Recovery of Disturbed Endothelium-Dependent Flow in the Collateral-Perfused Rabbit Ischemic Hindlimb After Administration of Vascular Endothelial Growth Factor
Background Disturbed endothelium-dependent blood flow has been shown to be a feature of native collateral vessels. Recent studies have shown that recombinant angiogenic growth factors augment collateral development in animal models of hindlimb ischemia. We therefore investigated the hypothesis that the administration of an angiogenic growth factor, in this case vascular endothelial growth factor (VEGF), may promote recovery of disturbed endothelium-dependent blood flow in a rabbit model of hindlimb ischemia.
Methods and Results Ischemia was induced by ligation of the external iliac artery and excision of the femoral artery in one limb of New Zealand White rabbits (day 0). Flow velocity was measured using a Doppler guide wire at rest and after administration of serotonin and acetylcholine. Blood flow (in mL/min) was calculated assuming a circular lumen geometry. In untreated control animals with an ischemic limb, serotonin administered at day 10 or 40 produced a decrease in hindlimb blood flow (71±2% and 33±6% reduction from baseline, respectively); in contrast, among animals treated with a single 500-μg bolus dose of VEGF administered selectively into the internal iliac artery at day 10 and studied at day 40, serotonin produced an increase in flow (119±8% from baseline; P<.05 versus control animals). Acetylcholine induced only a moderate increase in flow in control animals (152±15% at day 10 and 177±14% at day 40) in contrast to a profound increase among VEGF-treated animals studied at day 40 (254±25%; P<.05 versus control animals).
Conclusions To our knowledge, these findings constitute the first demonstration of successful pharmacological modulation of disturbed endothelium-dependent flow in the arterial circulation subserved by collateral vessels. This physiological benefit complements previously reported anatomic findings suggesting a favorable impact of angiogenic growth factors on collateral-dependent limb ischemia.
In obstruction of a major artery, blood flow to the ischemic tissue is often dependent on collateral vessels. When spontaneous development of collateral vessels is insufficient to allow normal perfusion of the tissue at risk, residual ischemia occurs. A growing body of evidence1 2 3 4 5 indicates that abnormal vascular reactivity may limit the facilitatory effects of collateral vessels on tissue perfusion. Previous studies have demonstrated that this abnormal reactivity is, at least in part, the consequence of dysfunctional endothelium.1 2 6 The magnitude of increased flow that is predictably induced by acetylcholine in normal limbs, for example, is significantly blunted in ischemic limbs perfused by collateral vessels.1 The response to serotonin may be even more striking: Although serotonin typically increases flow in normal limbs, it leads to a dramatic reduction in flow when administered to ischemic limbs perfused via collaterals.
Recent studies from our laboratory have shown that a single intra-arterial bolus of vascular endothelial growth factor (VEGF), a specific endothelial cell mitogen,7 augments collateral development in a rabbit model of hindlimb ischemia.8 9 Persistent impairment of endothelium-dependent vasorelaxation suggests that the physiological benefit of such augmented neovascularity is seriously limited. Accordingly, we investigated the hypothesis that administration of an angiogenic growth factor, in this case VEGF, may promote recovery of endothelium-dependent flow in a rabbit model of hindlimb ischemia. The results of these in vivo experiments demonstrate that endothelium-dependent responses are essentially restored 30 days after administration of a single intra-arterial bolus of VEGF. These findings thus constitute a physiological complement to previously published anatomic studies and support the notion that VEGF administration may confer functional benefit in the setting of lower-extremity ischemia.
We used a rabbit ischemic hindlimb model that has been described previously.10 All protocols were approved by St Elizabeth’s Institutional Animal Care and Use Committee. Male New Zealand White rabbits (weight, 3.8 to 4.2 kg) (Pine Acre Rabbitry) were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8 mg/kg) after premedication with xylazine (2 mg/kg). A longitudinal incision was then performed, extending inferiorly from the inguinal ligament to a point just proximal to the patella. The limb in which the incision was performed—right versus left—was determined randomly at the time of surgery by the operator. Through this incision, with surgical loops, the operator dissected free the femoral artery along its entire length; all branches of the femoral artery, including the inferior epigastric, deep femoral, lateral circumflex, and superficial epigastric, also were dissected free. After dissection of the popliteal and saphenous arteries distally, the external iliac artery and all of the mentioned arteries were ligated with 4-0 silk (Ethicon). Finally, the femoral artery was completely excised from its proximal origin as a branch of the external iliac artery to the point distally where it bifurcates to form the saphenous and popliteal arteries. Excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent on collateral vessels issuing from the internal iliac artery (Fig 1⇓).
Six rabbits did not undergo limb surgery; these rabbits were used to perform dose-response experiments in normal, nonischemic limbs.
The remaining 21 rabbits underwent surgical resection of the femoral artery as described. Nine of these rabbits were studied after surgery at day 10 only: 5 rabbits were used to assess the baseline (ischemic) response to serotonin and acetylcholine, whereas 4 rabbits were used to document the effect of pretreatment with the S2 antagonist ketanserin on the administration of serotonin. Twelve animals, at day 10 after surgery, received either VEGF (n=6) or saline (n=6) administered as a single intra-arterial bolus into the internal iliac artery of the ischemic limb. A 3F end-hole infusion catheter (Tracker-18, Target Therapeutics) was introduced into the right common carotid artery through a small cutdown and positioned under fluoroscopic guidance in the proximal segment of the internal iliac artery of the ischemic limb. VEGF (500 μg in 3 mL of saline containing 0.1% albumin) was then selectively delivered into the internal iliac artery as a bolus over 1 minute. The catheter was washed with 3 mL of saline containing 0.1% albumin. Six rabbits that received an identical volume of saline with 0.1% albumin but without recombinant VEGF protein administered over 1 minute were used as control animals. Vascular reactivity was studied in all of these 12 animals at day 40 (ie, 30 days after the administration of VEGF or saline). In 3 animals, vascular reactivity was also studied 10 days after surgery before the administration of VEGF (n=2) or saline (n=1); these animals were used to confirm that the findings documented for the groups studied at day 10 and the group studied at day 40 could be reproduced sequentially in the same rabbit.
On the day of the experiment, each rabbit was anesthetized with ketamine (10 mg/kg) and acepromazine (0.2 mg/kg) after premedication with xylazine (2 mg/kg). Supplemental anesthetic doses were not required, and spontaneous ventilation was maintained throughout the study.
A 3F end-hole infusion catheter (Tracker-18) was inserted into the left common carotid artery and advanced to the abdominal aorta.
As previously described,11 12 13 14 an 0.018-in Doppler guide wire (Cardiometrics, Inc) was advanced through the 3F infusion catheter to the proximal segment of the internal iliac artery supplying the ischemic limb. The Doppler wire recorded a real-time spectral analysis of the Doppler signal, from which the average peak velocity ([APV] the temporal average of the instantaneous peak velocity waveform) was calculated and displayed on-line. The cross-sectional area of this wire (0.164 mm2) is well suited for measurement of blood flow velocity in small vessels without compromising the flow profile downstream from its tip. Previous validation studies have established that the APV recorded with this instrument correlates closely with flow, determined by electromagnetic flowmeters, for lumen diameters varying between 0.79 and 4.76 mm in straight vascular segments.11 The size (1.26 to 2.12 mm; Table⇓) and straight orientation of the internal iliac artery from which the Doppler sample was recorded thus are both appropriate for the Doppler wire and suggest that velocity and flow can be assessed with reasonable accuracy. Moreover, because the tip of the Doppler wire remained in the same position throughout the experiment, changes in velocity and velocity-derived flow after infusion of serotonin or acetylcholine appear to constitute reliable indexes of the vasoreactivity of the collateral circulation in this animal model.
A second catheter (Tracker-18) was introduced into the left common carotid artery through the same cutdown and advanced under fluoroscopic guidance to the origin of the common iliac artery of the ischemic limb with a 0.014-in guide wire (Hi-Torque Floppy II, Advanced Cardiovascular Systems). This catheter was used for infusion of vasoactive drugs, for direct measurement of intra-arterial blood pressure via connection to a pressure transducer (model 78534C, Hewlett Packard), and for selective angiography of the ischemic limb. The use of the same catheter precluded graphic display of intra-arterial blood pressure during drug infusion; thus, blood pressure was determined before and immediately after drug administration. Angiography was performed immediately after drug administration with 1 mL of contrast media (Isovue-370, Squibb Diagnostics). Serial images of the ischemic limb were recorded on 105-mm spot film at a rate of two films per second for 5 seconds.
Vascular Reactivity Studies
Serotonin creatine sulfate and acetylcholine chloride were administered intra-arterially via a constant infusion pump (0.5 mL/min) at dosages of 0.15, 1.5, and 15 μg · kg−1 · min−1, each for 2 minutes. A 5-minute interval was used between each dose to reestablish basal hindlimb blood flow values. After serotonin and acetylcholine infusion, an intra-arterial bolus dose of nitroglycerin (50 μg/kg) was administered to assess endothelium-independent vasomotor reactivity. In 4 animals at day 10, ketanserin (100 μg/kg) was administered as an intra-arterial bolus 5 minutes after a 2-minute infusion of serotonin (1.5 μg · kg−1 · min−1); 5 minutes after ketanserin administration, a second infusion of serotonin (1.5 μg · kg−1 · min−1) was given.
The angiographic luminal diameter of the internal iliac artery in the ischemic limb at baseline and after drug infusion was determined with an automated edge-detection system that has been previously validated in vivo.15 The film selected for analysis was scanned with a high-resolution video camera; the signal produced by the video camera was digitized and displayed on a video monitor. Centerlines were traced manually for a 10-mm-long segment beginning immediately distal to the tip of the Doppler wire. The contours were subsequently detected automatically on the basis of the weighted sum of first- and second-derivative functions applied to the digitized brightness information. The vascular diameter was then measured at the site of the Doppler sample volume (5 mm distal to the wire tip11 ). Cross-sectional area was calculated assuming a circular lumen.
Doppler-derived flow was calculated as QD=(πd2/4) (0.5×APV), where QD is Doppler-derived time average flow (mL/min), d is vessel diameter, and APV is time average of the spectral peak velocity.11 The mean velocity was estimated as 0.5×APV by assuming a time-averaged parabolic velocity profile across the vessel. The Doppler-derived flow calculated in this fashion has been shown to correlate with flow measurements determined by electromagnetic flowmeters both in vitro and in vivo.11
Acetylcholine chloride and serotonin creatine sulfate were obtained from Sigma Chemical Co. Fresh stock solutions were prepared immediately before each experiment. Nitroglycerin was obtained from Solopak Laboratories. The 165–amino acid homodimeric species of recombinant human VEGF was purified from transfected Chinese hamster ovary cells as previously described.16 The purity of the material was assessed with a silver-stained SDS-PAGE gel and by the presence of a single NH2-terminal amino acid sequence.
All results are expressed as mean±SEM. Statistical significance was evaluated using unpaired Student’s t test for comparisons between two mean values and by ANOVA followed by Scheffé’s procedure for more than two mean values. A value of P<.05 was considered to indicate statistical significance.
Vascular Reactivity in the Nonischemic Rabbit Hindlimb
A dose-response experiment was performed in six nonischemic rabbits to assess the changes in flow (in mL/min) during serotonin and acetylcholine infusion in nonischemic limbs. Previous work from our laboratory9 established that most of the flow (>80%) to the normal distal limb is provided by the external iliac artery. To establish the normal dose-response curve and thereby the dose of acetylcholine and serotonin to be used, we performed these experiments in the external iliac artery. (Because most of the blood flow in the ischemic limb of this animal model is provided by the internal iliac artery, analyses of flow after administration of VEGF or saline to the ischemic limb were performed in the internal iliac artery.) Fig 2⇓ shows that for serotonin, both dosages of 0.15 and 1.5 μg · kg−1 · min−1 induced a modest increase in flow (116% of baseline flow for 1.5 μg · kg−1 · min−1), whereas 15 μg · kg−1 · min−1 produced a decrease in hindlimb blood flow. For acetylcholine, maximal flow (211% of baseline flow) was observed after administration of 1.5 μg · kg−1 · min−1. Dosages of 1.5 μg · kg−1 · min−1 of serotonin and of 1.5 μg · kg−1 · min−1 of acetylcholine were therefore used for all subsequent experiments in the ischemic hindlimb; these dosages of serotonin and acetylcholine had no significant impact on systemic arterial blood pressure (not shown).
Vascular Reactivity in the Rabbit Ischemic Hindlimb
The vasomotor responses recorded after administration of serotonin and acetylcholine on day 10 after surgery (immediately before administration of VEGF) and day 40 (in both VEGF-treated and control animals) are summarized in the Table⇑. No statistically significant differences were observed among the three groups for resting flow in the ischemic limb (day 10, 17.7±1.9 mL/min; day 40 saline, 22.5±0.7 mL/min; day 40 VEGF, 19.4 mL/min; P=NS). Accordingly, the responses to serotonin, acetylcholine, and nitroglycerin were each expressed as percentage of resting flow.
Fig 3⇓ shows the effect of serotonin on ischemic hindlimb blood flow. At day 10, serotonin induced a dramatic decrease in flow (71% reduction in resting flow). At day 40, in control animals, a decrease in flow was again observed after infusion of serotonin, although the reduction in flow observed at this later time point (33% reduction in resting flow) was significantly (P<.01) less severe than that observed at day 10. The response recorded in animals treated with VEGF at day 10 and evaluated at day 40 differed markedly from that of the control animals: serotonin produced an increase in flow (119% of resting flow; P<.001 versus control animals). The response of the ischemic limb to serotonin in VEGF-treated rabbits was similar to that observed in the normal (nonischemic) rabbit hindlimb (Fig 2⇑).
Fig 4⇓ illustrates representative angiographic findings in a rabbit studied at day 10 (before administration of VEGF) and at day 40 (30 days after VEGF). The initial study performed at day 10, immediately before VEGF administration, disclosed angiographically evident vasoconstriction in response to serotonin. The animal was restudied at day 40, ie, 30 days after receiving a single bolus of VEGF; the same dose of serotonin at this later time point produced no reduction in luminal diameter of the angiographically visible vessels.
As shown in Fig 5⇓, a bolus injection of the S2 antagonist ketanserin prevented the decrease in flow observed in response to serotonin at day 10 (72% reduction in resting flow without pretreatment versus a 9% increase in resting flow for pretreatment with ketanserin; P<.05). Fig 6⇓ is a representative example of a rabbit studied at day 10. Angiographically evident vasoconstriction was observed after the first administration of serotonin; the second administration of serotonin, 5 minutes after ketanserin pretreatment, had no appreciable effect on the angiographically visible vessels.
Fig 7⇓ shows the response of the ischemic limb to acetylcholine. At day 10, acetylcholine induced a moderate increase in hindlimb blood flow (152% of resting flow). In control animals, at day 40, the response was similar (177% of resting flow). In VEGF-treated animals, however, the response to acetylcholine was considerably more marked (254% of resting flow; P<.05 versus control animals).
The endothelium-independent responses to nitroglycerin are shown in Fig 8⇓. There were no significant differences between the day-10 group (198% of baseline flow) and the day-40 control group (239% of baseline flow). The response to nitroglycerin in the day-40 VEGF-treated group (293% of baseline flow) was significantly (P<.05) higher than that observed at day 10.
Recent studies have established that intra-arterial administration of VEGF augments the development of collateral vessels in the rabbit ischemic hindlimb.8 9 The present study provides important additional information by showing for the first time that the in vivo response of the collateral circulation to serotonin and acetylcholine is improved 30 days after a single intra-arterial administration of VEGF. These data suggest that VEGF not only augments neovascularity in this animal model but also facilitates the recovery of endothelium-dependent flow responses in the collateral-fed circulation of the ischemic extremity.
Previous studies have established that long-term perfusion through native coronary collateral vessels produces endothelial dysfunction in the recipient, downstream, reconstituted vasculature.1 4 The endothelium-dependent vasomotor response of limbs perfused via collaterals has likewise been shown to be abnormal. In a canine model of hindlimb ischemia-associated collateral development, Orlandi et al1 demonstrated a paradoxical decrease in flow in response to serotonin and a blunted increase in flow in response to acetylcholine. The sensitivity of limb collateral vessels to serotonin in particular has been documented in multiple species and has been shown to be of prolonged duration (for a review, see Hollenberg3 ). The findings observed in our experimental model are consistent with these previous reports. The administration of 1.5 μg · kg−1 · min−1 serotonin (a dosage that produced an increase in flow in normal, nonischemic limbs) resulted in a marked reduction in ischemic hindlimb blood flow at day 10. Although the decrease in flow in response to serotonin was less marked among saline-treated ischemic limbs at day 40, the response was still abnormal.
The protective effect of ketanserin pretreatment in this model suggests that serotonin-induced vasoconstriction was, as previously suggested,1 17 mediated by direct activation of S2 receptors in the medial vascular smooth muscle. Vasodilation in response to serotonin is believed to result from endothelium-dependent relaxation via activation of S1-like receptors that mediate the release of endothelium-derived relaxant factor (EDRF).18 In the case of dysfunctional endothelium, endothelium-dependent relaxation can no longer occur, and thus the direct, vasoconstrictor response of the medial smooth muscle cells to serotonin is predominant.17 19
Release of EDRF from endothelial cells plays a similarly major role in the vascular relaxation produced by acetylcholine.20 Although no in vitro study has specifically demonstrated that endothelial dysfunction occurs in limbs perfused via collaterals, several studies have suggested that arterioles perfused by myocardial collaterals lose their ability to release EDRF in response to various agonists.2 4 6 Moreover, previous analyses of endothelium-dependent flow in the lower extremities of primates21 and humans22 have suggested that in contrast to the coronary arteries, muscarinic receptors are limited to the endothelial cells of the hindlimb or lower-limb peripheral arteries and are absent from medial smooth muscle. Taken together, these observations strongly suggest that dysfunctional endothelium may lead to a reduction in the release of EDRF and a corresponding reduction in flow augmentation after administration of acetylcholine; competing vasoconstriction at the level of the arterial media, however, is insufficient to induce a reduction in flow compared with baseline.
The basis for impaired endothelium-dependent flow responses in limbs perfused by collateral vessels remains enigmatic. Studies of coronary collaterals have suggested two possible explanations.4 The first involves the possibility that the collateral circulation fails to develop at a rate sufficiently rapid to prevent ischemic damage to endothelial cells of the recipient, downstream, reconstituted microvasculature. The second suggests that receptor-mediated production or release of EDRF may be regulated by perfusion pressure within the recipient vasculature; compromised perfusion pressure, with or without a pulsatile character, may further compromise deranged endothelium-dependent flow. In vitro analyses of the collateral vessels themselves, in the case of the coronary circulation, have disclosed an appropriate response to endothelium-dependent agonists such as acetylcholine. In contrast, an abnormal response (reduced vasodilation to acetylcholine) was documented in microvessels (100 to 200 μm) perfused by mature collaterals compared with microvessels of the normal arterial circulation; the response of the microvessels perfused by mature collateral vessels to direct smooth muscle dilators (eg, nitroglycerin) was not reduced (it was, in fact, slightly enhanced). It therefore is possible that perfusion through collateral vessels selectively alters the recipient, downstream arterial circulation, reducing endothelial cell membrane receptor affinity, number, or interaction with second-messenger systems in the recipient vessels. The biosynthetic pathway, release, or degradation of EDRF itself appeared not to be implicated, due to the fact that the response to the calcium ionophore A23187 (which causes vasodilation by releasing EDRF through nonreceptor, non–second-messenger–mediated pathways) was preserved intact. Thus, membrane signaling within the endothelium may be impaired, similar to that described in regenerated endothelium after balloon denudation.19
The results of the present study demonstrate an improvement in both endothelium-independent and -dependent hindlimb blood flow 30 days after a single intra-arterial bolus of VEGF. The endothelium-independent increase in flow after administration of nitroglycerin is in agreement with previous studies from our laboratory demonstrating an increase in flow reserve (as assessed with papaverine, a direct smooth muscle vasodilator23 ) in VEGF-treated animals.9 This augmented response to endothelium-independent vasodilators is likely to represent a direct consequence of the increase in collateral vessel development induced by VEGF.8 9 Although supporting the concept of therapeutic angiogenesis, these previous studies provided no information concerning endothelium-dependent function of collateral circulation that develops in response to VEGF.
The results summarized in Figs 3⇑ and 7⇑ demonstrate that endothelium-dependent flow also is markedly improved in response to VEGF. Acetylcholine administered at day 40 induced a much greater increase in flow in VEGF-treated than in control animals. The response to serotonin administered at the same time point was even more striking: Although a decrease in flow was observed in control animals, an increase in flow was observed in VEGF-treated animals.
At least three mechanisms could explain an improvement in endothelium-dependent flow responses of the collateral-dependent limb after VEGF therapy. The first possibility relates to the characteristics of flow and perfusion pressure in arterioles distal to collaterals. We have previously demonstrated that VEGF therapy produces a significant increase in the calf blood pressure of the ischemic limb8 ; it is entirely possible that such improved perfusion pressure may lead to repair of dysfunctional endothelium in the collateral-perfused distal vasculature. A second and intriguing possibility relates to a direct improvement of endothelial function by VEGF. In the case of bFGF, for example, in vitro studies have recently demonstrated that endothelial function in the coronary microcirculation perfused via collateral vessels is preserved by long-term administration of this endothelial cell mitogen.24 The fact that VEGF may also modulate qualitative aspects of endothelial cell function25 suggests that it, too, may directly repair endothelial cells presumed to be damaged by protracted ischemia in the collateral-dependent limb and thereby restore normal endothelium-dependent flow. Third, the possibility that the documented improvement in endothelium-dependent flow is the result of the newly formed, VEGF-induced collateral vessels cannot be discounted. The precise basis for improved endothelium-dependent vasomotor reactivity in response to VEGF remains to be clarified.
In conclusion, the findings reported in the present study indicate that a single intra-arterial bolus of angiogenic growth factor restores, in large part, the responses of the ischemic hindlimb to endothelium-dependent vasodilators. These results may have important clinical implications. The hypersensitivity to serotonin of the collateral circulation is not limited to animal models. Platelet activation releases vasoactive quantities of serotonin in vitro,26 and the S2-receptor antagonist ketanserin dilates limb collaterals in >50% of patients with advanced atherosclerosis.27 Ketanserin can also improve symptoms of intermittent claudication and limb perfusion in selected patients with peripheral artery disease,28 suggesting that abnormal reactivity may in large part limit the beneficial consequences of collaterals in humans. VEGF, via both previously documented anatomic8 9 effects and currently demonstrated physiological effects on lower-extremity collateral circulation, may therefore constitute an efficacious therapeutic approach for patients with lower-limb ischemia.
This work was supported in part by grants HL-40518 and Academic Award in Vascular Medicine HL-02824 from the National Heart, Lung, and Blood Institute, National Institutes of Health (Bethesda, Md). We gratefully acknowledge the assistance of Mickey Neely in the preparation of the manuscript.
- Received December 19, 1994.
- Accepted January 31, 1995.
- Copyright © 1995 by American Heart Association
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