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Am J Physiol Heart Circ Physiol 288: H759-H768, 2005. First published October 7, 2004; doi:10.1152/ajpheart.00786.2004
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VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Pamela G. Lloyd,1 Barry M. Prior,1 Han Li,1 Hsiao T. Yang,1 and Ronald L. Terjung1,2,3

1Department of Biomedical Sciences, College of Veterinary Medicine, 2Department of Medical Pharmacology and Physiology, College of Medicine, and 3Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

Submitted 3 August 2004 ; accepted in final form 4 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from ~70 to ~100 min (at 15–20 m/min, twice per day) and produced a large (~75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (~2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (~25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.

kinase insert domain-containing receptor; Flt; receptor tyrosine kinase inhibition; treadmill running; vascular endothelial growth factor

THE VASCULATURE of skeletal muscle can remodel itself to enhance oxygen delivery to the active muscle. Remodeling occurs when the existing vasculature becomes insufficient to meet tissue metabolic demands, secondary to either increased muscle activity (exercise training) or vascular obstruction (peripheral arterial insufficiency). Two distinct types of vascular remodeling can occur in skeletal muscle. "Arteriogenesis" results in an increase in the number and/or size of collateral vessels delivering flow to the muscle, whereas "angiogenesis" results in an increase in the number of capillaries important for blood/muscle oxygen exchange (41, 50).

Arteriogenesis and angiogenesis differ on several levels. Arteriogenesis involves the enlargement of existing arteries and involves both endothelial and smooth muscle cell proliferation (40). The expanded cross section of the collateral vasculature results in increased blood flow capacity to downstream tissue. This effect can be clearly seen in animal models of peripheral arterial insufficiency (17, 19). Although the improvement in blood flow can relieve ischemia in the downstream tissues, ischemia is not thought to be the initiating stimulus for arteriogenesis because the tissue in which the remodeling collateral vessels are found is not itself ischemic (17, 19). Rather, shear stress is thought to be the major stimulus for arteriogenesis (40).

Angiogenesis involves the growth of new capillaries within the muscle and is accomplished by endothelial cell proliferation and migration and by intussusception, a process of capillary splitting (29). Although the enhanced muscle capillarity can improve oxygen delivery to individual myocytes, it does not result in an overall increase in muscle blood flow, because the major resistance in the circuit is upstream to the capillaries. Ischemia is thought to be a major stimulus for angiogenesis, although flow dynamics and tissue tension are also implicated (cf. Refs. 5, 19, and 29).

Previous studies have suggested that the most frequently studied VEGF (VEGF-A) plays a predominant role in angiogenesis (20, 35). Some studies have also implicated VEGF in arteriogenesis (3, 6, 7, 25, 56), although other studies disagree (9, 17). Numerous studies have shown that exercise training elevates VEGF mRNA in active muscle (4, 12, 14, 21). We have recently shown that VEGF and its receptor, VEGF-R1, are upregulated in a collateral vessel postocclusion (28). Thus we hypothesized that VEGF signaling is an important mediator of both angiogenesis and arteriogenesis in response to exercise training of ischemic muscle. To test this hypothesis, we treated both sedentary and exercise-trained rats with a VEGF-R tyrosine kinase inhibitor [ZD4190, AstraZeneca (16, 45)] and assessed arteriogenesis, angiogenesis, and collateral artery angiogenic growth factor mRNA expression. Our findings suggest that VEGF signaling is indeed important in both angiogenesis and arteriogenesis but that the dependence of these processes on VEGF signaling differs. In addition, our data indicate that other VEGF family ligands [e.g., placental growth factor (PlGF)] may be important in mediating training-induced arteriogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study Design

Rats were divided into six main experimental groups: sedentary + vehicle (Sed, n = 9), sedentary + VEGF-R antagonist (Sed + ZD4190, n = 8), exercise trained + vehicle (TR, n = 8), exercise trained + VEGF-R antagonist (TR + ZD4190, n = 9), exercise trained + VEGF (TR + VEGF, n = 11), and exercise-trained + VEGF + VEGF antagonist (TR + VEGF + ZD4190, n = 14). All rats in these groups had bilateral ligation of the femoral arteries to induce peripheral arterial insufficiency for the 2-wk period of the study. An additional group of nonligated sedentary animals [control (Cont), n = 8] was used for normalization of PCR results.

Animal Care

Male Sprague-Dawley rats (~350 g, Taconic Farms; Germantown, NY) were housed two per cage in temperature-controlled rooms (21°C) with a 12:12-h light-dark cycle and had free access to rat chow and water. Before the study, rats were familiarized with the treadmill by walking at 20 m/min, 15% grade, for ~5 min. This familiarization procedure, which was carried out 2 times/day for 5 days, does not induce peripheral adaptations in rats (50, 51, 53). The experimental protocols used in this study were approved by the Animal Care and Use Committee of the University of Missouri and conformed to National Institutes of Health guidelines.

Surgical Procedures

Both femoral arteries were ligated ~5–6 mm distal to the inguinal ligament in all groups except the Cont group. Surgery was performed under ketamine-acepromazine anesthesia (100 mg-0.5 mg/kg ip). The fur was shaved from the inner thighs, and the surgical site was cleaned with Betadine. Via small incisions in the skin, both the right and left femoral arteries were isolated and completely occluded by ligation with 3-0 surgical silk. When the surgeries were complete, the incisions were closed as previously described, and the animals were placed in their cages to recover (50, 51, 52).

VEGF Delivery

At the same time, animals in the VEGF treatment groups were also implanted with an osmotic minipump for delivery of the VEGF165 isoform. A polyethylene (PE)-60 catheter was inserted into the left jugular vein to establish the route for VEGF delivery and connected to an osmotic pump (Alzet model 2002, Alza; Palo Alto, CA). The pump delivered 15 µg·kg–1·day–1 via a constant flow rate of 0.50 ± 0.02 µl/h over 14 days. Osmotic pump preparation was conducted according to the manufacturer’s instructions. The pumps were filled with either vehicle solution (10% sodium citrate to prevent coagulation and 1.6% glycerol to stabilize protein, in PBS) or vehicle plus VEGF. The dead space of the catheter was filled with the same solution as its connected pump. The pump was placed in a tunnel under the subcutaneous tissue at the back of the neck; this placement did not hamper limb movement during treadmill exercise.

VEGF-R Inhibitor Delivery

Beginning on the day of femoral artery occlusion, rats in the VEGF-R antagonist groups were treated with the receptor kinase inhibitor ZD4190 (AstraZeneca). This compound effectively inhibits both VEGF-R1 and -R2 kinase activity (IC50 = 700 and 30 nM, respectively) and VEGF-stimulated endothelial cell proliferation (at 50 nM) (16, 45). ZD4190 was dissolved in a solution of 1% polysorbate 80 and given to the rats by daily gavage (1.0 ml) at a dose of 12.5 mg·kg–1·day–1. Initial work verified that 12.5 mg·kg–1·day–1 was as effective at inhibiting the increase in collateral blood flow and muscle capillarity as the higher dose of 50 mg·kg–1·day–1 (n = 4); thus these TR + VEGF animals were pooled into a single group for further analyses. Rats in the vehicle groups received a daily gavage of 1 ml of 1% polysorbate 80.

Exercise Training

Rats began exercising on the treadmill 24 h postsurgery and continued for ~14 days. Performance was determined as the length of time running before fatigue on the treadmill, twice per day. Initially, the rats walked at a very modest rate of 15 m/min at 15% grade until fatigued. Fatigue was defined as the inability to keep up with the treadmill speed and was preceded by a change in gait with exaggerated hops (50–53). As the rats’ ability to exercise increased, the protocol was modified. When the rats were able to exercise for an hour at 15 m/min, the speed was increased to 20 m/min after the first 45 min. This occurred within 5–7 days of running for most of the rats. The timing of the increase in speed was moved back 15 min each time the rats were able to complete the hour at the new level of intensity. A few rats were able to run for an entire hour at 20 m/min; the speed was left constant, and they were allowed to continue a while longer until fatigued.

Blood Flow Determination

Fourteen days after femoral artery ligation, rats were surgically prepared for blood flow measurement, as previously described (50, 51, 53). Briefly, rats were anesthetized with ketamine and then instrumented with a PE-50 catheter in the aortic arch for measurement of mean arterial pressure and heart rate and for infusion of radioactive microspheres. A second catheter was placed in the caudal artery and used to monitor caudal blood pressure and collect reference blood samples. Rats were allowed to recover for >4 h after instrumentation. The rats were alert, active in their cage, and able to run on the treadmill at the speeds necessary for blood flow determination without difficulty (51, 53, 55).

After the recovery period, blood flow to the hindlimbs and other tissues was measured by infusing radiolabeled microspheres (~500,000 85Sr and 141Ce, 15 ± 0.1 µm diameter, New England Nuclear; Boston, MA) (46–48). Blood flow was measured during treadmill exercise at two different speeds (20 and 25 m/min, 15% grade). This protocol allowed us to verify that maximal collateral-dependent blood flow was being measured. Because upstream resistance determines blood flow to collateral-dependent muscle when downstream resistance in the active muscle is minimal, the finding that blood flow is the same at both speeds indicates that maximal collateral-dependent flow has been measured.

Blood flow was measured first while the rats ran at low speed (20 m/min). After the rats had been running for 1 min, microspheres bearing the first radiolabel were delivered via the aortic catheter. Meanwhile, beginning 10 s before microsphere infusion and continuing throughout microsphere administration (total time, 1 min 30 s), a reference blood sample was collected from the caudal catheter (flow rate, 0.5 ml/min). After microsphere delivery, the aortic catheter was flushed with saline over ~20 s. Careful deliberate infusion of microspheres into the ascending aorta provides a valid measure of blood flow to the distal limbs. Rats were then allowed to rest for ~5 min. During this period, blood pressures and heart rates returned to preexercise levels. The procedure was then repeated with the rats running at 25 m/min, using microspheres with the second radiolabel.

After microsphere infusion, the rats were anesthetized with pentobarbital sodium (60 mg/kg ip). Samples of the perforating artery were collected for measurement of collateral vessel diameter, using a x50 dissecting microscope, and for use in RT-PCR (see RT-PCR). Muscle samples were also dissected from one limb for histological analysis (see below). Rats were then killed with an overdose of pentobarbital sodium, and tissues were dissected from the other hindlimb for blood flow measurement. These samples were counted in a gamma counter (Wallac Wizard 1480 Autogamma Counter; Turku, Finland) along with the reference blood samples. Muscle blood flow (in ml·min–1·100 g–1) was calculated as follows:

where RBS is the reference blood sample and CPM is counts per minute. To provide evidence that proper mixing of microspheres occurred within the circulation, the right and left kidney blood flows were compared. Blood flows to the proximal, distal, and total hindlimb were calculated by summing blood flows to individual tissues of the limb (50, 51, 53).

Determination of Collateral Artery Diameter and Wall Thickness

The perforating artery is a major collateral vessel in the medial thigh area that enlarges after femoral artery occlusion (28). A segment of the perforating artery (5–8 mm in length nearest its insertion into the distal femoral/popliteal artery) was collected after microsphere infusion and immediately placed in ice-cold (4°C), calcium-free, MOPS-buffered physiological saline solution containing (in mM) 140.0 NaCl, 4.7 KCl, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS, pH 7.4. The artery was then cannulated on both ends with glass micropipettes filled with the same calcium-free smooth muscle "relaxing" solution and secured with 11-0 ophthalmic sutures. After cannulation, the vessel was transferred to a stage of an inverted microscope (Nikon Diaphot 200, x10 magnification) attached to a monitor (Sony), video micrometer (Microcirculation Research Institute, Texas A&M University; College Station, TX), and a Macintosh/Maclab data-acquisition system. This system continually monitored and recorded the luminal diameter of the vessel maintained at 37°C. To simulate conditions in vivo, the perfusion reservoirs attached to each end of the artery were gradually elevated to raise the intraluminal pressure to >100 cmH2O. Maximum luminal diameter was reached when imposition of a higher intraluminal pressure (~100–120 cmH2O) resulted in no further change in vessel diameter. At this time, both maximal luminal diameter and vessel wall thickness were measured and recorded.

Histological Analysis

After microsphere infusion, a section from the center of the white gastrocnemius muscle was collected, mounted on a cork with Tris-buffered saline tissue freezing medium (Triangle Biomedical Sciences; Durham, NC), and frozen in liquid N2-cooled isopentane for histological analysis. Frozen sections (10 µm) were cut using a cryostat (CM 1850, Leica). The sections were fixed in acetone, stained for alkaline phosphatase activity to identify capillaries (34), and then counterstained with metanil yellow. Images of each muscle were captured with a digital charge-coupled device camera (Spot, Diagnostic Instruments; Sterling Heights, MI) attached to a microscope (Nikon Eclipse E600, Nikon; Torrance, CA). With the use of Adobe Photoshop software, a composite image covering the entire section was produced for each muscle. A grid was overlaid on the image, and 20 nonoverlapping fields were selected for analysis. An image was acquired in each field (actual image area, ~0.06 mm2). The number of myocytes and the number of capillaries surrounding each myocyte were counted using Adobe Photoshop and recorded for calculation of capillary contacts per fiber.

RNA Isolation

Total RNA was isolated from the collateral artery (perforating), obtained from the contralateral limb from that used for in vitro artery measurement described above, using TRIzol (Life Technologies; Frederick, MD) according to the manufacturer’s instructions, with the addition of 10 µl of a 20 mg/ml glycogen solution to aid in RNA recovery. Total RNA was treated with DNase (DNA Free, Ambion; Austin, TX) to remove contaminating genomic DNA.

RT-PCR

RT was performed on RNA samples using TaqMan reagents and protocols (Applied Biosystems; Foster City, CA). Random hexamers were used to prime the reaction. The resulting cDNA samples were then diluted 20-fold with Tris-EDTA buffer (Sigma Chemical; St. Louis, MO) and stored at –80°C until use. Real-time quantitative PCR was performed on the ABI Prism 7000 instrument using TaqMan reagents and protocols (Applied Biosystems). Primer and probe sequences used for VEGF, KDR, Flt, endothelial nitric oxide (NO) synthase (eNOS), angiopoietin-1, angiopoietin-2, Tie-2, and monocyte chemoattractant protein (MCP)-1 have been described previously (21). Sequences for PlGF and VEGF-B were as follows: PlGF forward primer, 5'-TGTCCTTCTGAGTCGCTGTAGTG-3'; PlGF reverse primer, 5'-GCTGTCTTTAGCGCCACACA-3'; PlGF probe, 5'-CAGACCCTCGTCACCACAGCA-3'; VEGF-B forward primer, 5'-TGACGATGGCCTGGAGTGT-3'; VEGF-B reverse primer, 5'-ACTGGATCATGAGGATCTGCATT-3'; and VEGF-B probe, 5'-TGCCCATTGGGCAACACCA-3'. Primer and probe sets for 18S rRNA and rat GAPDH were purchased from Applied Biosystems. All data were normalized to the signal of 18S rRNA to correct for differences in total RNA concentration.

Statistical Analyses

Data are expressed as means ± SE. ANOVA was used to assess the main treatment effects of ligation, ZD4190, and physical training and any ZD4190-training interactions. Repeated-measures ANOVA was applied to data where appropriate. P values <0.05 were considered significant. Differences between groups were identified using Fisher’s least-significant difference test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
VEGF-R Inhibition

The VEGF-R antagonist was well tolerated by the animals. Of the 55 rats, one ZD4190-treated animal and one vehicle-treated animal showed histological evidence of muscle damage (inflammatory cell infiltration, focal fiber atrophy). Because both animals were exercise trained, this is suggestive of overuse injury. Thus ZD4190 administration is not implicated in the muscle damage. No effects on body weight, hindlimb weight, heart rate, or blood pressure were detected (Tables 1 and 2), and ZD4190-treated animals had normal exercise capacity. This implies that the acute effects of VEGF (e.g., in acute regulation of NO) are not important in cardiovascular regulation. In sedentary rats, antagonist administration had no effect on hindlimb blood flow (Table 3).


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  		Table 1. Body and hindlimb tissue weight

 

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  		Table 2. Blood pressure and heart rate before and during collateral blood flow determination

 

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  		Table 3. Hindlimb blood flow

 
Exercise Training

Daily running time increased rapidly over the first week of the training program, from 67 ± 2.3 min on day 1 to 108 ± 0.5 min on day 7 (Fig. 1). Thereafter, running time remained relatively constant, but running speed continued to increase.



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  		Fig. 1. Total daily exercise time. Running speed was progressively increased throughout the course of the training program (n = 25–27). Line was drawn by hand to aid the eye. Values are means ± SE. Some error bars are obscured by the data points.

 
There was no effect of ZD4190 on body weight. However, body weights of TR rats and TR + VEGF rats were significantly less (~15%, P < 0.05) than those of Sed rats, a typical response of male rats to exercise training (Table 1).

Effect of Training and VEGF-R Antagonism

Collateral blood flow. Although there were differences in blood flows to the hindlimbs of animals among groups (cf. Table 3), tissues that include the proximal hindlimb are not completely collateral dependent. Therefore, it is more appropriate to focus on the response of the calf muscles, which exhibited significant increases in the animals that were exercised daily (~70% in the TR group and ~80% in the TR + VEGF group; cf. Fig. 2 and Table 3). VEGF administration did not further increase collateral blood flow in combination with daily exercise. Blood flows to individual muscles that comprise the distal hindlimb were uniformly elevated by exercise training (cf. Table 4).



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  		Fig. 2. Effect of training and VEGF receptor (VEGF-R) antagonist treatment on blood flow to the collateral-dependent calf muscles (gastrocnemius-plantaris-soleus group, means ± SE). Exercise training (TR, n = 8) significantly increased collateral-dependent flow over values in sedentary (Sed, n = 9) rats. ZD4190 treatment completely blocked the exercise-induced increase in flow (TR + ZD4190, n = 9). Treatment of exercising rats with VEGF did not enhance the exercise-induced increase in flow (TR + VEGF, n = 8) or rescue the increase in ZD4190-treated rats (TR + VEGF + ZD4190, n = 12). *P < 0.01 vs. Sed; {dagger}P < 0.01 vs. TR; {ddagger}P < 0.01 vs. TR + VEGF.

 

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  		Table 4. Individual muscle tissue blood flow

 
As illustrated in Fig. 2, treatment with the VEGF-R antagonist ZD4190 completely blocked the training-induced increase in collateral blood flow. Because blood pressures were not different, the results are the same if the data are expressed as conductances. Blood flows to the total hindlimb, proximal hindlimb, distal hindlimb, and gastrocnemius-plantaris-soleus group in TR + ZD4190 rats were not significantly different from those in Sed rats (Fig. 2 and Table 3). The absence of any improvement in collateral blood flow to the calf muscles in the TR + VEGF group that was given ZD4190 attests to the effective inhibition of VEGF’s actions, because VEGF independently will significantly increase calf muscle blood flow (56). Thus the VEGF signaling pathway appears to be of crucial importance in the training-induced increase in collateral blood flow.

Kidney and nonhindlimb muscle blood flow. As typical when microspheres are well mixed in aortic blood flow upon delivery, there was an excellent match in blood flow across kidneys (ratio of flows in left kidney to right kidney = 1.08 ± 0.022, n = 107 measurements). There was a main treatment effect of training to increase renal blood flows (P < 0.025; Table 5. However, this effect of training was dependent on the absence of VEGF-R inhibition (i.e., there was a significant interaction, P < 0.05). Thus there is a correspondence between greater renal blood flow and an improvement in collateral blood flow to the calf muscles. Training had no effect on blood flow to nonhindlimb muscle (abdominal muscle, psoas, diaphragm). Thus the effect of training to increase blood flow was specific to the tissue at risk of ischemia after femoral occlusion.


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  		Table 5. Kidney and trunk muscle blood flow

 
Muscle capillarity. Training induced angiogenesis within the active muscle, as demonstrated by an increase in capillarity (22–25% increase in capillary contacts per fiber) in the white gastrocnemius (Fig. 3). This increase in capillarity with training is not due to an increase in frequency of capillaries adjoining multiple fibers (unpublished observations). Furthermore, VEGF exhibited no influence on muscle capillarity. Therefore, corresponding animals that were kept sedentary, trained, or given ZD4190 were combined for clarity of illustration of VEGF-R inhibition. Treatment with ZD4190 reduced the training-induced increase in capillary contacts per fiber. VEGF treatment did not rescue the training effect. The number of capillary contacts per fiber in ZD4190-treated rats was significantly lower than that in TR rats but remained significantly greater than that in Sed rats (P < 0.01). Thus, in contrast to its effect on blood flow, ZD4190 did not completely abolish the increase in capillarity produced by training.



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  		Fig. 3. Capillarity (capillary contacts per fiber, means ± SE) in white gastrocnemius muscle. Muscle capillarity was significantly higher in TR animals (n = 17) than in Sed rats (n = 16). Treatment with ZD4190 (n = 17) partially blocked the increase in capillary induced by training. However, capillarity was still increased in ZD4190-treated trained rats compared with Sed animals. *P < 0.01 vs. Sed; {dagger}P < 0.01 vs. TR.

 
Collateral vessel enlargement. In the absence of VEGF-R inhibition, TR animals exhibited a trend (P < 0.10) toward a greater luminal diameter of the collateral vessel compared with the Sed group. ZD4190 administration eliminated this trend because collateral vessel sizes among all three VEGF-R-inhibited groups were similar to that observed in the Sed group (cf. Table 6). Exercise training significantly increased (P < 0.001) vessel wall area, and this effect was eliminated with ZD4190 (cf. Table 6). Thus the absence of a change in collateral vessel size is consistent with the absence of a blood flow increase with ZD4190 administration.


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  		Table 6. Collateral vessel inner diameter, wall thickness, and wall area

 
Effect of Ligation, Training, and VEGF-R Antagonism

Angiogenic gene expression in collateral arteries. There was no appreciable change in VEGF or VEGF-R2 (KDR) mRNA expression in response to any of the treatments. On the other hand, expression of mRNA for the VEGF-R1 (Flt-1) was not affected by ligation alone but was increased in the TR and TR + VEGF groups. This increase was not affected by ZD4190 treatment (Fig. 4). There was a main treatment effect of ligation to increase the mRNA expression of PlGF, the ligand specific for VEGF-R1. This tended to be less robust in the presence of ZD4190 administration (cf. Fig. 4). eNOS mRNA expression was increased in vessels of Sed rats relative to nonligated sedentary Cont animals (Fig. 5). Similar increases in eNOS mRNA were found in TR and TR + VEGF animals. ZD4190 treatment tended to further increase eNOS mRNA, although the effect was only significant in the TR + VEGF + ZD4190 group.



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  		Fig. 4. Expression of VEGF, VEGF-R1 (Flt-1), VEGF-R2 (kinase insert domain-containing receptor, KDR), and platelet growth factor (PlGF) mRNA in the collateral artery. Values are expressed as the fold change from the control (Cont) nonoccluded group. Values are means ± SE; n = 7–8 Cont, 9 Sed, 5 TR, 8–9 TR + VEGF, 7–8 Sed + ZD4190, 7–8 TR + ZD4190, and 8–12 TR + VEGF + ZD4190 rats. *P < 0.05 vs. Cont; {dagger}P < 0.05 vs. Sed + ZD4190.

 


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  		Fig. 5. Expression of endothelial nitric oxide synthase (eNOS) mRNA in the collateral artery. Values are expressed as the fold change from the Cont nonoccluded group. Note that eNOS mRNA was elevated by ligation alone, with no further effect of training. ZD4190 treatment further enhanced eNOS expression in the trained groups. Values are means ± SE. The sizes of each group are as described in Fig. 4. *P < 0.05 vs. Cont; {dagger}P < 0.05 vs. Sed; {ddagger} P < 0.05 vs. Sed + ZD4190; §P < 0.05 vs. TR + VEGF.

 
No effect of ligation, training, or VEGF-R antagonist treatment was found for VEGF-B, angiopoietin-1, angiopoietin-2, the angiopoietin receptor Tie-2, or MCP-1 mRNA expression relative to Cont animals (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our findings demonstrate the importance of VEGF-R signaling in vascular adaptations to exercise training and reveal intriguing potential differences in the signaling pathways regulating collateral vessel enlargement (arteriogenesis) and enhanced muscle capillarity (angiogenesis). They implicate stimuli induced by muscle contractions, because short-term VEGF-R inhibition did not modify vascular function in the absence of exercise training (i.e., in animals limited to normal cage activity). It is important to recognize that occlusion of the femoral artery does not produce hindlimb ischemia in these animals at rest, because the measured flow reserve (54, 55) is greater than three to five times greater than the blood flows measured in resting quiescent muscle (24). As a result, there is no increase in capillarity (22), typical of ischemic muscle. Thus there is no opportunity for VEGF inhibition to alter muscle capillarity in our short-term study when the animals are kept sedentary. This is in contrast to the more long-term absence of VEGF expression, which leads to an appreciable diminution of muscle capillaries, apoptosis of myocytes and endothelial cells, and the resulting increased potential for focal ischemia (37).

Training-Induced Arteriogenesis Requires Normal VEGF-R Function

We induced peripheral arterial insufficiency in rats by bilateral ligation of the femoral arteries, which removes ~85–90% of the flow reserve to the distal hindlimb (55). Although the main route of flow is blocked by femoral ligation, small collateral vessels exist, which allow some flow to bypass the obstruction. This limited flow [20–25 ml·min–1·100 g–1 (55)] is sufficient to meet the needs of resting quiescent calf muscle (24). Thus the animals do not experience ischemia at rest nor its pathological complications (19, 33, 55). Although flow can be increased somewhat [to ~30–35 ml·min–1·100 g–1 (54, 55)] by modulation of collateral tone (39), any larger increase in collateral flow is related to structural expansion of the collateral vasculature (19, 50). Indeed, vascular casts demonstrate enlargement of the collateral network after vascular obstruction (18, 19, 44, 50), and histological evidence for endothelial and vascular smooth muscle cell proliferation (19, 33) indicates that this enlargement reflects arteriogenesis and not simple vascular dilatation. Thus the 70–80% increase in blood flow to the collateral-dependent calf muscles that we observed after exercise training (cf. Fig. 3) is related to enlargement of the collateral network via arteriogenesis.

Treatment of rats with the VEGF-R antagonist ZD4190 completely blocked the training-induced increase in collateral blood flow. Demonstration that ZD4190 adminstration effectively inhibits VEGF signaling comes from the absence of an increase in collateral blood flow in rats given VEGF (Fig. 2). This suggests that intact VEGF-R signaling is essential for arteriogenesis in response to training and expands our understanding of the arteriogenic effects of VEGF, beyond that apparent with exogenous delivery (56), upregulation by gene transfer (13), and reversal by neutralizing antibody (8), to a physiological condition where endogenous stimuli are operant. The increase in collateral blood flow, observed with training in the absence of VEGF-R tyrosine kinase inhibition, is coincident with enlargement of a representative collateral vessel [i.e., greater wall cross-sectional area and luminal diameter evaluated ex vivo (28)]. Enlargement of this collateral vessel increases over the duration of training (28) and was evident in this study [wall cross-sectional area (P < –0.05) and luminal diameter (P < 0.10)] with only 2 wk of daily exercise. Not unexpectedly, elimination of the increase in collateral blood flow, with VEGF-R tyrosine kinase inhibition, was coincident with elimination of the enlargement of the isolated collateral vessel (cf. Table 6). This absence of vessel remodeling implies that the endothelial and vascular smooth muscle proliferation, typically observed with arteriogenesis (19, 33), and other coordinated events necessary for arteriogenesis to proceed [e.g., extracellular matrix remodeling (38, 42)], did not occur.

How the VEGF-R tyrosine kinase signaling is instrumental in the exercise-induced enlargement of collateral vessels has yet to be explored. The two receptors, VEGF-R1 (Flt-1) and VEGF-R2 (KDR), exhibit different specificities for the VEGF family of growth factors. Furthermore, each receptor can exhibit distinct effects. Most attention has been focused on VEGF-R2, because it is generally thought to be the major mediator of the mitogenic and angiogenic effects of VEGF (11). However, recent evidence suggests that the role of VEGF-R1 in angiogenesis and vascular remodeling may be more significant than previously thought. For example, Luttun et al. (23) recently reported that PlGF, which specifically activates VEGF-R1, was able to induce angiogenesis in ischemic mouse myocardium and an antibody to VEGF-R1 was able to inhibit VEGF-stimulated angiogenesis in corneal and Matrigel implants. More important to the role of VEGF-R1 in arteriogenesis, Pipp et al. (27) have demonstrated that PlGF is as effective as VEGF at improving hindlimb ischemia by enhancing collateral vessel enlargement. The increase in mRNA expression of PlGF that we observed in the isolated collateral vessels of occluded animals (cf. Fig. 4) is consistent with the expectation that PlGF is important in this vascular remodeling. However, there is no clear pattern in the upregulation of PlGF mRNA in relation to the outcome of collateral blood flow, because the greatest increases in PlGF mRNA were observed in both high- and low-blood flow groups (e.g., Sed vs. TR + VEGF; Fig. 4). Interestingly, the mRNA abundance for VEGF-R1 was uniformly greater in those groups that exercised daily. If this increased expression results in a greater receptor protein abundance, as observed in ischemic muscle (26), there could be an enhanced capacity to respond to VEGF and/or PlGF. This is consistent with the idea that the VEGF-R1 pathway may contribute to training-induced remodeling of collateral vessels. The exercise training effect would have been eliminated by ZD4190, because it inhibits receptor tyrosine kinase activity and not the binding to the receptor (16). Alternatively, increased shear stress, which is expected to be enhanced in the collateral vessels after occlusion and more so during the daily exercise periods, could provide a stimulus for vascular remodeling, because VEGF-R2 (36) and eNOS (46) mRNAs are upregulated by shear stress. Although the abundance of VEGF-R2 mRNAs was similar across groups in this study, the elevation in eNOS mRNA with occlusion could lead to a greater responsiveness to VEGF-mediated vascular remodeling, as NO is important in the signaling pathway. For example, inhibition of normal NO production eliminates the VEGF-induced (56) and exercise training-induced (22) increases in collateral blood flow. This dependence on NO and the present inhibition by ZD4190 implies that NO is essential, but not sufficient, for arteriogenesis. Furthermore, the interaction between enhanced shear stress and NO is implied by the exaggerated upregulation of eNOS in the exercised groups that received ZD4190. Recall that collateral vessel enlargement was not observed in animals subjected to VEGF-R kinase inhibition (Table 6). In the absence of an enlarged vessel caliber, any increase in blood flow through these vessels above rest should prompt a greater shear stress. Thus absence of collateral vessel enlargement should sustain a greater shear stress any time the animals were exercised, and hence the exaggerated upregulation in eNOS mRNA in the ZD4190-treated animals. The benefit of an improved capacity for NO production would not be realized, however, because VEGF-R kinase inhibition (cf. Table 6) would preempt the NO-dependent VEGF-induced arteriogenesis (56). Thus alterations in mRNA expression in the collateral vessels implicate eNOS, VEGF-R1, and PlGF in collateral vessel enlargement; however, their specific roles remain to be evaluated experimentally.

VEGF-R Antagonism Partially Blocks Training-Induced Angiogenesis in Active Muscle

VEGF has been identified as a key mediator of physiological angiogenesis in a wide variety of circumstances (11), including exercise training (2). VEGF mRNA is upregulated with muscle contractions and immediately after exercise (4, 14, 15). The magnitude of VEGF upregulation with acute exercise lessens over the days of training (31), roughly in parallel with the increase in muscle capillarity induced by ischemic exercise (21); however, VEGF protein and the number of VEGF-positive capillaries remain elevated in the ischemic muscle (26). Furthermore, the increase in microvascular density observed after 3 days (23%) of treadmill walking or 7 days (30%) of muscle contractions was eliminated when VEGF upregulation was inhibited or when VEGF was scavenged with an antibody (1, 2). Thus the actions of VEGF appear to be vital to the angiogenic adaptation in active muscle by exercise. The present results extend these observations to demonstrate the importance of VEGF-R1 and/or -R2 tyrosine kinase activity. The loss of the increased capillarity in response to training was therefore expected. However, our findings suggest that the training-induced adaptation of increased muscle capillarity is more complicated than simply a function of VEGF availability and receptor signaling. Although ZD4190 significantly reduced the magnitude of the angiogenic response, it did not eliminate the increase in capillarity. Thus a careful evaluation of the data set and experiment is warranted.

Recent work has enhanced our understanding of the process of angiogenesis in adult skeletal muscle. Angiogenesis occurs via sprouting of new capillaries from existing ones and/or via intussusception, the production of new capillaries by longitudinal splitting of existing capillaries. Increases in muscle blood flow, calculated to increase shear stress, lead to an increase incapillarity primarily via intussusception (32). This is in contrast to conditions that involve muscle overload such as chronic muscle stretch (10, 57) and muscle contractions (5) where sprouting angiogenesis contributes to the increase in capillaries. Whereas matrix metalloproteinase (MMP)-2 and membrane-type 1 MMP are upregulated during vascular remodeling in muscle subjected to overload, they are not when capillaries are increased via intussusception by higher flow (32). There does not appear to be a similar distinction in VEGF upregulation, because both high flow conditions and muscle overload stimuli, including treadmill exercise, increase VEGF. Thus the absence of complete elimination of angiogenesis with VEGF-R inhibition does not reveal a distinct process of angiogenesis by either sprouting or intussusception. Our results, however, could be explained by the relative affinities of the receptors for VEGF in combination with the receptor selectivity for ZD4190 tyrosine kinase inhibition. In recombinant enzyme assays, ZD4190 is a more potent inhibitor of VEGF-R2 tyrosine kinase activity (IC50 = 29 nM) than VEGF-R1 tyrosine kinase (IC50 = 708 nM) (45). Thus VEGF stimulation of endothelial cell proliferation and migration via VEGF-R2 signaling should be effectively eliminated relative to VEGF-R1. Furthermore, even though shear stress can activate the VEGF-R2 pathway via integrin-receptor interaction that is independent of ligand activation (43), this also would be inhibited by ZD4190, because it is the tyrosine kinase activity that is inhibited by ZD4190 and not VEGF binding to the receptor. Thus it is reasonable to conclude that any reduction in capillarity as observed in the TR + ZD41490 group (cf. Fig. 3) is assignable first to inhibition of VEGF-R2 tyrosine kinase activity.

In a manner consistent with the above interpretation, it is possible that the VEGF-R1 tyrosine kinase activity remained relatively active. The IC50 for inhibition by ZD4190 is less effective, and the affinity of VEGF-R1 for VEGF is much higher [an apparent Kd of 0.6 nM (30)]. Thus the significant increase in muscle capillarity that remained with ZD4190 treatment (cf. Fig. 3) could be due to VEGF-activated VEGF-R1 tyrosine kinase signaling, which has recently been shown possible (23). Alternatively, VEGF-R1 activation could have occurred by PlGF, as PlGF induced angiogenesis in the ischemic mouse myocardium (23). PlGF mRNA (21) and protein (26) are upregulated in ischemic muscle. Similar to the circumstances where Luttun et al. (23) observed PlGF-induced angiogenesis, the white muscle region of the gastrocnemius, which we used to evaluate angiogenesis, is most susceptible to ischemia during exercise (22). Thus our results can be interpreted as due to differing interactions between the VEGF-Rs and their ligands. Alternatively, ZD4190 could have completely inhibited both receptor tyrosine kinase pathways and the residual angiogenesis with training could have been caused by some VEGF-R-independent angiogenic signal that has yet to be described. This is in contrast to the complete elimination of the training-induced increase in collateral blood flow (arteriogenesis) with VEGF-R kinase inhibition.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-37387 (to R. L. Terjung), HL-10485 (to B. M. Prior), and HL-10406 (to P. G. Lloyd).


   ACKNOWLEDGMENTS
 
We thank Jane Chen for excellent technical assistance; Nancy Swick, Brian Gamel, and Lin Sun for care in exercising the animals; Dr. Stephen R. Wedge, AstraZeneca, for the ZD4190; and Dr. Andrew Protter, Scios, for the VEGF.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. L. Terjung, Dept. of Biomedical Sciences, E102 Veterinary Medicine Bldg., 1600 E. Rollins Rd., Univ. of Missouri, Columbia, MO 65211 (E-mail: TerjungR{at}missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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