Basic Fibroblast Growth Factor Restores Endothelium-Dependent Responses After Balloon Injury of Rabbit Arteries
Background After experimental angioplasty, partial or complete reendothelialization of the denuded surface occurs; the function of the regenerated endothelium has, however, been shown to be abnormal. Basic fibroblast growth factor (bFGF) is mitogenic for endothelial cells in vitro and in vivo. We investigated whether chronic administration of bFGF in a rabbit model of balloon denudation might not only improve endothelial regrowth but also restore normal physiological responses to endothelium-dependent agonists.
Methods and Results Thirty-nine New Zealand White rabbits underwent balloon denudation of the right iliac artery. Twenty rabbits received intravenous administration of bFGF (2.5 μg twice a week for 2 weeks). Nineteen rabbits receiving saline injections served as controls. Animals were killed on day 28 for assessment of reendothelialization and neointimal thickening and for analysis of in vitro vasoreactivity. Animals in the bFGF group had a significantly (P<.005) greater degree of reendothelialization than controls (115±13 versus 55±6 mm2). Neointimal thickening was similar in the two groups. Four weeks after denudation, endothelium-independent responses did not differ significantly between the two groups. In contrast, the maximal endothelium-dependent acetylcholine-induced relaxation of the bFGF-treated animals (Emax, 40±7%) was significantly greater than that of the control group (Emax, 11±9%; P<.05).
Conclusions Systemic administration of bFGF restores, in large part, the responses of previously denuded arterial segments to endothelium-dependent vasodilators. Angiogenic growth factors may help to reestablish normal endothelial cell function in patients who have undergone angioplasty.
The response of arterial segments to experimental endoluminal injury has been studied extensively during the past few years. Blood vessel balloon denudation induces proliferation and migration of smooth muscle cells and accumulation of a large amount of extracellular matrix.1 2 Endothelium plays a fundamental role in controlling vessel tone and, in addition, may regulate the growth of the underlying smooth muscle cells.3 4 5 6 After experimental angioplasty, reendothelialization of the denuded surface occurs within weeks and may be either complete7 8 or incomplete,5 9 10 depending on the animal model studied. The function of the regenerated endothelium, however, has been shown to be abnormal8 11 12 ; the relaxant effect of endothelium-dependent vasodilators remains impaired in injured vessels even if complete reendothelialization has occurred.8 11
Previous studies have demonstrated the feasibility of using recombinant angiogenic growth factors such as bFGF or VEGF for increasing the rate and the extent of endothelial regrowth after arterial injury.13 14 However, although these studies clearly demonstrated a beneficial anatomic effect, the function of the regenerated endothelium was not assessed.
It has recently been shown that the administration of VEGF in an animal model of hindlimb ischemia is associated with an improvement of endothelium-dependent responses of the collateral-perfused arterial bed15 ; similar results have been reported for bFGF in a model of myocardial ischemia.16 These findings suggest that angiogenic growth factors, in addition to having an effect on endothelial cell growth, may also modulate endothelial cell function. We thus designed the present study to test the hypothesis that chronic administration of an angiogenic growth factor, in this case bFGF, in a rabbit model of balloon denudation might not only improve endothelial regrowth but also restore normal physiological responses to endothelium-dependent agonists.
A total of 39 male New Zealand White rabbits (2.5 to 3.0 kg) were used. Twenty rabbits received four intravenous boluses of human recombinant bFGF (2.5 μg bFGF in 1 mL 0.5% albumin per injection) on days 1, 4, 7, and 10 after balloon denudation (see below). The dose of bFGF was chosen because it has already been demonstrated to be effective in enhancing collateral development in the rabbit.17 Nineteen rabbits receiving vehicle injections served as controls. Animals in both groups were then killed on day 28 for assessment of the degree of reendothelialization, assessment of the degree of neointimal thickening, and analysis of in vitro vasoreactivity. All experiments were conducted in compliance with the position of the American Heart Association on research animal use.
Rabbits fed a normal diet were anesthetized with ethyl carbamate (1 g/kg IV). After exposure of the right femoral artery, a 3F Fogarty balloon catheter was passed retrogradely to the junction of the aorta with the iliac artery and inflated until contact was made with the endothelium. The iliac artery was denuded by advancement and withdrawal of the catheter three times. The catheter was then removed, the femoral artery was ligated, and 250 mg amoxicillin was given locally in the groin incision. The investigator performing the procedure was blinded to the nature of the treatment.
Macroscopic Evaluation of Reendothelialization
Eleven animals (five controls, six receiving bFGF) were used for macroscopic evaluation of the degree of reendothelialization. Thirty minutes before they were killed, the animals received an intravenous injection of 5 mL 1% Evans blue dye18 to identify the remaining denuded area. A catheter inserted into the aorta was used to perfuse PBS in situ at a pressure of 110 mm Hg until the effluent ran clear. The iliac arteries (from the aortoiliac bifurcation to the inguinal ligament) were then harvested, dissected free, incised longitudinally, pinned to a cork board, and photographed with a dissecting microscope. Planimetric analysis was then performed with a computerized sketching program by one analyst, who was blinded to the treatment regimen. The reendothelialized area was defined macroscopically as the area that was not stained with Evans blue.
Eighteen animals (nine in each group) were used for histological analysis of neointimal thickening. A catheter was introduced into the aorta through the right carotid artery, and the iliac arteries were perfusion-fixed with 4% paraformaldehyde (in PBS) at a pressure of 110 mm Hg over a period of 30 minutes to maintain the vessels in their in vivo dimensions for subsequent histological analysis. After further immersion fixation (in 4% paraformaldehyde for 24 hours), each iliac artery was cut into three 5-mm segments, which were embedded in paraffin. Cross sections of vessels were cut and stained with orcein-van Gieson’s stain or with Mallory’s phosphotungstic hematoxylin. Morphometric analysis of the histological cross sections was performed by use of digital microscopic planimetry (SMC 2002, Bioblock Scientific). For each iliac segment, one cross section was analyzed. Neointimal and medial areas were measured for each section and averaged for each artery. The ratio of intimal area to medial area was calculated for each artery. All measurements were performed by a pathologist unaware of the study design. Regenerated endothelium was identified by immunostaining with platelet–endothelial cell adhesion molecule-1 (PECAM-1/CD-31)–related antigens (Dako Co) as previously described.19
In Vitro Vasoreactivity
Ten animals (five in each group) were used for assessment of vasoreactivity. Four weeks after unilateral balloon denudation, the two iliac arteries were removed and placed in iced oxygenated Krebs-Henseleit solution consisting of the following (in mmol/L): NaCl 118, KCl 4.6, NaHCO3 27.2, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.75, Na2EDTA 0.03, and d-glucose 11.1 (pH 7.35 to 7.45). Intravenous heparin (1000 IU) was given before removal of the vessels to prevent coagulation. Vessels were cleaned of surrounding fat and connective tissue and cut into rings 4.5 to 5 mm long. Rings were then suspended in organ chambers (Radnoti Glass Technology) filled with 40 mL warmed (37°C) and oxygenated (95% O2/5% CO2) Krebs-Henseleit solution. Rings were connected to force transducers, and changes in isometric force were recorded continuously. During a 60-minute period, the vascular rings were stretched to 3.0 g, previously determined as the optimal point of their length-tension relation. The output from the transducers was amplified by signal conditioners and sent to an Intel 486–based computer (Kenitec) for analog-to-digital conversion. In each animal, the nondenuded contralateral iliac artery served as a control. For each iliac artery, two rings were studied. The contractile response to a depolarizing concentration of KCl (70 mmol/L) provided a measure of maximal contractile responsiveness in each ring. All of the rings were then preconstricted with phenylephrine (10−9 to 3×10−5 mol/L). Endothelium-dependent or -independent relaxant effects then were established when the constrictor response to phenylephrine was stable. First, the relaxant response to acetylcholine (10−8 to 3×10−5 mol/L) was investigated. When the maximal responses produced by this agonist were stable, the rings were washed and allowed to stabilize at a resting tension. The smooth-muscle relaxant sodium nitroprusside (10−9 to 3×10−5 mol/L) then was given to vessels preconstricted with phenylephrine (3×10−5 mol/L).
Human recombinant bFGF, ethyl carbamate, phenylephrine hydrochloride, acetylcholine, and sodium nitroprusside were purchased from Sigma Chemical Co. bFGF (2.5 μg/mL) was dissolved in 0.5% albumin. Drugs used for vasomotor experiments were dissolved in 0.9% NaCl solution. Gases were purchased from the Compagnie Française des Produits Oxygénés and were within a tolerance of 1% of the desired mixture.
Data are given as mean±SEM. Statistical evaluation of the data (comparisons between control animals and bFGF-treated animals) was performed with unpaired Student’s t test. Relaxations to the vasodilator agents are expressed as percentages of the initial contraction to phenylephrine. To analyze vasodilation, we determined the Emax, expressed as percent relaxation of the contraction to phenylephrine, and the EC50.
In this animal model, injury with the balloon catheter resulted in complete denudation of the arterial surface (not shown). Four weeks later, a significant degree of reendothelialization was observed (Fig 1⇓). The extent of reendothelialization, however, was markedly superior in the bFGF group versus the control group at 4 weeks after balloon injury. The reendothelialized area in the control group measured 55±6 mm2; in contrast, the reendothelialized area in the bFGF group measured 115±13 mm2 (P<.005) (Fig 1⇓).
The degree of neointimal thickening was similar in the two groups (neointimal area: control, 0.32±0.03 mm2; bFGF, 0.31±0.05 mm2; P=NS; neointima-to-media ratio: control, 0.84±0.16; bFGF, 0.99±0.13; P=NS). Histological analysis of nondenuded arteries showed a normal appearance of the vessel wall with no neointimal thickening in both the control and the bFGF-treated groups (not shown).
In Vitro Vasoreactivity
Nondenuded iliac arteries. In nondenuded arteries, endothelium-independent responses to phenylephrine and sodium nitroprusside and endothelium-dependent responses to acetylcholine did not differ significantly in bFGF-treated and control animals (Table⇓, Fig 2⇓).
Previously denuded iliac arteries. Four weeks after denudation, endothelium-independent responses did not differ significantly between the two groups. The maximal tension induced by phenylephrine (3×10−5 mol/L) was not different between groups (Table⇑). Similarly, there were no differences between the two groups in maximal vasodilation to sodium nitroprusside (Emax, 102±4% for bFGF-treated animals versus 105±3% for control animals) (Fig 2⇑).
In contrast, the maximal endothelium-dependent acetylcholine-induced relaxation of the bFGF-treated animals (Emax, 40±7%) was significantly greater than that of the control group (Emax, 11±9%; P<.05) (Fig 2⇑). In addition, the Emax and the sensitivity (EC50) to acetylcholine in the bFGF group were restored to the level of nondenuded vessels, with Emax values of 40±7% and 43±6% (P=NS) and EC50 values of 6.4±2.5×10−7 and 4.2±2.2×10−7 mol/L (P=NS), respectively.
Our reendothelialization studies provide evidence for an effect of bFGF on endothelial regrowth; bFGF-treated animals had a higher degree of reendothelialization at 4 weeks than controls. These results are in agreement with the studies of Lindner et al13 that established clear evidence for the mitogenic effect of bFGF on endothelial cell replication in vivo and further demonstrated that total endothelial cell regrowth could be achieved in a rat carotid model of balloon denudation by administration of bFGF. Similar results have been obtained with acidic FGF in the same model.20
We observed a similar degree of neointimal thickening in control and bFGF-treated animals 4 weeks after injury. Previous experimental studies support the notion that certain functions of the endothelium, such as production of NO, are critical to the prevention of luminal narrowing by neointimal thickening.12 21 Accelerated reendothelialization may thus reduce neointimal formation. On the other hand, bFGF, which is a potent growth factor for smooth muscle cells,22 may directly enhance neointimal formation. Previous studies have already investigated the effect of FGFs on the neointimal response to injury and have provided discordant results. Lindner et al,23 using high doses of bFGF (12 μg/d for 2 weeks in a rat model), found a significant increase in neointimal thickening. By contrast, Bjornsson et al,20 using low doses of acidic FGF (a mitogen for smooth muscle cells in vitro24 ) in the same model, observed an inhibition of neointimal thickening. Recently, Lazarous et al25 gave systemic bFGF to dogs after femoral artery balloon denudation, and there was no increase in intimal thickening. These results, taken together with the results of the present study, suggest that the final effect of FGF on neointimal thickening may be the consequence of a balance between stimulatory and inhibitory mechanisms on smooth muscle cell growth; factors such as the dose used, the duration of the treatment, and the animal model studied may explain discrepancies between studies. In the present study, only one dose of bFGF was used; further dose-response studies may provide a better understanding of the effects of bFGF on neointimal thickening. A recent study by Asahara et al14 demonstrated that a single local administration of the angiogenic factor VEGF is sufficient to facilitate endothelial repair in a rat model of balloon injury; in this study, the degree of neointimal thickening at 2 weeks and 4 weeks after balloon injury was correspondingly attenuated to a statistically significant degree in arteries treated with VEGF versus controls. These results have been attributed to a beneficial effect of VEGF on endothelial cells, ie, reendothelialization, without detrimental effect on smooth muscle cells, ie, proliferation, because VEGF high-affinity binding sites are limited to endothelial cells.
To the best of our knowledge, the present study constitutes the first demonstration that an angiogenic growth factor may also restore normal physiological responses of an injured artery to endothelium-dependent agonists. The improvement of endothelium-dependent relaxation in bFGF-treated animals is well demonstrated by the fact that acetylcholine relaxed previously denuded arterial segments to the same extent as nondenuded segments. It appears unlikely that this improved response to acetylcholine observed after treatment with bFGF is due to an increase in the sensitivity of vascular smooth muscle cells to NO generated in endothelial cells. Indeed, the response to sodium nitroprusside, a direct NO donor, is not modified by bFGF administration. The normalized endothelium-dependent responses observed after bFGF treatment are probably not solely related to endothelial regrowth. Previous studies performed in rabbit iliac arteries have demonstrated persistent abnormal endothelium-dependent responses even in the case of complete endothelial regrowth8 ; this suggests that bFGF, in addition to its effects on endothelial cell growth, might also modulate some qualitative aspects of endothelial cells and restore normal physiological responses to endothelium-dependent agonists. Two recent studies demonstrated that the administration of angiogenic growth factors may restore normal endothelium-dependent responses in arterial beds perfused via collaterals.15 16 Chronic administration of bFGF maintained receptor-mediated endothelial function in the coronary microcirculation perfused via collateral vessels soon after gradual coronary occlusion.16 Similarly, a single intra-arterial bolus of VEGF restored, in large part, endothelium-dependent responses in an ischemic limb model.15 Further studies will be needed to characterize the effect of angiogenic growth factors in other models of endothelial dysfunction.
Finally, it should be pointed out that, in the present study, endothelium-dependent responses were improved in the absence of inhibition of neointimal thickening. Previous reports have shown a statistically significant correlation between intimal thickness and impairment of endothelium-dependent relaxation,8 26 leading to the hypothesis that intimal thickness can act as a barrier for NO, a factor with a very short half-life. Furthermore, most of the drugs that improved endothelium-dependent relaxation reduced neointimal thickening.12 27 In the present study, bFGF restored normal endothelium-dependent relaxation with no effect on intimal thickening. This suggests that the action of bFGF is solely at the endothelial level and that the observed degree of neointimal thickening does not alter NO diffusion.
In conclusion, the findings reported in the present study indicate that systemic administration of bFGF restores, in large part, the responses of previously denuded arterial segments to endothelium-dependent vasodilators. These results may have important clinical implications. Studies performed after coronary angioplasty in men have demonstrated abnormal response of previously dilated sites to endothelium-dependent agonists such as serotonin28 and acetylcholine.29 Angiogenic growth factors, via both previously documented anatomic13 14 effects and physiological effects on previously denuded arteries described here, may therefore help to reestablish normal endothelial cell function in patients who have undergone angioplasty.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|EC50||=||concentration required to cause half-maximal relaxation|
|VEGF||=||vascular endothelial growth factor|
This study was supported in part by the Centre Hospitalier et Universitaire de Lille, CIVIS project No. 93-04. Thibaud Meurice was supported by the French Federation of Cardiology.
- Received September 14, 1995.
- Revision received October 11, 1995.
- Accepted October 24, 1995.
- Copyright © 1996 by American Heart Association
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