., et al. Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury. Circulation (1996) 94:1074–1082.[Abstract/Free Full Text]
  6. Masaki I., Yonemitsu Y., Yamashita A., et al. Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res (2002) 90:966–973.[Abstract/Free Full Text]
  7. Deindl E., Buschmann I., Hoefer I.E., et al. Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit. Circ Res (2001) 89:779–786.[Abstract/Free Full Text]
  8. Helisch A., Schaper W. Angiogenesis and arteriogenesis—not yet for prescription. Z Kardiol (2000) 89:239–244.[CrossRef][ISI][Medline]
  9. Epstein S.E., Kornowski R., Fuchs S., Dvork H.F. Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects. Circulation (2001) 104:115–119.[Free Full Text]
  10. Mellin T.N., Mennie R.J., Cashen D.E., et al. Acidic fibroblast growth factor accelerates dermal wound healing. Growth Factors (1992) 7:1–14.[Medline]
  11. Bjornsson T.D., Dryjski M., Tluczek J., et al. Acidic fibroblast growth factor promotes vascular repair. Proc. Natl. Acad. Sci. USA (1991) 88:8651–8655.[Abstract/Free Full Text]
  12. Lopez J.J., Edelman E.R., Stamler A., et al. Angiogenic potential of perivascularly delivered aFGF in a porcine model of chronic myocardial ischemia. Am J Physiol (1998) 274:H930–H936.[ISI][Medline]
  13. Sellke F.W., Li J., Stamler A., et al. Angiogenesis induced by acidic fibroblast growth factor as an alternative method of revascularization for chronic myocardial ischemia. Surgery (1996) 120:182–188.[CrossRef][ISI][Medline]
  14. Linemeyer D.L., Menke J.G., Kelly L.J., et al. Disulfide bonds are neither required, present, nor compatible with full activity of human recombinant acidic fibroblast growth factor. Growth Factors (1990) 3:287–298.[Medline]
  15. Ortega S., Schaeffer M.T., Soderman D., et al. Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor. J. Biol. Chem. (1991) 266:5842–5846.[Abstract/Free Full Text]
  16. Hershey J.C., Baskin E.P., Glass J.D., et al. Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis. Cardiovasc Res (2001) 49:618–625.[Abstract/Free Full Text]
  17. Takeshita S., Rossow S.T., Kearney M., et al. Time course of increased cellular proliferation in collateral arteries after administration of vascular endothelial growth factor in the rabbit model of lower limb insufficiency. Am J Pathol (1995) 147:1649–1660.[Abstract]
  18. Zaida A.M., Hudlicka O., Tyler K.R., Wright A.J. The effect of long-term vasodilation on capillary growth and performance in rabbit heart and skeletal muscle. Cardiovasc Res (1984) 18:724–732.[Abstract/Free Full Text]
  19. Baffour R.J., Berman J., Garb J.L., et al. 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. J Vasc Surg (1992) 16:181–191.[CrossRef][ISI][Medline]
  20. Hendricks D.L., Pevec W.C., Shestak K.C., et al. A model of persistent partial hindlimb ischemia in the rabbit. J Surg. Res. (1990) 49:453–457.[CrossRef][ISI][Medline]
  21. Mackie B.G., Terjung R.L. Blood flow to different skeletal muscle fiber types during contraction. Am J Physiol Heart Circ Physiol (1983) 245:H265–H275.[Abstract/Free Full Text]
  22. Yang H.T., Feng Y., Allen L.A., Protter A., Terjung R.L. Efficacy and specificity of bFGF increased collateral flow in experimental peripheral arterial insufficiency. Am J Physiol Heart Circ Physiol. (2000) 278:H1966–H1973.[Abstract/Free Full Text]
  23. Tabata H., Silver M., Isner J.M. Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA. Cardiovasc Res (1997) 35:470–479.[Abstract/Free Full Text]
  24. Lederman R.J., Mendelsohn F.O., Anderson R.D., et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet (2002) 359:2053–2058.[CrossRef][ISI][Medline]
  25. Yang H.T., Deschenes M.R., Ogilvie R.W., Terjung R.L. Basic fibroblast growth factor increases collateral blood flow in rats with femoral arterial ligation. Circ. Res. (1996) 76:62–69.
  26. Ito W.D., Arras M., Scholz D., et al. Angiogenesis but not collateral growth is associated with ischemia after femoral artery occlusion. Am J Physiol. (1997) 42:H1255–H1265.
  27. Deindl E., Hoefer I.E., Fernandez B., et al. Involvement of the fibroblast growth factor system in adaptive and chemokine-induced arteriogenesis. Circ Res (2003) 92:561–568.[Abstract/Free Full Text]
  28. van Royen N., Piek J.J., Buschmann I., et al. Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease. Cardiovasc Res (2001) 49:543–553.[Abstract/Free Full Text]
  29. Arras M., Ito W.D., Sholz D., et al. Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J. Clin. Invest. (1998) 101:40–50.[ISI][Medline]
  30. Battler A.M., Scheinowitz A., Bor D., et al. Intracoronary injection of basic fibroblast growth factor enhances angiogenesis in infarcted swine myocardium. J. Am. Coll. Cardiol. (1993) 22:2001–2006.[Abstract]
  31. Lindner V., Lappi D.A., Baird A., Majack R.A., Reidy M.A. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res. (1991) 68:106–113.[Abstract/Free Full Text]
  32. Yang H.T., Ogilvie R.W., Terjung R.L. Heparin increases exercise-induced collateral blood flow in rats with femoral artery ligation. Circ Res. (1995) 76:448–456.[Abstract/Free Full Text]
  33. Yang H.T., Ogilvie R.W., Terjung R.L. Exercise training enhances basic fibroblast growth factor-induced collateral blood flow. Am J Physiol Heart Circ Physiol. (1998) 274:H2053–H2061.[Abstract/Free Full Text]

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