Journal of Vascular Surgery
Volume 28, Issue 5 , Pages 919-928, November 1998

Regulation of new blood vessel growth into ischemic skeletal muscle☆☆☆★★★

Presented at the 1997 Annual Scientific Session of the Western Surgical Association, Colorado Springs, Colo, Nov 16–19, 1997.

Sacramento, Calif, and Tampa, Fla

Received 21 January 1998; accepted 30 April 1998.

Article Outline

Abstract 

Purpose: In a rabbit model, transposition of a muscle pedicle flap to an ischemic hind limb has been shown to result in the development of new blood vessels that connect the arterial circulation of the flap to the circulation of the limb. The hypothesis that exogenous recombinant basic fibroblast growth factor (bFGF) would enhance the development of this new blood supply was examined and the regulation of bFGF in this process was investigated. Methods: The right common iliac artery was ligated in 12 male New Zealand white rabbits. An abdominal wall muscle flap based on the left inferior epigastric artery was transposed to the right thigh. bFGF in phosphate-buffered saline (PBS) at 3 ng/h (n = 6), or PBS alone (n = 6), was infused for 7 days via mini-osmotic pumps with an infusion catheter positioned at the flap-muscle interface. The flap-muscle interface was immunostained with anti-α–actin antibody to determine blood vessel density (number of vessels/mm) and with anti-bFGF antibody to evaluate bFGF distribution. RNA was isolated from these sections, and polymerase chain reaction (PCR) was used to examine endogenous bFGF messenger RNA (mRNA) expression. Results: Blood vessel density was significantly increased in animals receiving exogenous bFGF (22.0 ± 10.6 vessels/mm vs. 10.7 ± 8.8 vessels/mm, P = .009). In the controls, neovessels were arranged in clusters with endogenous bFGF concentrated around these clusters. In bFGF-treated animals, vessels were diffusely scattered throughout the flap-limb interface, corresponding to the distribution pattern of infused bFGF. There was no difference in bFGF mRNA expression between the control and the bFGF-treated groups. Conclusion: Exogenous bFGF infusion significantly augmented new blood vessel development at the flap-limb interface. Endogenous bFGF was up-regulated around the newly developed microvessels in control animals, and vessel growth correlated with the diffuse distribution of exogenous bFGF, implicating bFGF as an important factor in angiogenesis. Exogenous bFGF did not affect bFGF mRNA expression, suggesting that the regulation of bFGF is not under autocrine control. (J Vasc Surg 1998;28:919-28.)

 

Techniques to induce the development of a new arterial blood supply should allow limb salvage in patients with severe arterial insufficiency and nonreconstructable vessels. Several attempts to supply an indirect source of arterial inflow via tissue transfer to ischemic limbs have been reported.1, 2, 3, 4, 5, 6, 7, 8, 9 Although these reports suggest that the transposition of a vascularized tissue flap may result in neovascularization of the ischemic recipient bed, the technique has not been generally accepted because of a lack of long-term success.

Recently, the use of specific growth factors promoting vascular development has been examined in animal models of hind limb and myocardial ischemia.6, 10, 11, 12, 13, 14 Angiogenic growth factors that have been shown to enhance vascular development include basic fibroblast growth factor (bFGF), endothelial cell growth factor (ECGF), and vascular endothelial growth factor (VEGF). bFGF is a multifunctional, heparin-binding growth factor that has been isolated from a wide variety of tissue and tumor sources and is known to be a potent endothelial cell mitogen. bFGF is synthesized by endothelial cells and stored in the extracellular matrix in association with heparan sulfate proteoglycans.15 This intimate association with the extracellular matrix protects bFGF from degradation16, 17 and makes bFGF available during local proteolysis. bFGF has been demonstrated to be important in the process of angiogenesis, a necessary component of embryonic development, wound repair, and tumor growth.18, 19

Previous studies of ischemic limbs have documented the hypertrophy or recruitment of pre-existing (ie, collateral) vessels in response to bFGF, ECGF, or VEGF, resulting in increased vessel density in a limb distal to an arterial ligation.6, 10, 11, 12, 13, 14

We used an established model of persistent hind limb ischemia that is unique because it allows for the study of newly developing blood vessels across a tissue interface, rather than simply collateral development.20 Enhancement of this process may lead to treatment modalities for severely ischemic limbs in which direct revascularization is not possible. The hypothesis that exogenous bFGF will augment new blood vessel growth was examined and the role of bFGF in the control of new blood vessel development was investigated.

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Materials and methods 

Animal model 

The model of chronic hind limb ischemia has been described.20, 21 This model has been shown to consistently reduce resting hind limb perfusion to 30% of baseline for at least 7 days.21 When a well perfused abdominal wall muscle flap is transposed to the ischemic hind limb, new blood vessels that connect the arteries of the flap with the native circulation of the limb have been shown to develop; these vessels have been demonstrated angiographically20 and histologically.22 These new vessels have been shown to increase the resting perfusion of the ischemic limb by 45%.20

The present study design was approved by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis. The guidelines of the National Institutes of Health (publication number 86-23, “Guide for the Care and Use of Laboratory Animals,” revised 1985) were followed throughout the course of the study. Twelve New Zealand white rabbits weighing 2.5 to 3.5 kg were anesthetized with an intramuscular injection of 0.5 mg/kg acepromazine, 5 mg/kg xylazine, and 75 mg/kg ketamine. The animals also received 10 mg/kg enrofloxacin intramuscularly prior to operation. An intravenous line for infusion of normal saline was placed in a dorsal ear vein. Hind limb blood pressure was determined using a pulse sensor (RTBP096, Kent Scientific Corporation, Litchfield, Conn). After occlusion of arterial flow with a pediatric blood pressure cuff proximal to the pulse sensor probe, the systolic arterial pressure was determined to be the cuff pressure at which a pulsatile waveform reappeared on a strip chart recorder as the cuff was slowly released.

A midline laparotomy was performed using sterile technique. The right common iliac artery was isolated at its origin, ligated, and transected. Hind limb blood pressure was measured both before and after surgery to confirm arterial ligation. A muscle flap based on the left deep inferior epigastric artery was developed; the terminal arborization of this artery determined the distal edge of the flap. The flaps measured 9.3 ± 0.8 cm by 2.1 ± 0.2 cm. The left epigastric muscle flap was sutured to the anteromedial right thigh (Fig. 1).

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  • Fig. 1. 

    Diagrammatic illustration of the surgical procedure performed on the rabbits. A muscle flap based on the left deep inferior epigastric artery is sutured onto muscle of the right thigh after ligating the right common iliac artery. (Courtesy of Pevec WC, Hendricks D, Rosenthal MS, Shestak KC, Steed DL, Webster MW: Revascularization of an ischemic limb by use of a muscle pedicle flap: A rabbit model. J Vasc Surg 1991;13:385–90.)

Mini-osmotic infusion pumps (Alza model 2ML1, Alza Corp., Palo Alto, Calif) were secured in the peritoneal cavity. A polyethylene catheter (PE-60, Fisher Scientific, Santa Clara, Calif) extended from the pump to the muscle-flap interface on the right thigh. The abdominal muscles were reapproximated at the midline after flap dissection.

The mini-osmotic pumps delivered a solution at a continuous rate of 10 μL/h for 7 days. Before surgery, the animals were assigned to either the experimental or the control group. The 6 experimental animals had infusion, via the mini-osmotic pumps, of recombinant human bFGF (Gibco-cat. no. 13256-029, Grand Island, NY) in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin at a rate of 3 nanograms of bFGF per hour (10 μL/h) for 7 days (bFGF group). bFGF has been shown to retain its biological activity when stored in microcapsules at 37°C for up to 4 weeks.23 The 6 control animals had infusion of PBS with 0.1% bovine serum albumin at 10 μL/h for 7 days (control group).

On day 7, all rabbits were clinically evaluated for hind limb function and muscle atrophy. Function was evaluated by observing the posture of the hind limb at rest and during hopping. Muscle atrophy was determined by measuring the thigh and calf circumferences of both hind limbs. The animals were then reanesthetized, and hind limb blood pressure was measured. The rabbits were exsanguinated by a catheter placed in the common carotid artery and were killed with an overdose of ketamine and potassium chloride. The pelvis and hind limb tissues were perfusion fixed with 1000 ml of 10% buffered formalin, infused at a pressure of 100 mm Hg through a 14-gauge catheter placed in the infrarenal aorta. A block of tissue, including the interface between the muscle flap and underlying limb muscle with the infusion catheter remaining in place, was harvested and placed in 10% formalin solution.

Recombinant human bFGF 

The preparation of recombinant human basic FGF was supplied as lyophilized powder. The bFGF was reconstituted by means of 0.1% bovine serum albumin in PBS, which had been filter-sterilized through a 0.22 μm millipore filter. The infusion pumps were loaded using sterile technique with 0.5 μg bFGF diluted to a volume of 2 ml with 0.1% bovine serum albumin in PBS.

Blood vessel density 

The harvested tissue blocks were dehydrated through a graded series of alcohol and then paraffin-embedded. Histologic sections (6 μm thick) perpendicular to the flap-limb muscle interface were cut at 5 mm intervals. The sections were immunostained for α-actin antibody. This antibody recognizes only the α-actin isoform of smooth muscle cells and does not react with striated cardiac or skeletal muscle cells. Anti-α–actin antibodies (Boehringer Mannheim Biotechnology, Indianapolis, Ind) were applied in a 1:200 dilution, and the sections were stained using standard immunoperoxidase staining procedure with a commercial kit (Vectastain, Vector Laboratories, Burlingame, Calif) and counterstained with hematoxylin and eosin. In our previous experience with this rabbit model, we found the more conventional endothelial markers, factor VIII and various lectins, to be very nonspecific.22 The most specific immunostaining was achieved against α-actin.

After staining, the slides were examined, using light microscopy, to determine microvascular density. Histologic sections were cut 5 mm and 10 mm from the end of the flap (near the placement of the infusion catheter). Blood vessels were identified by tubular morphology with a single layer of endothelial cells, the presence of intraluminal red blood cells, and positive α-actin staining.22 For each section, the total number of new blood vessels in the interface between the abdominal wall flap and the thigh muscle was counted by 2 independent blinded observers and normalized per linear distance of the flap-limb interface (number of blood vessels/mm).

Distribution of bFGF 

Immunoperoxidase staining of the sections with antibodies specific to bFGF was used to assess the expression of endogenous bFGF and the radius of diffusion of the exogenous growth factor. bFGF staining was correlated to the location of enhanced angiogenesis. The primary anti-bFGF-antibody (developed in chickens) was donated by a private source (Dr J. Sasse, Tampa, Fla) with secondary immunoperoxidase staining by standard techniques using a commercial kit (Vectastain, Vector Laboratories, Burlingame, Calif).

bFGF mRNA expression 

Polymerase chain reaction (PCR) was used to examine the expression of bFGF mRNA in the sections. Total RNA was isolated from 25 sections (each 6 μm thick) using the guanidine isothiocyanate method.24 The RNA was phenol extracted and then ethanol precipitated with transfer RNA (tRNA) as carrier. The resulting pellet was dissolved in 30% formamide. The RNA was probed for bFGF. The following primers were specific for sense bFGF: (5' primer) 5'-AAG AGC GAT CCG CAC ATC AA-3' and (3' primer) 5'-GGA TAG CTT TCT GTC CAG GT-3', and for β-actin: (5' primer) 5'-GTC GCC CTG GAC TTC GAG C-3' and (3' primer) 5'-GGT ACA TGG TGG TGC CGC CA-3'. bFGF mRNA was specifically amplified utilizing primers that span intron/exon boundaries, thus discriminating between mRNA and genomic DNA amplification. Bovine brain RNA was used as a positive control because it is a rich source of bFGF.25 Primers for β-actin, a protein expected to be present in most, if not all, cell types in the tissue samples, were used to normalize the amount of RNA between samples. The band intensity was normalized by adjusting the RNA concentration so all samples amplified with the same efficiency. RNA from both the control and the bFGF-treated groups was first subjected to enzymatic digestion with either RNAse or DNAse. The signal was identified by means of this treatment as originating from mRNA and not from contaminating genomic DNA. Equal amounts of total RNA from the sections were reverse transcribed to complementary DNA (cDNA) using 2.5 μmol of random hexamers as primers. An aliquot of cDNA from the reverse transcription reaction was used as the template for the PCR reaction. The cDNA was denatured by means of a 3 minute cycle at 97°C, followed by 45 cycles of 1 minute at 94°C, 30 seconds of annealing at 58°C, and 1.5 minutes extension at 72°C using a DNA thermal cycler (#480, Perkin-Elmer Cetus, Branchburg, NJ). Twelve μl of the PCR reaction were subjected to electrophoresis on a 2.5% agarose gel, and DNA was stained with ethidium bromide and visualized with UV light.

Data analysis 

Values are reported as mean ± SD. Blood vessel density was compared using the Mann-Whitney test for the comparison of nonparametric data. Other results were analyzed using a two-tailed Student's t-test for unpaired comparisons between 2 means. A P value less than .05 was considered statistically significant. All calculations were performed using a computerized statistical package (Abacus Concepts, Berkeley, Calif.).

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Results 

Clinical assessment 

On day 7, the animals were examined for evidence of muscle atrophy. There was no difference in body weight between the control group and bFGF-treated group (Table I).

Table I. Comparison of physiologic parameters between rabbits in control and bFGF-treated groups.
Control (n = 6)bFGF-treated (n = 6)P
Day 0
Weight (kg) 2.9 ± 0.32.9 ± 0.3.78
Thigh circumference (cm)L13.7 ± 1.213.6 ± 1.2.91
R13.6 ± 1.314.0 ± 1.3.65
Calf circumference (cm)L10.1 ± 0.910.3 ± 0.8.63
R10.1 ± 1.310.7 ± 1.3.46
Preoperative BP (mm Hg)L58 ± 860 ± 7.66
R55 ± 461 ± 10.23
Postoperative BP (mm Hg)L42 ± 2150 ± 15.49
R00—
Day 7
Weight (kg) 2.9 ± 0.22.9 ± 0.3.87
Thigh circumference (cm)L13.6 ± 1.013.8 ± 0.5.79
R13.6 ± 1.013.8 ± 0.9.68
Calf circumference (cm)L10.2 ± 1.210.2 ± 1.0.90
R10.1 ± 0.99.9 ± 1.1.76
BP (mm Hg)L47 ± 647 ± 9.97
R00—

BP, Blood pressure.

Also on day 7, there were no differences observed in either thigh or calf circumference between the ischemic limb and the contralateral nonischemic limb in controls and bFGF-treated animals. On day 0, blood pressure was undetectable in the ischemic right hind limb in all animals, and it remained undetectable on day 7, confirming arterial ligation and limb ischemia for the duration of this study. This is consistent with a previous study using this animal model in which Hendricks et al. demonstrated persistent hypotension in the experimental limb, with only 2 of 20 animals having detectable pulsatile arterial flow at 7 days postligation.21

Effects of local infusion of bFGF on blood vessel density 

Local infusion of bFGF at 3 ng/h for 7 days resulted in increased blood vessel density at the flap-muscle interface. An average of 22.0 ± 10.6 vessels/mm were seen in the bFGF-treated animals, compared with 10.7 ± 8.8 in the controls (P = .009) (Fig. 2).

The sections examined were within 3 to 5 mm from the end of the osmotic pump delivery catheter, where the concentration of growth factor would be expected to be the highest. In control sections, the new microvessels were observed to develop in clusters, with other areas in the muscle-flap interface relatively devoid of the new vessels (Fig. 3).
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  • Fig. 3. 

    Histology of vessel density in the muscle-flap region of control and bFGF-treated animals. A, The flap-limb interface of a control animal, 50× magnification (hematoxylin and eosin stain). Microvessels can be seen, but are scarce and arranged in clusters. B, The flap-limb interface of a bFGF-treated animal, 50× magnification (hematoxylin-eosin stain). The distribution pattern of microvessels is diffuse, and vessel density is increased.

In comparison, the microvessels developed in a more random, diffuse pattern throughout the interface in the bFGF-treated sections. The blood vessels observed at the flap-muscle interface were more mature than capillaries as they stained with antibodies to α-actin, a smooth muscle cell protein.

Distribution of bFGF 

Staining with the anti-bFGF antibody showed diffuse extracellular staining throughout the muscle-flap interface of animals given the exogenous bFGF as compared with control animals, confirming that exogenous bFGF was being delivered to the area of interest (Fig. 4).

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  • Fig. 4. 

    Immunohistochemical staining for bFGF at the muscle-flap interface in control and bFGF-treated animals. A, Control tissue, 40× magnification, showing intense pericellular staining with minimal staining in the muscle-flap interface. Brown stain indicates positive reaction for bFGF. B, bFGF-treated tissue, 40× magnification, has both pericellular staining and diffuse extracellular staining throughout the flap-limb interface.

Sections 5 to 10 mm distal to the infusion catheter demonstrated little or no interface staining, suggesting that exogenous bFGF did not diffuse far from the catheter (data not shown). In contrast, sections from the muscle-flap interface of control animals demonstrated extracellular bFGF only surrounding clusters of microvessels, with minimal extracellular staining elsewhere throughout the interface. In both control and experimental animals, bFGF was seen within the nuclei of endothelial cells.

Expression of bFGF mRNA 

PCR was performed to determine whether the addition of exogenous bFGF affected the expression of endogenous rabbit bFGF mRNA. A single amplified product of 273 base pair (bp) was detected, which corresponds to endogenous rabbit bFGF mRNA (Fig. 5).

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  • Fig. 5. 

    Actin and bFGF mRNA expression analyzed by PCR. Lane 1—DNA marker; control animal, lanes 2–5: lane 2—positive control, lane 3—DNAse treated, lane 4—RNAse treated, lane 5—negative control; bFGF-treated animal, lanes 6–9: lane 6—positive control, lane 7—DNAse treated, lane 8—RNAse treated, lane 9—negative control; lane 10—bovine brain. The primer pair used is shown above each panel. Arrow indicates position of correct PCR fragment.

The PCR product's identity was confirmed by means of DNA sequencing (data not shown). The amplified product was caused specifically by bFGF mRNA and not contaminating genomic DNA, because the treatment of the samples before reverse transcription with RNAse destroyed the signal, and DNAse treatment did not affect the signal. Endogenous bFGF mRNA expression was detected in both the control animals and those treated with exogenous bFGF. No differences in expression were detected. The similar intensity of the β-actin signals indicate that equivalent amounts of RNA were tested in both the control and the bFGF-treated PCR reactions. Bovine brain was used as a positive control because of the known high endogenous bFGF concentration.

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Discussion 

A previously developed rabbit model of hind limb ischemia was used to examine the effects of exogenous bFGF on the development of new blood vessels between a well-perfused muscle flap and ischemic limb muscle.20 An animal model of reproducible, persistent limb ischemia is necessary for studying angiogenesis in ischemic skeletal muscle. Common iliac artery division in the rabbit has been shown to produce immediate limb ischemia with a decrease in resting hind limb perfusion to 31% of baseline, which persists for 7 days and has been documented by radiolabled microsphere studies.20, 21 By postligation day 17, microsphere measurements show recovery of the perfusion to 80% of baseline. Thus, for at least 7 days, this model provides profound hind limb ischemia.

Contrast injected into the artery of the flap was shown by means of angiography to opacify the native hind limb vasculature, confirming that connections had developed between the arteries of the flap and the circulation of the limb. By means of microsphere measurements, these new arterial connections were demonstrated to be hemodynamically significant, increasing resting muscle perfusion by 45%.20 Histologic confirmation of microvessel development at the interface between the flap and the ischemic limb was documented in a subsequent report.22 The vessels could be detected at 7 days, but not 3 days, suggesting these were newly developed vessels. Also, the vessels stained for proliferating cell nuclear antigen (PCNA), which binds only to nuclei of proliferating cells, establishing that these were neovessels. Thus, this is a unique in vivo model for studying new blood vessels that develop across a tissue interface where no vessels had previously existed.

bFGF is a cationic, 18 to 23 kd, 154 amino acid polypeptide, first localized in 1974, and known to be mitogenic for vascular endothelial cells.26, 27, 28 The molecular mechanisms responsible for this mitogenic effect have not been completely defined. bFGF is widely distributed in the body and is synthesized in considerable amounts by endothelial cells.29 It is not secreted via the normal cellular secretory pathways, as it lacks the necessary signal peptide sequence. The biologic half-life of bFGF is only a few minutes in vivo.30 It is bound in an inactive state to glycosaminoglycans, mainly heparan sulfate, in the basement membrane and subendothelial cell extracellular matrix.31, 32 This “stored” growth factor is available for rapid mobilization during endothelial cell injury.33 There is evidence suggesting that ischemia stimulates the release of various angiogenic agents, including bFGF.34, 35

Angiogenesis is necessary not only for normal growth and development, but also for tissue repair and wound healing. Angiogenesis is initiated in both acute and chronic conditions. Tissue injury leads to hypoxia, which has been suggested as a powerful stimulus for angiogenesis.14, 26 Hypoxia has also been shown to induce expression of VEGF, another potent angiogenic growth factor.37 Clinical studies of patients with proliferative diabetic retinopathy have demonstrated the effects of ischemia on localized neovascularization, as well as the potent effect of bFGF on retinal endothelial mitosis.36 The formation of new blood vessels involves the interaction of many basic cellular activities, including capillary basement membrane destruction and endothelial cell migration, division, and organization into tubular structures.18

Previous animal studies of hind limb ischemia have demonstrated enhanced collateral artery development with the intramuscular or intravascular administration of angiogenic growth factors: bFGF,11 ECGF,10 and VEGF.12, 13 Repeated intramuscular administration of bFGF11 and intramuscular and intravascular administration of VEGF13 has been shown to increase collateral arterial development in experimental models of hind limb ischemia created by iliac artery ligation. Neovascularization has also been described in models of chronic myocardial ischemia exposed to bFGF.6, 14 In pilot experiments for this study, bFGF was systemically infused via the jugular vein. After 1 week, there was no difference in vessel density (data not shown). This result was not surprising, given bFGF's avidity for binding to extracellular matrix proteins and endothelial receptors. It would be expected that massive systemic doses would be required to overcome the nonspecific binding to extracellular matrix proteins to see a local effect. In other pilot studies, systemic and local infusions of VEGF resulted in no enhanced angiogenesis. Although this may have been caused by an improper dose of VEGF, a species effect with the isoform used, or another technical reason, the dramatic response to local infusion of bFGF led to its use in the current study.

Because of bFGF's avidity for binding to extracellular matrix proteins and to widely distributed receptors and its short biologic half-life in the presence of proteases, it would be expected that a prolonged local effect would require either repeated local injections or a continuous local infusion. This was the rationale for delivering bFGF to the flap-limb interface via a local infusion pump. Edelman et al23 showed that bFGF retained biologic activity when protected in microcapsules at 37°C for 4 weeks. Mayer et al,38 Cuevas et al,39 and Epply et al40 showed angiogenic activity was increased by bFGF when infused via mini-osmotic pumps for 7 to 28 days.

Neovascularization of ischemic tissue occurred in this model. Local infusion of exogenous bFGF, at a dose of 3 ng/h for 7 days, significantly augmented new blood vessel development between an independently perfused muscle pedicle flap and ischemic skeletal muscle. This dose for bFGF administration was based on other studies of local infusions of bFGF.14, 38, 39 A 7-day experiment was chosen because this model has been documented to produce hind limb ischemia for at least 7 days.21 bFGF is an “early” growth factor. Prior work with this model showed no histologic microvessels at 3 days, but mature microvessels, with smooth muscle cells in the media and positive staining for proliferating cell nuclear antigen, at 7 days.22 Ibukiyama showed the peak of angiogenesis with exogenous bFGF occurred within 1 week after administration.41 Thus, a 7-day model was considered to be most appropriate.

bFGF is a known mitogen and chemoattractant for endothelial cells.18, 42 Further, bFGF and hypoxia have been shown to synergistically increase mRNA for VEGF, a potent angiogenic factor, in rabbit vascular smooth muscle cells.43 A new vascular system has been shown to develop between host and graft in skeletal muscle implanted into the cheek pouch of a hamster.44 These vessels grew from the graft to the host and functioned in 4 days. It is speculated that the addition of bFGF increases the intrinsic response of new microvessels budding to bridge the gap between the perfused muscle flap and the ischemic hind-limb muscle.

Angiogenesis was documented in all animals by means of direct histologic evidence of new blood vessel development. Exogenous bFGF doubled the microvessel density at the flap-limb interface, with the new blood vessels dispersed randomly throughout the interface. In contrast, sections from the control group had only isolated clusters of microvessels. This type of heterogenous angiogenesis is similar to that described by Weidner and Folkman in their studies of breast carcinoma, in which neovascular “hot spots” were associated with metastatic foci.45, 46 The processes responsible for cluster formation are not understood and are not addressed in this study. However, this is currently an area of active investigation.

Immunostaining confirmed that bFGF was delivered to the flap-limb interface, and that it diffused only a short distance (5 to 10 mm) from the tip of the infusion catheter. The extracellular matrix diffusely stained for bFGF to a distance 5 to 10 mm from the catheter tip; large numbers of new microvessels were diffusely distributed in this same region (Fig. 4, B). In contrast, fewer new microvessels developed in the control animals, and these vessels developed in inhomogeneous clusters. In control animals, bFGF stained only in the vicinity of these clusters of microvessels (Fig. 4, A). Although there is no specific antibody to distinguish exogenous from endogenous bFGF, these findings (increased numbers of microvessels in the region of exogenously supplied bFGF) strongly suggest that exogenous bFGF enhanced angiogenesis.

Similar endogenous bFGF mRNA expression was found in both the control animals and the bFGF-treated animals. This suggests that the expression of the endogenous bFGF was not regulated in an autocrine fashion. If bFGF is regulated in either a positive or negative autocrine manner, variances in bFGF mRNA expression should be observed in bFGF-treated samples. Perhaps bFGF expression is regulated by an alternative growth factor or cytokine induced by ischemia. Because of the semiquantitative nature of the PCR performed in this study, the amount of mRNA cannot be quantified. Further studies using in-situ hybridization are needed to identify and quantitate the specific cells expressing the angiogenic growth factor in the muscle-flap interface of this model. Also planned is infusion of antibodies to bFGF receptors. A decrease in angiogenesis in response to such antibodies would be compelling evidence of the central role of bFGF in angiogenesis in this model.

The physiologic relevance of the new blood vessels induced was not addressed. However, previous microsphere perfusion determinations demonstrated that resting limb perfusion was increased by 45%, presumably via the vascular connections between the flap and the ischemic limb.20 If the dose and area of distribution of bFGF can be increased, a greater angiogenic response that may have a more significant physiologic effect can be expected. In addition, physiologic and long-term data are required to assess whether the enhanced angiogenesis in this model represents a persistent, functioning, new vascular system, or simply a temporary, non-functional, neovascular response.

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Conclusions 

Local infusion of exogenous bFGF, a potent angiogenic mitogen, significantly increased new blood vessel development. Immunostaining confirmed that bFGF was delivered to the muscle-flap interface. In the bFGF-treated group, the pattern of vessel development correlated with the diffusion of bFGF. bFGF mRNA expression was not downregulated after exogenous bFGF infusion. In-situ hybridization studies to quantitate and localize endogenous bFGF production are in progress, as are assays for bFGF receptors. These studies should further define the role of bFGF in the control of new blood vessel ingrowth into ischemic skeletal muscle.

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References 

  1. Goldsmith HS. Salvage of end stage ischemic extremities by intact omentum. Surgery. 1980;88:732–736
  2. Hoshino S, Hamada O, Iwaya F, Takahira H, Honda K. Omental transplantation for chronic occlusive arterial disease. Int Surg. 1979;64:21–29
  3. Hoshino S, Nakayama K, Igari T, Honda K. Long-term results of omental transplantation for chronic occlusive arterial disease. Int Surg. 1983;68:47–50
  4. Maurya SD, Singhal S, Gupta HC, Elhence IP, Sharma BD. Pedicled omental grafts in the revascularization of ischemic lower limbs in Buerger's disease. Int Surg. 1985;70:253–255
  5. Unger EF, Sheffield CD, Epstein SE. Creation of anastomoses between an extracardiac to coronary anastomoses in a canine model. Am J Physiol. 1991;260:H1625–H1634
  6. Unger EF, Sheffield CD, Epstein SE. Creation of anastomoses between an extracardiac artery and the coronary circulation: Proof that myocardial angiogenesis occurs and can provide nutritional blood flow to the myocardium. Circulation. 1990;82:1449–1466
  7. Shestak KC, Hendricks DL, Webster MW. Indirect revascularization of the lower extremity by means of microvascular free-muscle flap—a preliminary report. J Vasc Surg. 1990;12:581–585
  8. Cohen LB. Limb salvage in the patient with severe peripheral vascular disease: The role of microsurgical free tissue transfer. Plast Reconstr Surg. 1987;79:389–395
  9. Casten DF, Alday ES. Omental Transfers for revascularization of the extremities. Surg Gynecol Obstet. 1971;132:301–304
  10. Pu L, Sniderman AD, Arekat Z, Graham AM, Brassard R, Symes JF. Angiogenic growth factor and revascularization of the ischemic limb: Evaluation in a rabbit model. J Surg Res. 1993;54:575–583
  11. Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collateral by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model: Dose-response effect of basis fibroblast growth factor. J Vasc Surg. 1992;16:181–191
  12. Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, et al.  Site-specific therapeutic angiogenesis after systemic administration of vascular endothelial growth factor. J Vasc Surg. 1995;21:314–325
  13. Takeshita S, Pu L, Stein LA, Sniderman AD, Bunting S, Ferrara N, et al.  Intramuscular administration of vascular endothelial growth factor induces dose-dependent collateral artery augmentation in a rabbit model of chronic limb ischemia. Circulation. 1994;90(Pt 2):II-228–II-234
  14. Landau C, Jacobs AK, Haudenschild CC. Intrapericardial basic fibroblast growth factor induces myocardial angiogenesis in a rabbit model of chronic ischemia. Am Heart J. 1995;129:924–931
  15. Gospodarowicz D. Humoral control of cell proliferation: The role of fibroblast growth factor in regeneration, angiogenesis, wound healing, and neoplastic growth. In:  Marchesi VT editors. Membranes and Neoplasia: New Approaches and Strategies. New York: : Alan R. Liss; 1976;p. 1–19
  16. Saksela O, Moscatelli D, Rifkin DB. The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells. J Cell Biol. 1987;105:957–963
  17. Flaumenhaft R, Moscatelli D, Saksela O, Rifkin DB. Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long term stimulation of plasminogen activator production and DNA synthesis. J Cell Physiol. 1991;260:H1625–H1634
  18. Folkman J, Klagsburn M. Angiogenic factors. Science. 1987;235:442–444
  19. McGee GS, Davidson JM, Buckley A, et al.  Recombinant basic fibroblast growth factor accelerates wound healing. J Surg Res. 1988;45:145–153
  20. Pevec WC, Hendricks D, Rosenthal MS, Shestak KC, Steed DL, Webster MW. Revascularization of an ischemic limb by use of a muscle pedicle flap: A rabbit model. J Vasc Surg. 1991;13:385–390
  21. Hendricks DL, Pevec WC, Shestak KC, Rosenthal MC, Webster MW, Steed DL. A model of persistent partial hind limb ischemia in the rabbit. J Surg Res. 1990;49:453–457
  22. Pevec WC, Ndoye A, Brinsky JL, Wiltse S, Cheung AT. New blood vessels can be induced to invade ischemic skeletal muscle. J Vasc Surg. 1996;24:534–544
  23. Edelman ER, Mathiowitz E, Langer R, Klagsbrun M. Controlled and modulated release of basic fibroblast growth factor. Biomaterials. 1991;12:619–626
  24. Woodall CJ, Watt NJ, Clements GB. Simple technique for detecting RNA viruses by PCR in single sections of wax embedded tissue. J Clin Pathol. 1993;46:276–277
  25. Gospodarowicz D. Purification of a fibroblast growth factor from bovine pituitary. J Biol Chem. 1975;250:2515–2520
  26. Gospodarowicz GD. Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature. 1987;325:257–259
  27. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization and biological function of fibroblast growth factor. Endocr Rev. 1987;8:95–114
  28. Klagsbrun M. The fibroblast growth factor family: Structural and biological properties. Prog Growth Factor Res. 1989;1:207–235
  29. Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D. Cappillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature. 1987;325:257–259
  30. Whalen GE, Shing Y, Folkman J. The fate of intravenously administered bFGF and the effect of heparin. Growth Factors. 1989;1:157–164
  31. McNeil D, Muthukrishnan L, Warder E, D'Amore PA. Growth factors are released by mechanically wounded endothelial cells. J Cell Biol. 1989;109:811–822
  32. Abraham JA, Mergia A, Whang JL, et al.  Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science. 1986;233:545–548
  33. Klagsbrun M. Angiogenic factors: Regulators of blood supply-side biology. New Biol. 1991;3:745–749
  34. Casscells W, Speir E, Sasse J, et al.  Isolation, characterization, and localization of heparin-binding growth factors in the heart. J Clin Invest. 1990;85:433–441
  35. Phillips HS, Hains J, Leung DW, Ferrara N. Vascular endothelial growth factor is expressed in rat corpus luteum. Endocrinology. 1990;127:965–968
  36. De Juan E, Shefansson E, Ohira A. Basic fibroblast growth factor stimulates 3H-thymidine uptake in retinal venular and capillary endothelial cells in vivo. Invest Ophthalmol Vis Sci. 1990;31:1238–1244
  37. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–845
  38. Mayer E, Cardoso PFG, Puskas JD, et al.  The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heteroptic rat tracheal isografts. J Thorac Cardiovasc Surg. 1992;104:180–188
  39. Cuevas P, Gonzalez AM, Carceller F, Baird A. Vascular response to basic fibroblast growth factor when infused onto the normal adventitia or into the injured media of the rat carotid artery. Circ Res. 1991;69:360–369
  40. Eppley B, Doucet M, Connolly D, Feder J. Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit. J Oral Maxillofac Surg. 1988;46:391–398
  41. Ibukiyama C. Angiogenesis. Angiogenic therapy using fibroblast growth factors and vascular endothelial growth factors for ischemic vascular lesions. Jpn Heart J. 1996;37:285–300
  42. Klagsbrun M, D'Amore P. Regulators of angiogenesis. Annu Rev Physiol. 1991;53:217–239
  43. Stavri GT, Zachary IC, Baskerville PA, Martin JF, Erusalimsky JD. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Circulation. 1995;92:11–14
  44. Faulkner JA, Wess SW, McGeachie JK. Revascularization of skeletal muscle transplanted into the hamster cheek pouch: Intravital and light microscopy. Microvasc Res. 1983;26:49–64
  45. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1–8
  46. Weidner N, Folkman J, Pozza F, et al.  Tumor angiogenesis: A new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst. 1992;84:1875–1887

☆ From the Department of Surgery (Drs Bush, Pevec, and Pearson), and the Department of Pathology (Drs Ndoye and Cheung), University of California Medical Center, Davis, and the Department of Molecular Biology (Dr Sasse), Shriners Childrens Hospital, Tampa.

☆☆ Supported in part by the University of California Davis Faculty Research Grant (1996–1997).

★ Reprint requests: William C. Pevec, MD, Department of Surgery, University of California Davis Medical Center, 4301 X Street, Room 2330, Sacramento, CA 95817.

★★ 24/1/91610

PII: S0741-5214(98)70070-9

Journal of Vascular Surgery
Volume 28, Issue 5 , Pages 919-928, November 1998

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