CYP4A metabolites of arachidonic acid and VEGF are mediators of ...

Am J Physiol Heart Circ Physiol 284: H1528–H1535, 2003. First published January 9, 2003; 10.1152/ajpheart.00406.2002.

CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis Sandra L. Amaral, Kristopher G. Maier, Daniela N. Schippers, Richard J. Roman, and Andrew S. Greene Department of Physiology and Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Submitted 23 May 2002; accepted in final form 9 December 2002

Amaral, Sandra L., Kristopher G. Maier, Daniela N. Schippers, Richard J. Roman, and Andrew S. Greene. CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis. Am J Physiol Heart Circ Physiol 284: H1528–H1535, 2003. First published January 9, 2003; 10.1152/ajpheart.00406.2002.—Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis induced by electrical stimulation in skeletal muscle. Less is known about the role of arachidonic acid metabolites in the control of growth of blood vessels in vivo. The present study examined the role of 20-hydroxyeicosatetraenoic acid (20-HETE) on the angiogenesis induced by electrical stimulation in skeletal muscle. The tibialis anterior and extensor digitorum longus muscles of rats were stimulated for 7 days. Electrical stimulation significantly increased the 20-HETE formation and angiogenesis in the muscles, which was blocked by chronic treatment with N-hydroxy-N⬘-(4-butyl-2methylphenol)formamidine (HET0016) or 1-aminobenzotriazole (ABT). Chronic treatment with either HET0016 or ABT did not block the increases in VEGF protein expression in both muscles. To analyze the role of VEGF on 20-HETE formation, additional rats were treated with VEGF-neutralizing antibody (VEGF Ab). VEGF Ab blocked the increases of 20-HETE formation induced by stimulation. These results place 20-HETE in the downstream signaling pathway for angiogenesis and show that both VEGF and 20-HETE are involved in the angiogenesis induced by electrical stimulation in skeletal muscle. vessel growth; vascular endothelial growth factor; electrical stimulation

PHYSIOLOGICAL ANGIOGENESIS is a complex process involving an interplay between cells, extracellular matrix molecules, and soluble factors that culminates in cell migration, proliferation, and tube differentiation of endothelial cells. The process of angiogenesis involves preexisting vessels, which send out capillary sprouts to produce new vessels (14). Several cytokines and growth factors have been established to modulate angiogenesis in vitro and in vivo, and, among these factors, vascular endothelial growth factor (VEGF) has been considered the most potent angiogenic inducer (9). Recent studies from our laboratory have further supported the role of VEGF as an important regulator of angiogenesis in

Address for reprint requests and other correspondence: A. S. Greene, Medical College of Wisconsin, 8701 Watertown Plank Rd., PO Box 26509, Milwaukee, WI 53226-0509 (E-mail: [email protected]). H1528

skeletal muscle, because treatment with a VEGF-neutralizing antibody blocked the angiogenic response to electrical stimulation and exercise (3, 4). Arachidonic acid (AA) metabolites have been implicated in endothelial cell migration, tube formation, and angiogenesis (29, 30, 34). A recent study provided evidence for the expression of cytochrome P-450A (CYP4A) ␻-hydroxylase in skeletal muscle cells and arterioles of rat cremaster muscle (18). This enzyme is responsible for the formation of both a vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), and a vasodilator, epoxyeicosatrienoic acid (EET). It has been shown that 20-HETE, which can be formed by the actions of enzymes in either the CYP4A and CYP4F families, plays a role in myogenic activation of small arterioles of the cerebral (15, 16) and renal (15, 22) circulations. More recently, Frisbee et al. (10) and Kunert et al. (19) have demonstrated that 20-HETE contributes to the vasoconstrictor responses to elevations in transmural pressure and PO2 in skeletal muscle resistance arterioles. Much less is known about the role of 20-HETE in the control of growth of blood vessels. Recent studies have suggested that norepinephrine and angiotensin II (ANG II) stimulate the synthesis and release of 20HETE in vascular smooth muscle cells (25) and that cytochrome P-450 inhibitors block activation of the MAPK system and the mitogenic effects of norepinephrine and ANG II on cultured vascular smooth muscle (VSM) cells. Because 20-HETE serves as a second messenger for the vasoactive and mitogenic actions of ANG II (2) and the local renin-angiotensin system plays a critical role in angiogenesis induced by electrical stimulation (3), the present study examined the role of 20-HETE in this response. MATERIALS AND METHODS

Animal surgery. All protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. The rats were housed in the Animal Resource Center of the Medical College of Wisconsin and were given food and water ad libitum. Thirty-two male Sprague-Dawley rats, 7–8 wk old, were anesthetized with an 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.

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ANGIOGENESIS MEDIATED BY 20-HETE AND VEGF

intramuscular injection of a mixture of ketamine (100 mg/kg) and acepromazine (2 mg/kg). A subcutaneous incision was made over the thoracolumbar region, and a miniature battery-powered stimulator, which was previously designed and validated for chronic studies by our laboratory (21), was implanted and secured in place. Another incision was made in the skin and fascia covering the lateral side of the knee joint (over the region of the common peroneal nerve) of the right hindlimb. A pair of electrodes was guided under the skin from the stimulator and secured to the muscles surrounding the knee in close proximity to the common peroneal nerve (22). The electrodes were locally secured into place using biocompatible acrylic cement (Loctite; Rocky Hill, CT) and distally with a fine suture (size 5-0, Ethicon; Somerville, NJ). The skin over both incisions was sutured closed, and the rats were allowed to recover before the initiation of the stimulation period the following day. Experimental protocols and tissue preparation. After a 24-h recovery period, the implanted stimulator was activated by momentary closure of the magnetic reed switch using a small hand-held magnet. The stimulator produced electrically induced muscle contractions in the lower leg muscles by stimulating the common peroneal nerve with square wave impulses of 0.3-ms duration, 10-Hz frequency, and 3-V potential (21). Contractions of the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were automatically initiated at 9 AM each day and sustained for 8 h/day over a consecutive 7-day period. At the end of the stimulation period, the animals were euthanized by an overdose of pentobarbital sodium (100 mg/kg ip), and the EDL and TA muscles were harvested for analysis as previously described (13, 33) The rats were divided in four groups. To evaluate the role of 20-HETE in contributing to the VEGF protein expression and skeletal muscle angiogenesis, nine rats in group I received two daily intraperitoneal injections of a potent and selective inhibitor of the CYP4A enzymes [N-hydroxy-N⬘(4-butyl-2-methylphenol)formamidine (HET0016), Taisho Pharmaceutical (24)] at a dose of 1 mg/kg each injection during the period of electrical stimulation. This dose was chosen based on our previous results (17). In that study, a dose of 10 mg/kg iv produced plasma concentrations that far exceeded (10 times higher) the effective inhibitory concentration of HET0016 in plasma for many hours. To compare the effects of HET0016 with a more commonly used, but less specific inhibitor, four rats were treated with 1-aminobenzotriazole (ABT; group 2) at a dose of 50 mg 䡠 kg⫺1 䡠 day⫺1 ip during the period of electrical stimulation. To determine the contribution of VEGF to the angiogenesis induced by electrical stimulation, six rats in group 3 were treated with 3 mg/kg ip injections of a monoclonal VEGFneutralizing antibody (Texas Biotechnology; Houston, TX) during the period of electrical stimulation. The protocol for administration of VEGF-neutralizing antibody was modified from Zheng et al. (36), and this dose was based on our previous results (3). After the stimulation period was started, the rats received intraperitoneal injections on days 3, 5, and 7 (0.6 mg/100 g). In group 4, rats were treated with either the vehicle for HET0016, lecithin (n ⫽ 9), or saline for VEGF antibody, PBS (n ⫽ 4). Because there was no significant difference in the results obtained in rats treated with either vehicle, the results from those groups were pooled. Measurement of urinary excretion of 20-HETE. On the last day of electrical stimulation, rats were placed in a metabolic cage that efficiently separates urine from food. Just before the start of the urine collection, food was withdrawn to avoid contamination of the urine sample, and the 24-h control and AJP-Heart Circ Physiol • VOL

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treated urine samples were collected into a glass bottle packed with ice. The concentration of 20-HETE in the urine samples was measured using a fluorescent HPLC assay, as previously described (23). After the addition of 25 ng of an internal standard [20–5(Z),14(Z)-hydroxyeicosadienoic acid (WIT-002), Taisho Pharmaceutical; Saitama, Japan] the samples were acidified to pH 4 with formic acid and extracted with 1 ml of ethyl acetate, and the organic phase was dried using argon gas. The samples were redissolved in 1 ml of 20% acetonitrile and loaded onto a Sep-Pak Vac column (Waters; Milford, MA). The column was washed twice with 1 ml of 30% acetonitrile, and the fraction containing HETEs and EETs was eluted with 400 ␮l of 90% acetonitrile. The samples were diluted in water, applied to a Sep-Pak Vac column, eluted with 500 ␮l of ethyl acetate, and then dried down. The lipid fraction was labeled with 20 ␮l of acetonitrile containing 36.4 mM 2-(2,3-napthalimino)ethyl trifluoromethanesulfonate. N,N-diisopropylethylamine (10 ␮l) was added to catalyze the reaction. Excess dye was removed using Sep-Pak Vac extraction (23), and the samples were dried under argon, resuspended in 100 ␮l of methanol, and analyzed by reverse-phase HPLC (Waters) using a fluorescence detector (model number L-7480; Hitachi, Naperville, IL). The amount of 20-HETE in the sample was determined by comparing the area of the 20-HETE peak with that of the internal standard. Tissue harvest and morphological analysis of vessel density. The stimulated and contralateral muscles were removed, weighed, and rinsed in physiological salt solution. A 300-mg sample was taken from the rostral portion of the TA muscle and frozen in liquid nitrogen for Western blot (100 mg) and HPLC (200 mg) analysis for measurement of VEGF protein expression and 20-HETE formation, respectively. The remaining TA and EDL muscles were lightly fixed in a 0.25% formalin solution overnight. The muscles were sectioned via a manual microtome to a thickness of ⬃100 ␮m by securing the tendons and slicing parallel to the longitudinal orientation of the muscle fibers. From every animal, two slices of each EDL muscle and three slices of each TA muscle were made. The slices were than immersed for 2 h in a solution of 25 ␮g/ml rhodamine-labeled Griffonia simplicifolia I (GS-I) lectin [Sigma; St. Louis, MO (13)]. Immediately after this 2-h exposure to GS-I lectin, the muscles were rinsed in physiological solution. The rinsing procedure was repeated after 15 and 30 min, and the muscles were rinsed in physiological saline solution for 12 h (overnight, at 4°C). On the next day, the slices were mounted on microscope slides with a water-soluble mounting medium consisting of toluene and acrylic resin (SP ACCU-MOUNT 280, Baxter Scientific). The labeled sections were visualized using a video fluorescent microscope system (Olympus ULWD CD Plan, ⫻20 objective, 1.6 cm working distance and 0.4 numerical aperture) with epi-illumination, as previously described (33). In the present study, 10–15 and 20–25 representative fields were selected for study from each EDL and TA muscle slice, respectively. Each field was converted to a digitized image (DT2801 Data Translation; Marlboro, MA) and stored as an 8-bit/pixel image file with a resolution of 512 ⫻ 512 pixels. Morphometric analysis of the scanned histochemical sections was done as previously described (33). Vessel-grid intersections have been previously demonstrated to provide an accurate and quantitative estimate of vessel density (33). Western blot analysis to detect the presence of VEGF protein. The 100-mg TA muscle specimens were homogenized, and the protein was suspended in potassium buffer (10 mM). Five micrograms of protein (as determined by a protein assay kit, Bio-Rad; Hercules, CA) from the TA and a tumor cell line known to express VEGF at high levels (C6, American Type

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Culture Collection, 107-CCL) were separated on a 12% denaturing polyacrylamide gel. The gels were transferred to a nitrocellulose membrane, which was blocked overnight in 5% nonfat dry milk diluted in Tris-buffered saline (50 mM Tris and 750 mM NaCl, pH 8) with 0.08% Tween 20 (Bio-Rad). The blots were then incubated with a polyclonal antibody to a peptide derived from the human VEGF sequence (1:1,000 dilution, clone G143-850, Pharmingen) for 2 h at room temperature. Washed blots were then incubated with goat antimouse secondary antibody at a dilution of 1:1,000 for 1 h at room temperature and then subjected to a SuperSignal West Dura chemiluminescence substrate (Pierce; Rockford, IL) detection system. Membranes were exposed to X-ray film (Fuji Medical; Stamford, CT) for 15⬃30 s and developed using a Kodak M35 X-Omat processor. For the quantitative VEGF analysis, film was always exposed for a period of time that ensured that all signals were within the linear range of the detection of the film. The VEGF band intensity was quantified using a morphometry imaging system (Metamorph, Universal Imaging; West Chester, PA), and values are expressed as a percentage of the C6 tumor cell standard. Muscle preparations for measurement of 20-HETE. The 100–200 mg of frozen TA muscle were homogenized in a solution containing 1 ml of acidified water and 50 ␮l of an internal standard, WIT-002, which was synthesized and kindly provided by Taisho Pharmaceutical. Ethyl acetate (3 ml, Fisher Scientific; Pittsburgh, PA) was added to the mixture and gently vortexed. The homogenized tissues were then centrifuged at 3,000 revolutions/min for 2 min. With the use of a glass Pasteur pipet, the top layer was removed and transferred to a sterile glass vial, and the samples were dried under nitrogen and stored at ⫺80°C. Labeling of samples and fluorescent detection of 20-HETE. Detection of 20-HETE was performed as previously described (22). Samples, which were extracted and dried under argon, were resuspended in 20 ␮l of acetonitrile containing 36.4 mM 2-(2,3-napthalimino)ethyl trifluoromethanesulfonate, and N,N-diisopropylethylamine (10 ␮l) was added as a catalyst. The sample was reacted for 30 min at room temperature, dried under argon, resuspended in 1 ml of 40% acetonitrilewater, and applied to a Sep-Pak Vac column. The column was washed with 6 ml of 50% acetonitrile-water solution to remove unreacted dye, eluted with 500 ␮l of ethyl acetate, dried under argon, and resuspended in 100 ␮l of the HPLC mobile phase [methanol-water-acetic acid, 82:18:0.1 (vol/vol)]. A 25-␮l aliquot of the derivatized sample was separated on a 4.6 ⫻ 250-mm Symmetry C18 reverse-phase HPLC column (Waters) isocratically at a rate of 1.3 ml/min using methanolwater-acetic acid [82:18:0.1 (vol/vol)] as the mobile phase. Fluorescence intensity monitored using an in-line fluorescence detector (model number L-7480, Hitachi; Naperville, IL) at medium gain sensitivity. The amount of 20-HETE in the sample was determined by comparing the area of the 20-HETE peak with that of the internal standard (WIT-002). Data analysis and statistics. For each muscle, the vessel counts of all the selected fields (10–15 scans ⫻ 2 slices for each EDL muscle; 20–25 scans ⫻ 3 slices for each TA muscle) were averaged to a single vessel density. Vessel density was expressed in terms of the mean number of vessel-grid intersections per microscope field (0.224 mm2). For each experimental group, the measured vessel density and 20-HETE formation of the stimulated muscle was compared with its unstimulated counterpart as well as with age-matched controls. All values are presented as means ⫾ SE. The significance of differences in values measured in the same animal was evaluated using a two-factor ANOVA (drug ⫻ stimulation) with repeated measures on one factor (stimulation). AJP-Heart Circ Physiol • VOL

Significant differences were further investigated using a post hoc test (Tukey’s). RESULTS

To evaluate the effect of the blockade of CYP4A enzymes, we measured the urinary 20-HETE excretions in rats treated with HET0016 for 7 days. A representative HPLC chromatogram illustrating the separation of 20-HETE is presented in Fig. 1. As shown in Fig. 1, there are other peaks very close to 20-HETE. On the basis of comigration of standards, we identified the preceding peak in the chromatogram as 19-HETE and the one after 20-HETE peak as 18-HETE. The next peak is 16-HETE, followed by 15-HETE. To analyze the area of each peak, we subtracted the shouldering peaks by using a deconvolution procedure (Hitachi software), as described in MATERIALS AND METHODS. As shown in Fig. 2, chronic treatment with HET0016 significantly reduced (by 36%) the 24-h urinary 20HETE excretion compared with the control group, which was treated with lecithin (P ⬍ 0.05). Seven days of electrical stimulation significantly increased the 20-HETE formation in skeletal muscle, as demonstrated in Fig. 3 (from 69.52 ⫾ 31.3 to 177.58 ⫾ 54.4 ng/g muscle for unstimulated and stimulated muscles, respectively, P ⬍ 0.05). Treatment with HET0016 for 7 days did not change the basal formation of 20HETE in skeletal muscle (110.26 ⫾ 28.36 and 69.52 ⫾ 31.3 ng/g muscle for treated and control, respectively, P ⬎ 0.05; Fig. 3); however, chronic treatment with HET0016 completely blocked the increase of 20-HETE formation induced by electrical stimulation in skeletal muscle (from 110.26 ⫾ 28.3 to 102.1 ⫾ 22.3 ng/g muscle for the unstimulated and stimulated sides, respectively; Fig. 3). Electrical stimulation, as has been shown previously, produced an increase in vessel density in the control group, which was treated with lecithin (from 107.0 ⫾ 1.6 to 121.0 ⫾ 4.5 and from 100.4 ⫾ 8.4 to 132.0 ⫾ 9.9 number of vessel intersections for the EDL

Fig. 1. Representative reverse-phase HPLC chromatogram illustrating the separation of fluorescently labeled 20-hydroxyeicosatetraenoic acid (20-HETE) in samples from the tibialis anterior (TA) muscle. WIT-002, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid.

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Fig. 2. Effects of treatment with a selective cytochrome P-450A (CYP4A) inhibitor [N-hydroxy-N⬘-(4-butyl-2-methylphenol)-formamidine (HET0016)] on 20-HETE urine formation in rats after 7 days of the stimulation protocol. Values are means ⫾ SE of 5 rats treated with lecithin and 5 rats treated with HET0016. *P ⬍ 0.05 vs. lecithin.

and TA, respectively, P ⬍ 0.05). As shown in Fig. 4, chronic treatment with HET0016 completely blocked the increase in vessel density induced by 7 days of electrical stimulation in skeletal muscles (from 116.0 ⫾ 1.0 to 118.0 ⫾ 10.1 and from 105.7 ⫾ 4.9 to 110.5 ⫾ 1.1 number of vessel intersections for the EDL and TA, respectively). Chronic inhibition of 20-HETE formation using ABT also attenuated the increase in vessel density induced by electrical stimulation in skeletal muscle (from 111 ⫾ 7.4 to 121 ⫾ 4.35 and from 99.7 ⫾ 4.72 to 119.5 ⫾ 4.51, number of vessel intersections for the EDL and TA, respectively). Because VEGF has been shown to play an important role in the angiogenesis of skeletal muscle, we performed Western blot analysis to verify the effects of HET0016 or ABT on VEGF protein expression. Figure 5 shows a representative Western blot (A) and the quantitative densitometry (B) used to compare the responses of VEGF protein expression after 7 days of stimulation in all of the animals treated with HET0016 or control. As shown in Fig. 5, VEGF protein levels

Fig. 3. Effects of treatment with a selective CYP4A inhibitor (HET0016) on 20-HETE formation in the muscle of rats after 7 days of the stimulation protocol. PBS, phosphate-buffered saline. Values are means ⫾ SE of 5 rats treated with lecithin and 5 rats treated with HET0016. *P ⬍ 0.05 vs. the unstimulated side. AJP-Heart Circ Physiol • VOL

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Fig. 4. Changes in vessel density of the extensor digitorum longus (EDL) and TA muscles in control rats (n ⫽ 4), those treated with a selective CYP4A inhibitor (HET0016, 2 mg 䡠 kg⫺1 䡠 day⫺1 in lecithin, n ⫽ 4), and those treated with a nonselective CYP4A inhibitor [1-aminobenzotriazole (ABT), 50 mg 䡠 kg⫺1 䡠 day⫺1 in PBS, n ⫽ 4] after 7 days of the electrical stimulation protocol. Values are means ⫾ SE. Significance: *P ⬍ 0.05 vs. the unstimulated side.

were significantly increased by stimulation in control animals (P ⬍ 0.05). To compare the effects of HET0016 with another CYP4A inhibitor, we treated a group of animals with ABT for 7 days during the electrical stimulation period, and the results are presented in Fig. 5. Neither HET0016 nor ABT had any effect on baseline VEGF expression. The increases in VEGF protein expression induced by electrical stimulation were not blocked by HET0016 or ABT.

Fig. 5. A: Western blot of vascular endothelial growth factor (VEGF) in TA muscle unstimulated (U) or electrically stimulated (S) from rats treated with lecithin, HET0016 (2 mg 䡠 kg⫺1 䡠 day⫺1 in lecithin), and ABT (50 mg 䡠 kg⫺1 䡠 day⫺1 in PBS, n ⫽ 4) after 7 days of the electrical stimulation protocol. For each sample, 50 ␮g of total protein were loaded. C6 tumor cells were used as a control. B: quantitative densitometry of VEGF protein in the control group (n ⫽ 5) and those treated with the selective CYP4A inhibitor HET0016 (n ⫽ 7) after 7 days of the electrical stimulation protocol. Values are means ⫾ SE. *P ⬍ 0.05 vs. the unstimulated side.

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In a complementary experiment, rats were treated with VEGF-neutralizing antibody or PBS (control) to analyze the role of VEGF on 20-HETE formation. As shown in Fig. 6, treatment with VEGF antibody completely blocked the increases in 20-HETE formation induced by 7 days of electrical stimulation. DISCUSSION

Angiogenesis is a fundamental physiological process by which blood vessels are formed. Several candidates have been identified as being important for angiogenesis, including multiple growth factors, hormones, hypoxia, cytokines, and nitric oxide. Data from our laboratory have demonstrated that the renin-angiotensin system and VEGF contribute to the physiological angiogenesis in skeletal muscle induced by electrical stimulation (3) or exercise (4). In those studies, the expression of VEGF induced by exercise or electrical stimulation was completely blocked after treatments with angiotensin-converting enzyme inhibitors (captopril or lisinopril) or an angiotensin type 1 (AT1) receptor blocker (3, 4). However, the angiogenesis induced by electrical stimulation was only attenuated by those treatments, suggesting that another pathway could also be contributing to angiogenesis in this model. Recent studies have suggested that ANG II stimulated the formation of 20-HETE in renal microvessels (2, 8) and that blockade of the formation of 20-HETE attenuates the vasoconstrictor (2) and growth-promoting effects of ANG II in VSM cells. Thus the present study examined whether chronic inhibition of 20HETE formation attenuates the vessel growth induced by electrical stimulation. This study is the first demonstration that 20-HETE formation is increased in skeletal muscle after chronic electrical stimulation and that this AA metabolite plays an important role in angiogenesis because its pharmacological blockade by a specific inhibitor, HET0016, completely eliminated the increase in vessel

Fig. 6. Effects of treatment with VEGF-neutralizing antibody (VEGF Ab; 0.6 mg/100 g ip in PBS) on 20-HETE formation in the muscle of rats after 7 days of the stimulation protocol. Values are means ⫾ SE of 5 rats treated with PBS (control) and 4 rats treated with VEGF Ab. Values are expressed as a percentage of C6 tumor cells. *P ⬍ 0.05 vs. the unstimulated side. AJP-Heart Circ Physiol • VOL

density normally observed with electrical stimulation. Also, this is the first physiological study demonstrating the chronic effects of HET0016, a potent and selective inhibitor recently characterized by Miyata et al. (24). Chronic treatment with HET0016 decreased (by 36%) the 24-h urinary 20-HETE excretion compared with the control group, which was treated with lecithin (P ⬍ 0.05). This study also demonstrates the relationship between 20-HETE and angiogenesis induced by electrical stimulation of skeletal muscle in vivo. The chronic treatment with HET0016, a specific inhibitor of CYP4A activity, completely blocked the increase of vessel density induced by electrical stimulation (1.7% and 4.7% for EDL and TA muscles, respectively) compared with the control group (13% and 32% for EDL and TA muscles, respectively). We further analyzed the involvement of 20-HETE in angiogenesis using a second cytochrome P-450 inhibitor, ABT. ABT has been shown by several investigators to completely block the formation of EETs and 20-HETE in the kidney after both acute and chronic administration in rats (2, 27). Unlike HET0016, ABT only attenuated, but did not block, the increases in vessel density induced by electrical stimulation (9% and 20% for EDL and TA muscles, respectively). These results provide additional evidence that HET0016 is a specific inhibitor for blockade of 20HETE and is a highly effective inhibitor of stimulated increases in vessel density. Interestingly, in both the group treated with HET0016 and that treated with lecithin, there was a small difference in the percent increase of vessel density between EDL and TA muscles. This observation has been shown in several studies previously published by our group (3, 21); however, the reasons for differences are not clear. Both the TA and EDL muscles are flexor skeletal muscles containing a population of red and white fibers; therefore, they are mixed muscles (glycolytic and oxidative fibers). Also, both muscles are innervated primarily by branches of the peroneal nerve, which is electrically stimulated by the electrodes surgically implanted in our model. Several possible explanations could account for this variation in response: differences in the basal loading of the muscle, differences in flow change with stimulation, differences in the innervation, or differences in the degree of contraction. The mechanisms underlying the role of 20-HETE in angiogenesis induced by electrical stimulation in skeletal muscle are not completely understood. It has been shown that in addition to regulating vascular tone, 20-HETE may contribute to the mitogenic actions of vasoactive agents and growth factors. Recent reports have demonstrated that norepinephrine, ANG II, and some growth factors such as epidermal growth factor stimulate cytosolic phospholipase A2 (cPLA2) and AA release by activating Ca2⫹/calmodulin-dependent kinase II (26, 27, 35). Also, it has been demonstrated that CYP4A pathways stimulate MAPK pathways in VSM cells (25). Muthalif et al. (28) recently demonstrated that 20-HETE-induced VSM cell proliferation is medi-

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Fig. 7. Hypothesized pathways involving angiotensin II (ANG II), VEGF, and 20-HETE on angiogenesis induced by electrical stimulation in skeletal muscle.

ated by Ras and MAPK. These data, together with the results presented in this study, where treatment with HET0016 inhibited the angiogenesis in skeletal muscle induced by electrical stimulation, support the conclusion that 20-HETE may play a central role in the regulation of signaling molecules involved in cell proliferation and growth. The presence of CYP4A protein in rat microvessels and skeletal muscle fibers demonstrated by immunohistochemical experiments (18) gives further support for the action of 20-HETE in skeletal muscle. It has been shown that ANG II, the final and active product of the renin-angiotensin system, stimulates migration, hypertrophy, and hyperplasia of VSM cells (11) and angiogenesis (3, 12). Its transduction mechanisms include activation of phopholipases A, C, and D, L-type Ca2⫹ channels, and inhibition of adenylate cyclase (12). Several studies have suggested that AA release in response to various stimuli is mediated by cPLA2 by MAPK (26, 31, 32). Therefore, the cascade of events might include ANG II 3 Ca2⫹/calmodulin-dependent kinase II 3 cPLA2 3 AA 3 CYP4A 3 20HETE 3 Ras/MAPK 3 cell growth, proliferation, and differentiation. Because some studies have demonstrated that ANG II stimulates the production of 20HETE in different tissues (5, 8), and the content of ANG II in the muscle seems to be increased by electrical stimulation (unpublished data), and ANG II plays a critical role in stimulated angiogenesis in skeletal muscle (3), it may be reasonable to speculate that the participation of ANG II is part of an important pathway for the increased 20-HETE formation and angio-

genesis in stimulated muscles. Interestingly, it has been demonstrated that Dahl salt-sensitive rats, which have a diminished capacity to modulate ANG II (7) and to produce 20-HETE (22), do not increase vessel density in response to 7 days of electrical stimulation (3). Because we have shown that VEGF has a crucial role in angiogenesis in skeletal muscle induced by electrical stimulation (3), we further investigated the effects of blocking 20-HETE formation with HET0016 on the expression of VEGF. The results of this study demonstrated that the increase in VEGF protein expression induced by electrical stimulation was not blocked in rats treated with HET0016, which is consistent with the view that ANG II promotes angiogenesis by increasing the expression of VEGF, which in turn increases the production of 20-HETE (27). The signaling pathways are not completely understood; however, similar interactions have been demonstrated (1, 20) between 20-HETE and EEF involving protein kinase C. To further investigate the potential involvement of VEGF on 20-HETE formation, we performed another group of experiments in which rats were treated with VEGF-neutralizing antibody. After 7 days of chronic treatment with VEGF-neutralizing antibody, the increase of 20-HETE formation in the skeletal muscle induced by electrical stimulation was completely inhibited, suggesting that VEGF contributes to the elevation in 20-HETE levels induced by electrical stimulation. In this regard, it is interesting that other growth factors that activate the MAPK system, such as EGF or fibroblast growth factor (6, 20), stimulate the formation of 20-HETE and that CYP4A inhibition can attenuate the mitogenic actions of these growth factors. These results, combined with the observation that ABT and HET0016 did not block the increases in VEGF, suggest that 20-HETE is downstream in the pathway of angiogenesis induced by electrical stimulation. This relationship among ANG II, VEGF, and 20-HETE on the

Table 1. Body weight and unstimulated and stimulated EDL and TA muscle weight-to-body weight ratios Body Weight, g

Lecithin Unstimulated Stimulated HET0016 Unstimulated Stimulated ABT Unstimulated Stimulated PBS Unstimulated Stimulated VEGF Ab Unstimulated Stimulated

EDL Weight/ Body Weight

TA Weight/ Body Weight

235.0 ⫾ 4*

0.43 ⫾ 0.01 0.46 ⫾ 0.01

2.13 ⫾ 0.08 2.1 ⫾ 0.1

208.4 ⫾ 3

236.9 ⫾ 4*

0.44 ⫾ 0.01 0.40 ⫾ 0.02

2.0 ⫾ 0.04 1.97 ⫾ 0.07

4 4

186.7 ⫾ 5

235.8 ⫾ 8*

0.43 ⫾ 0.02 0.43 ⫾ 0.02

1.97 ⫾ 0.12 1.86 ⫾ 0.06

5 5

251.8 ⫾ 3

296.2 ⫾ 5*

5

250.2 ⫾ 3

277.5 ⫾ 9

n

Starting Weight

Ending Weight

9 9

205.4 ⫾ 4

9 9

Values are means ⫾ SE; n, number of muscles in each group. Extensor digitorum longus (EDL) and tibialis anterior (TA) muscle weights are given in units of grams per kilograms. HET0016, N-hydroxy-N⬘-(4-butyl-2-methylphenol)formamidine; PBS, phosphate-buffered saline; ABT, 1-aminobenzotriazole; VEGF Ab, VEGF-neutralizing antibody. * P ⬍ 0.05, significant difference vs. before stimulation. AJP-Heart Circ Physiol • VOL

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angiogenesis induced by electrical stimulation is summarized in Fig. 7. In Fig. 7, we demonstrate the participation of at least three main pathways on angiogenesis induced by electrical stimulation using pharmacological blockade: ANG II, using ANG II-converting enzyme inhibitors and AT1 receptor blockers (previously published, Refs. 3 and 4); VEGF, using the VEGF-neutralizing antibody; and 20-HETE, using HET0016 and ABT. In summary, the present study examined the role of 20-HETE in the angiogenesis induced by electrical stimulation and demonstrated that both VEGF and 20-HETE contribute to the angiogenesis induced by electrical stimulation in skeletal muscle. The authors thank Michael R. Kloehn, Bonnie Freudinger, and Lisa Henderson for expert technical assistance. This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-29587. S. L. Amaral was supported by Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo Fellowship 1998/13772-0, and K. G. Maier was supported by NHLBI Grant HL-10407-02. REFERENCES 1. Aiello LP, Bursell S, Clermont A, Duh E, Ishii H, Takagi C, Mori F, Ciulla T, Ways K, Jirousek M, Smith LEH, and King GL. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 46: 1473–1480, 1997. 2. Alonso-Garcia M, Maier KG, Greene AS, Cowley AW, and Roman RJ. Role of 20-hydroxieicosatetraenoic acid in the renal and vasoconstrictions actions of angiotensin II. Am J Physiol Regul Integr Comp Physiol 283: R60–R68, 2002. 3. Amaral SL, Linderman JR, Morse MM, and Greene AS. Angiogenesis induced by electrical stimulation is mediated by angiotensin II. Microcirculation 8: 57–67, 2001. 4. Amaral SL, Papanek PE, and Greene AS. Angiotensin II and VEGF are involved in angiogenesis induced by short-term exercise training. Am J Physiol Heart Circ Physiol 281: H1163– H1169, 2001. 5. Bautista R, Sanchez Hernandez J, Oyekan A, and Escalante B. Angiotensin II type AT2 receptor mRNA expression and renal vasodilation are increased in renal failure. Hypertension 38: 669–673, 2001. 6. Chen JK, Falck JR, Reddy KM, Capdevila J, and Harris RC. Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells. J Biol Chem 273: 29254–29261, 1998. 7. Cowley AW Jr, Roman RJ, Kaldunsky ML, Dumas P, Dickhout JG, Greene AS, and Jacob HJ. Brown Norway chromosome 13 confers protection from high salt to consomic Dahl S rat. Hypertension 37: 456–461, 2001. 8. Croft KD, McGiff JC, Sanches-Mendonza A, and Carrol MA. Angiotensin II releases 20-HETE from rat renal microvessels. Am J Physiol Renal Physiol 279: F544–F551, 2000. 9. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 280: C1358–C1366, 2001. 10. Frisbee JC, Roman RJ, Krishna M, Falck JR, and Lombard JH. 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats. Am J Physiol Heart Circ Physiol 280: H1066–H1074, 2001. 11. Gibbons GH, Pratt RE, and Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest 90: 456–461, 1992. 12. Greene AS and Amaral SL. Microvascular angiogenesis and the rennin-angiotensin system. Curr Hypertens Rep 4: 56–62, 2002. AJP-Heart Circ Physiol • VOL

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