INTRODUCTION

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BASIC FIBROBLAST GROWTH FACTOR (bFGF; FGF-2) and vascular endothelial growth factor (VEGF) are both potent angiogenic proteins capable of inducing vascular remodeling to improve collateral blood flow after occlusion of a peripheral artery. Enhanced vascular expansion in response to exogenous delivery of bFGF or VEGF has been demonstrated in several different acute-onset peripheral arterial insufficiency animal models, as judged by angiography (3, 5, 6, 25, 31) or measurements of collateral blood flow to active muscle (5, 31, 33). These growth factor treatments also generally improved the blood flow perfusion of the collateral-dependent tissue in these models, minimizing or eliminating tissue necrosis (5, 25) and improving muscle performance (29, 31).

Schaper and colleagues (2, 13) have pointed out that vascular expansion in adult organisms can occur through either of two mechanisms: capillary network expansion via the sprouting of new capillaries from existing capillaries (angiogenesis) or enlargement of existing vessels (arteriogenesis). In their rabbit model of femoral artery ligation, these investigators have demonstrated that angiogenesis occurs in the ischemic lower limb of the animals. Nonetheless, the basis of considerations of flow dynamics, they concluded that significant increases in collateral-dependent flow can only be provided by the development of muscular collateral conduits rather than capillaries. Because the formation of such conduits was observed in the nonischemic thigh region of the rabbits, they have proposed that it is increased shear stress, rather than ischemia, that drives the vascular remodeling that most affects collateral flow (2, 13). Angiogenesis and arteriogenesis are likely to share a number of processes, including endothelial cell proliferation, and both appear to be enhanced by the application of exogenous bFGF or VEGF (3, 5, 6, 25, 31).

Recent work (7, 10) has implicated the involvement of nitric oxide (NO) in the regulation of angiogenesis as well as in the individual processes of endothelial cell proliferation, migration, and differentiation. In addition, flow-induced vessel enlargement in animal models has been shown to be diminished in the presence of the NO synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), implicating NO in arteriogenesis as well (11, 27). A variety of studies have indicated that NO also plays a direct role in the mechanism of action of VEGF. VEGF has been shown to trigger the production of NO from the endothelium (15, 17, 28, 30). Endothelial cell proliferation (14, 17, 20, 22, 37), migration (19, 22, 37), and tube formation (20) in response to VEGF in vitro could all be inhibited by the addition of NOS inhibitors to the cultures. Ziche et al. (37) demonstrated that systemic administration of L-NAME could strongly reduce the angiogenic response induced in the avascular rabbit cornea by an implanted pellet containing VEGF. Finally, Murohara et al. (18) found that VEGF treatment was unable to improve blood flow in an ischemic hindlimb model involving endothelial NOS (eNOS) knockout mice, even though a similar application of VEGF had proven effective at augmenting blood flow in another mouse model with normal eNOS.

The dependence of bFGF activity on NOS is less clear. Babaei et al. (4) found that bFGF stimulated the production of NO by cultured endothelial cells and reported that endothelial cell tube formation in vitro in response to bFGF could be blocked by the addition of L-NAME, suggesting a role for NO in this process. On the other hand, NOS inhibition had no effect on bFGF-induced endothelial cell migration or proliferation (22) or rabbit cornea angiogenesis (37) in two of the studies that had documented a blockade of VEGF effects by these inhibitors. Thus, in at least some cases, bFGF and VEGF can produce similar effects via pathways that converge downstream from the point of involvement of NOS.

Because NO has been shown to be involved in flow-induced vessel enlargement (11, 27) and because arteriogenesis rather than angiogenesis has been proposed to play the critical role in enhancing blood flow in ischemic situations (2, 13), we hypothesized that bFGF, like VEGF, depends on NO production to produce an increase in collateral-dependent blood flow in vivo. To test this hypothesis, we utilized a rat model of experimental peripheral arterial insufficiency and analyzed the effects of exogenous bFGF and VEGF on collateral-dependent hindlimb blood flow in the presence or absence of systemic L-NAME.

	METHODS

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Experiment design. Rats were randomly divided into either L-NAME (n = 36) or non-L-NAME (n = 36) groups and started L-NAME feeding 2 days before bilateral femoral artery ligation. Within each group, rats were randomly assigned into vehicle control (n = 12), bFGF (5 µg · kg¹ · day¹ for 14 days, n = 12) or VEGF₁₂₁ (10 µg · kg¹ · day¹ for 14 days, n = 12) subgroups. Collateral-dependent blood flow was determined during treadmill running on day 16 after femoral artery occlusion. This was 2 days after the end of growth factor infusion, extensive time for clearance of bFGF and VEGF from the circulation. The experimental design permitted direct evaluation of the main treatment effects of NOS inhibition and the angiogenic growth factors bFGF and VEGF as well as the interaction between NOS inhibition and the growth factors.

Animal care. Seventy-two male Sprague-Dawley rats weighing ~325 g (supplied by Taconic Farms, Germantown, NY) were housed 2 rats per cage in a temperature (21°C)- and light (12:12-h dark-light cycle)-controlled room. Rats were fed Purina rat chow and water ad libitum. On arrival, all rats were accustomed to handling and treadmill walking at 20 m/min on a 15% grade for 5-10 min daily for ~5 days. The treadmill protocol included turning the treadmill on and off briefly to condition the rats to run at the front of the treadmill when the belt started moving. Our previous experiments (33-35) indicated that this treadmill conditioning protocol does not produce any detectable peripheral adaptations in the animals.

L-NAME administration. The NOS inhibitor L-NAME (Sigma; St. Louis, MO) was fed through the drinking water beginning 2 days before femoral artery ligation surgery and continuing throughout the study. L-NAME was dissolved in distilled water at a concentration of 2 mg/ml, as described by Guzman et al. (11), and was provided fresh daily. L-NAME consumption per rat was calculated daily from the water consumption.

Surgical preparation for femoral artery ligation and growth factor infusion. Under ketamine-acepromazine (100 mg per 0.5 mg/kg body wt) anesthesia, both the left and right femoral arteries were isolated and ligated with 3-0 surgical silk sutures ~5-6 mm distal to the inguinal ligament. In addition, a polyethylene (PE)-60 catheter connected to an osmotic pump (for 14 days; Alzet model 2002, Alza; Palo Alto, CA) was inserted into the left common iliac artery through the ligated femoral artery to establish the route for bFGF or VEGF₁₂₁ (Scios; Sunnyvale, CA) delivery. Topical antibiotic powder (Neo-Predef, Upjohn; Kalamazoo, MI) was placed on the wound before closure with skin clips.

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Blood flow determination. On day 16 after the pump installation and femoral artery ligation, rats from each group were surgically prepared for blood flow measurement under ketamine anesthesia, as done previously (33-35). Briefly, a PE-50 catheter was placed in the left carotid artery and advanced to the arch of the aorta for monitoring blood pressure and heart rate as well as infusing microspheres. A second catheter was placed in the caudal artery for monitoring caudal blood pressure and obtaining the reference blood sample during microsphere infusion. The arteries were catheterized early in the day. The fully conscious animals were run on the treadmill for blood flow determination after more than 4 h recovery, as described previously (33-35).

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Data analysis. All data are expressed as means ± SE. Analyses of variance were used to assess the main treatment effects of L-NAME and growth factors and the L-NAME-growth factor interactions. P < 0.05 was recognized as a significant difference. The treatment differences across groups were determined by Tukey's procedure (24).

	RESULTS

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NOS inhibition. In general, the animals showed good tolerance to L-NAME administration with acceptable running performance, although two animals were not continued on days 15 and 16 of the treatment period. L-NAME intake averaged 137 mg · kg¹ · day¹. The body weights of animals receiving bFGF and VEGF₁₂₁ infusion plus L-NAME administration were marginally but significantly less (~5-7%; Table 1) than non-L-NAME animals; however, the tissue weights of the hindlimbs were not different across the treatment groups (Table 1). L-NAME administration significantly increased preexercise systemic blood pressure by ~26% in all three subgroups (P < 0.001; Table 2). Heart rates were similar across the groups during preexercise and exercise conditions (Table 2).

Blood flow measurement. Blood flow measurements on ~10% of the animals were not possible due to difficulties in surgery and/or in obtaining a high-flow withdrawal rate of the reference sample. Appropriate microsphere distribution within the animals was evident by similar blood flow distributions between the left and right kidney with a ratio of 1.05 ± 0.02 (111 observations; blood flows to the left kidney divided by blood flows to the right kidney). Blood flows to the tissues of the left hindlimb were well matched to the blood flows of corresponding tissues in the right hindlimb for all treatment groups, with correlation coefficients between 0.986 and 0.994 and no significant differences between limbs (data not shown). Thus, for further analysis, blood flow values from the left and right side tissues were combined into one value for each tissue of each animal. Finally, blood flows during the second high-running-speed determination were not different from the first determination, indicating that the upstream resistance of the collateral vessels was the primary determinant of blood flow to the collateral-dependent calf muscles. The second blood flow determination was not obtained in a few animals because of unacceptable running performance during the high running speed, as indicated by the difference in group size between the low- and high-speed measurements given in Table 2. Although duplicate blood flows could not be verified in these animals, we accepted the flow determination at the low speed as meaningful, because speed did not change collateral-dependent blood flow. Including these values did not alter the findings of the study.

Growth factor-induced collateral blood flow. In the absence of L-NAME treatment, bFGF or VEGF₁₂₁ infusion for 2 wk markedly increased blood flows to the total, proximal, and distal hindlimbs (P < 0.001) compared with the vehicle control subgroup (Table 3). Blood flows to individual tissues comprising the hindlimb were similiarly changed (Table 4). VEGF₁₂₁ at 10 µg · kg¹ · day¹ induced a collateral blood flow increase that was similar to the increase induced by 5 µg · kg¹ · day¹ bFGF (Table 3). Collateral-dependent blood flow to calf muscles (gastrocnemius-plantaris-soleus muscle group) increased ~70% in growth factor-treated groups compared with vehicle controls (P < 0.001; Fig. 1 and Table 3). There was a significant interaction between the treatments, however, indicating that the influence of bFGF and VEGF₁₂₁ on collateral blood flow expansion was dependent on the absence of L-NAME treatment.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Collateral-dependent blood flow to calf muscle (gastrocnemius-plantaris-soleus muscle group) during treadmill running. Data are expressed as means ± SE. *Significantly different from non-NG-nitro-L-arginine methyl ester (L-NAME) vehicle group and L-NAME groups (P < 0.001). VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor.

NOS inhibition blunted growth factor-induced collateral blood flow increase. Under L-NAME administration, the increases in collateral blood flow induced by exogenous growth factors diminished in all hindlimb sections (total, proximal, and distal) (Table 3). Similarly, L-NAME feeding abolished the growth factor-induced increase in collateral-dependent blood flows in the calf muscle group (Table 3 and Fig. 1). As illustrated in Fig. 2, the results are similar when expressed as conductance.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Calculated collateral circuit conductance under maximal vasodilation induced by treadmill running. Data are expressed as means ± SE. *Significantly different from non-L-NAME vehicle and L-NAME groups (P < 0.001).

	DISCUSSION

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L-NAME inhibition of VEGF. VEGF is a dimeric molecule that normally occurs as a homodimer. Through alternative exon splicing, the VEGF gene can encode seven different isoforms of the monomer subunit, which extend 121, 145, 148, 165, 183, 189, and 206 amino acids in length. Among the various forms, the VEGF₁₂₁ homodimer is the only one that does not bind to heparin-like molecules, and it is therefore predicted to be the most freely diffusible (for reviews, see Refs. 9 and 12). Two previous studies have demonstrated that VEGF₁₂₁ gene therapy can produce increased blood flows in both rabbit hindlimb (26) and porcine coronary (16) models of ischemia. In addition, we have recently shown that the intravenous delivery of recombinant human VEGF₁₂₁ protein in the rat hindlimb ischemia model utilized here was also efficacious at producing an increase in collateral-dependent blood flow (unpublished observation). We therefore chose to use an infusion of recombinant human VEGF₁₂₁ in the present study.

L-NAME inhibition of bFGF. Although endothelial cell proliferation and migration in response to VEGF were blocked by NOS inhibition in the study of Shizukuda et al. (22), the inhibitor had no effect on the endothelial cell responses to bFGF. Similarly, Ziche et al. (37) found that NOS inhibition blocked corneal angiogenesis in a rabbit model in response to a VEGF-containing implant but did not significantly affect the angiogenic response to bFGF-containing implants. These reports indicated that bFGF-induced effects on the vascular architecture, unlike the effects of VEGF, are independent of NOS activity. In the present study, however, we found that NOS inhibition completely blocked the ability of exogenous bFGF to induce an increase in collateral-dependent blood flow (Table 3 and Figs. 1 and 2). While the reasons for these conflicting results are unclear, several possibilities merit consideration. It may be that an action of bFGF, not directly related to its angiogenic effects, was involved in stimulating the change in collateral flow. For example, a continuous NO-mediated vasodilation caused by bFGF infusion could have introduced hemodynamic stimuli over the 14-day period that effected vascular remodeling. At present, we cannot rule out this possibility; however, the exceptionally low dose of bFGF delivered [the rate of bFGF delivery per minute was ~270-fold less than the bolus dose identified by Cuevas et al. (8) that was ineffective at decreasing blood pressure] and its similar efficacy via systemic delivery (34) tend to lessen this likelihood. Alternatively, bFGF is capable of upregulating VEGF (21, 23), a response that could effect vascular remodeling. L-NAME would be expected to inhibit this indirect VEGF-mediated response. It is also possible that the opposing results reflect a difference in the mediators utilized by bFGF to promote angiogenesis versus arteriogenesis. Finally, the difference could reflect the tissue location of the vascular effects in the studies (hindlimb muscle vs. normally avascular cornea). Further work will be necessary to clarify this apparent conflict.

Collateral-dependent blood flow. Femoral artery ligation, as conducted in the present study, initially results in an acute drop in blood flow capacity of the calf muscles to ~20-25 ml · min¹ · 100 g¹ during treadmill exercise (34, 36). Over the course of 2 wk, we observed this flow capacity spontaneously recovers to ~50 ml · min¹ · 100 g¹, as seen in the present experiment (Table 3 and Fig. 1), likely due to the recruitment of the full caliber of existing collateral vessels. It is important to note that this normal spontaneous recovery in collateral blood flow was not affected by L-NAME treatment in the absence of growth factor administration (compare vehicle-treated L-NAME and non-L-NAME subgroups in Table 3 and Fig. 1). This result suggests that the mechanism for the recovery of blood flow that occurs in the absence of exogenous growth factors differs from that triggered by the added bFGF and VEGF₁₂₁ in that it is NOS independent. As developed below, we believe that this difference is due to the growth factor-induced structural enlargement of the vessels that function as collateral conduits, a process referred to as arteriogenesis (13).

Collateral circuit conductance. Blood flow to the calf muscle after occlusion of the femoral artery is collateral dependent (33-35). The actual blood flow that can be measured at any given time, however, does not represent the maximal collateral-dependent blood flow unless the resistance of the circuit is minimized. Collateral circuit resistance is comprised of two resistances in series: an upstream resistance of the collateral conduits that circumvent the obstruction (femoral artery occlusion) and a downstream resistance in the muscle. Minimizing downstream resistance was achieved in the present study by exercise, the most powerful stimulus for vasodilation. Lowering of vascular resistance of the calf muscles by treadmill running results in the upstream collateral resistance becoming the largest rate-determining resistance in the circuit (31). Verification that flows to the calf muscles were not further increased during the high running speed (cf. Table 3), which occurs in nonischemic active muscle (Ref. 1; Table 5, abdominal and psoas muscles), indicates that maximal collateral-dependent blood flows were determined. The increase in blood pressure established by L-NAME administration would increase the perfusion pressure at the head of the collateral vessels and, thereby, increase collateral blood flow downstream. This elevated perfusion pressure, however, did not confound the outcome of this experiment, because expressing the data in terms of collateral conductance (cf. Fig. 2) does not change the pattern of response from that observed when comparing actual measured calf blood flows (cf. Fig. 1). The increases in collateral conductance induced by bFGF and VEGF₁₂₁ were eliminated by L-NAME. Collateral conductances were calculated by assuming that the near-minimal resistance of the calf muscle (0.43 mmHg · ml¹ · min¹ · 100 g¹), which we measured during treadmill running previously (32), was also created during our ischemic exercise conditions of similar treadmill running. Knowing the measured blood flow to the calf muscles and the perfusion pressure at the head of the collateral vessels (taken as aortic pressure), we can calculate total circuit resistance. From this, we can subtract downstream muscle resistance of the active muscle to obtain the upstream resistance, which is then expressed as conductance of the upstream collateral conduits.