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(Circulation Research. 2004;94:167.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Smooth Muscle—Specific Expression of CYP4A1 Induces Endothelial Sprouting in Renal Arterial Microvessels

Miao Jiang, Alexandre Mezentsev, Rowena Kemp, Kihwan Byun, John R. Falck, Joseph M. Miano, Alberto Nasjletti, Nader G. Abraham, Michal Laniado-Schwartzman

From the Department of Pharmacology (M.J., A.M., R.K., A.N., N.G.A., M.L.-S.), New York Medical College, Valhalla, NY; Departments of Biochemistry and Pharmacology (K.B., J.R.F.), University of Texas Southwestern Medical Center, Dallas, Tex; and Center for Cardiovascular Research (J.M.M.), University of Rochester, Rochester, NY.

Correspondence to Michal Laniado-Schwartzman, PhD, Department of Pharmacology, New York Medical College, Valhalla, NY. 10595. E-mail michal_Schwartzman{at}nymc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid -hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell–specific promoter SM22 (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). ß-Galactosidase expression was detected in Ad-SM22-nLacZ–transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1–transduced A7r5 and PAC1 cells. Ad-SM22-4A1–transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79±1.77 versus 0.97±0.57 nmol 12-hydroxy lauric acid/10⁶ cells per h). Smooth muscle–specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04±0.29 and 13.43±2.84 ng/mg protein in Ad-SM22-nLacZ–transduced and Ad-SM22-4A1–transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63±3.66 mm in Ad-SM22-4A1–transduced vessels versus 1.79±0.89 mm in Ad-SM22-nLacZ–transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle–specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.

Key Words: angiogenesis • cytochrome P450 • 20-HETE • adenovirus

	Introduction

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The cytochrome P450 (CYP) 4A gene family encodes several CYP enzymes that are capable of hydroxylating the terminal -carbon and, to a lesser extent, the -1 position of saturated and unsaturated fatty acids as well as enzymes active in the -hydroxylation of various prostaglandins.^1,2 Among the CYP 4A enzymes, CYP4A1 has been characterized as the most efficient /-1 hydroxylase of short- and long-chain fatty acids, including lauric, palmitic, and arachidonic acids.^1,3 CYP4A1 expression and activity have been shown to localize in tissues such as liver, lung, kidney, and the vasculature.^4–7 Within the kidney, CYP4A1 expression has been detected along the nephron, with highest concentration in the proximal tubules^2,8,9 and in the vasculature.¹⁰ Systemic administration of CYP4A1 antisense oligonucleotides to Sprague-Dawley rats significantly decreased renal vascular CYP4A1 expression and activity, as measured by arachidonic acid conversion to 20-hydroxyeicosatetraenoic acid (20-HETE).¹¹ In the spontaneously hypertensive rats, administration of CYP4A1 antisense oligonucleotides decreased CYP4A1 expression, 20-HETE synthesis, and reactivity to phenylephrine in mesenteric arteries.¹² Collectively, these observations support the notion that CYP4A1 localized to the vasculature contributes, presumably via synthesis of 20-HETE, to the regulation of vascular function.

20-HETE, the -hydroxylation product of arachidonic acid, is a primary eicosanoid in several microcirculatory districts, most notably, the renal and cerebral microcirculation.^13,14 Its synthesis within the blood vessel wall is believed to be localized to the smooth muscle,¹⁵ except for the pulmonary circulation, where it has been shown to be expressed in the endothelium.¹⁶ It has been characterized as a potent vasoconstrictor of small arteries and arterioles in the rat kidney.¹⁴ It elicits vasoconstriction by inhibiting large-conductance K⁺_Ca channels, inducing depolarization,¹⁷ and additionally increasing intracellular [Ca²⁺]. This effect is most likely related to the activation of L-type Ca²⁺ channels.¹⁵ It has also been shown to mediate mitogenic signaling via mitogen-activated protein kinase activation in vascular smooth muscle cells^18,19 and to contribute to the angiogenic response via increased expression of vascular endothelial growth factor (VEGF).²⁰ As indicated above, 20-HETE production is not limited to the vasculature. It is formed along the nephron and has been shown to inhibit the activities of Na-K-ATPase and the Na-K-2Cl cotransporter in the proximal tubules and medullary thick ascending limb of the loop of Henle, respectively.^21–23 Of note, 20-HETE bioactivity in tubular and vascular tissues may lead to opposing functional outcomes. For example, in the renal tubule, it inhibits sodium reabsorption, yet it is also a renal vasoconstrictor, which could promote sodium retention and development of hypertension.²⁴ Therefore, tissue-specific manipulation of 20-HETE synthesis is imperative for studying its role in cellular processes.

In the present study, we constructed an adenovirus expressing the CYP4A1 cDNA under the control of the smooth muscle–specific promoter SM22. Such construct should allow specific expression of CYP4A1 in vascular smooth muscle cells and thereby could provide an excellent tool for investigating the role of this protein in the regulation of vascular function. The choice of CYP4A1 stems from numerous observations showing that the CYP4A1 protein, in its recombinant or purified form, is the most active among CYP4A proteins in catalyzing arachidonic acid -hydroxylation.^3,25 Another feature typical to CYP4A1 is its preference for catalyzing arachidonic acid at the -carbon; the ratio of to -1 hydroxylation is 6:1 to 10:1.³ The results showed that this construct directed a smooth muscle–specific functional expression of CYP4A1 and that the increased expression of CYP4A1 is associated with increased angiogenic activity of renal microvessels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of Recombinant Adenovirus
The CYP4A1 cDNA (2.1-kb EcoRI/BamHI fragment of CYP4A1-pBSK plasmid)³ was blunt ligated into the BamHI site of the adenovirus expression shuttle vector pCA3. The transgene was placed under the control of the mouse SM22 promoter (-445 nucleotides), which was cloned into a BglII site of pCA3 in place of the CMV promoter.²⁶ Recombinant shuttle plasmids were rescued into a serotype 5-replication–defective adenovirus (strain dl327) by homologous recombination in HEK 293 cells carrying the E1 gene of adenovirus. Putative recombinant plaques were purified twice in HEK cells. The titer was 1x10¹² pfu/mL. The construct was designated as Ad-SM22-4A1. The Ad-SM22-nLacZ virus was used as control. It was amplified in HEK 293 cells, and the titer was 1x10¹² pfu/mL.

Cell Cultures
HEK 293 cells (ATCC CRL 1573), rat thoracic aorta smooth muscle cells A7r5 (ATCC CRL 1444), and NIH3T3 (ATCC CRL 1658) fibroblasts were grown in DMEM supplemented with 10% FBS, 4 mmol/L L-glutamine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells were incubated at 37°C in a 5% CO₂ humidified atmosphere. Rat lung microvessel (RLMV) endothelial cells were cultured in MCDB131 medium with 10% FBS as previously described.²⁷ PAC1 smooth muscle cells were cultured as previously described.²⁶

Transduction of Cells With Adenovirus
Cells were plated in 60-mm culture dishes and allowed to grow until 60% confluent, at which time the cells were washed and incubated with different amounts of adenovirus in 2 mL of DMEM. Cells were gently rocked every 15 minutes over a 1.5-hour incubation period to facilitate uniform adsorption of virus to the cell monolayer. After 1.5 hours, complete medium was added, and the cells were allowed to grow for an extra 2 days. Cells were then harvested for mRNA and protein expression assays as described below.

Detection of ß-Galactosidase
Cultured cells plated in 60-mm dishes were transduced with Ad-SM22-nLacZ, as described above. Expression of ß-galactosidase (ß-gal) was detected by light microscopy as nuclear-localized blue staining using the ß-gal substrate, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), according to the manufacturer’s instructions (Stratagene).

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA (5 µg) isolated using Trizol reagent was subjected to cDNA synthesis and denaturation, followed by polymerase chain reaction (PCR), using SuperScript One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen). PCR reactions were cycled 30 times through a 5-minute denaturing step at 95°C, a 1-minute annealing step at 60°C, and a 1-minute extension step at 72°C. After the cycling procedure, a final 10-minute elongation step at 72°C was performed. PCR was also performed using the CYP4A1 plasmid (100 ng) as a template for positive control. An aliquot (10 µL) of each PCR reaction was separated on a 1% agarose gel, and PCR products were stained with ethidium bromide. The CYP4A1-specific primers were designed to amplify a 351-bp fragment and were as follows: 5'-CTC TTA CTT CGG AGA ATG GAG AA-3' (forward primer) and 5'-GACTTGGATACCCTTGGGTAAAG-3' (backward primer). The CYP4A1 full-length primers were designed to amplify a 2.1-kb fragment and were as follows: 5'-ACCATGAGCGTCTCTGCACTGA-3' (forward primer) and 5'-CAGGACACTGGACACTTTATTG-3' (backward primer). The primer inside the SM22 promoter was designed to amplify a 2.4-kb fragment with a CYP4A1 full-length backward primer and was as follows: 5'-GCATCTCAAAGCATGCAGAG-3'.

Western Blot Analysis
Cells were harvested using trypsin/EDTA and lysed in PBS (pH 7.4) containing 1% (vol/vol) NP40, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mmol/L PMSF. The lysate proteins (60 µg) were separated by electrophoresis on a large (16x20 cm) 8% SDS-polyacrylamide gel at 18 mA, 4°C for 18 to 20 hours. Proteins were transferred electrophoretically to a nitrocellulose membrane. The membrane was blocked, washed, and incubated overnight with goat anti-rat CYP4A1 polyclonal antibody (1:500; Gentest) at room temperature, washed, and then incubated with 1:5000 dilution of horseradish peroxidase–conjugated second antibody for 1 hour. Immunoreactive proteins were detected using the ECL Plus detection system (Amersham Life Sciences).

Enzyme Activity
Cultured VSMCs (T-175) transduced with either Ad-SM22-4A1 or Ad-SM22-nLacZ were washed twice with PBS and incubated with [1-¹⁴C] lauric acid (1 µCi; 57.0 mCi/mmol) and NADPH (1 mmol/L) in 0.2 mL of potassium phosphate buffer (100 mmol/L, pH 7.4) containing 10 mmol/L MgCl₂ for 1 hour at 37°C. In some experiments, cells were preincubated with the CYP4A inhibitor N-(4-butyl-2-methylphenyl)-N'-hydroxyformamidine (HET0016; 1 µmol/L)²⁸ for 5 minutes before the addition of the reaction mixture. The reaction was terminated by acidification, and lauric acid metabolites were extracted with ethyl acetate. Radiolabeled metabolites were separated by reverse-phase HPLC, as previously described.³ The identity of 12-hydroxy lauric acid was confirmed by its comigration with an authentic standard.

Isolation and Transduction of Renal Arteries
Animal experimentation was in accordance with institutional guidelines. Male Sprague-Dawley rats (7- to 9-week-old) were anesthetized with pentobarbital sodium (50 mg/kg body wt). Kidneys were excised, placed in ice-cold Tyrode buffer, and coronally sectioned. The renal papilla was removed to expose the microvessels. The interlobar arteries (100 µm) were microdissected and freed from cortical and connective tissue. The purity of the microdissected renal interlobar arteries was routinely examined by phase-contrast microscopy, as previously described.^10,11 After washing with endothelial cell basal medium-2 (Clonetics), microvessels were placed in a 48-well plate and incubated with 25 to 50 µL of either Ad-SM22-4A1 or Ad-SM22-nLacZ in 150 µL of endothelial cell basal medium-2 for 1.5 hours followed by addition of 600 µL endothelial cell medium-2 (Clonetics) and incubation for an additional 24 hours. Detection of ß-galactosidase was followed according to Lund et al.²⁹ Arterial segments were embedded in OCT (Sakura Finetechnical USA), sectioned, and counterstained with nuclear fast red. Increased expression of CYP4A1 in arteries transduced with Ad-SM22-4A1 was determined by measuring the levels of 20-HETE in the incubation medium by negative chemical ionization gas chromatography/mass spectrometry (NCI-GC/MS). Briefly, arterial segments were transduced with either Ad-SM22-nLacZ or Ad-SM22-4A1, as described above, and incubated for an additional 72 hours. The medium was collected, and [²H₂]-20-HETE (0.5 ng/mL) was added as an internal standard. 20-HETE was isolated by ethyl acetate extraction and HPLC separation and additionally derivatized to the pentafluorobenzyl ester, trimethylsilyl ether.¹⁰ NCI-GC/MS was performed on a HP6890 mass spectrometer (Hewlett-Packard) interfaced with a capillary gas chromatographic column (HP-5MS, 30 mx0.25 mmx0.25 µm, Agilent). Single ions were monitored; m/z 391 and 393 for the derivatized 20-HETE and [²H₂]-20-HETE, respectively. 20-HETE levels were quantified as described previously.¹⁰

Angiogenesis Assays
In vitro capillary tube–like formation was examined in RLMV endothelial cells grown on growth factor–reduced basement membrane Matrigel (BD Biosciences-Discovery Labware), as previously described.³⁰ Formation of tube-like structures was examined 24 hours after addition of 1 nmol/L of either 19-HETE, 20-HETE, or 20-HETE analogues, ie, N-(20-hydroxy-eicosa-5(Z),14(Z)-dienoyl)benzenesulfonamide (UMK-IV-101-30) and N-(20-hydroxy-eicosa-5(Z),14(Z)-dienoyl)-4-iodobenzenesulfonamide (UMK-V-26-36), which were designed to be less susceptible to autooxidation and ß-oxidation and, thereby, long lasting. VEGF (1 nmol/L) was used as a positive control and ethanol as the vehicle control. Cultures were photographed, and the length (micrometer) of the tube-like structures was quantified using Image Pro-Express Software (Cyber Media). The angiogenic activity was also examined ex vivo using renal arterial microvessels according to a method described by Zhu et al,³¹ with some modifications. Renal arterial microvessels were transduced with Ad-SM22-4A1 or Ad-SM22-nLacZ, as described above. After 24 hours, the vessels were placed onto fibronectin-coated 24-well plates (one vessel per well). Matrigel (250 µL) was added into each well on top of the vessel and allowed to solidify for 30 minutes, after which 500 µL endothelial cell medium-2 was added with and without HET0016 (1 to 10 µmol/L) or the 20-HETE analogue, UMK-IV-101-30 (1 µmol/L). Culture medium was changed every 2 days. The angiogenic response of the renal arteries was measured over time (at days 4, 7, and 14) by measuring the length of the neovessel sprouts and counting the branching points. To determine whether neovessel sprouts were composed of endothelial cells, renal arterial microvessels were placed in glass-bottom culture dishes and cultured as described above. At day 4, the lectin Ulex europeus (Sigma), which specifically binds to endothelial cells, was added to the medium in a final concentration of 10 µg/mL and incubated with the vessels for 1 hour at 37°C as described.³² Cultures were washed 3 times with MCDB 131 medium (GIBC-BRL), and endothelial cell fluorescence was examined by confocal microscopy.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adenovirus Construction
PCR analysis was performed to verify positive clones of the shuttle vector. Control CYP4A1 plasmid, amplified with the full-length primers and specific primers, was used as positive control. As seen in Figure 1, the full-length CYP4A1 primers amplified the predicted size of a 2.1-kb band, and the CYP4A1-specific primers amplified the predicted 350-bp band, thus indicating that CYP4A1 cDNA was cloned into the shuttle vector. To additionally verify the orientation of the inserted CYP4A1, a primer inside the promoter SM22, located 300 bp away from the CYP4A1 cDNA, was used as forward primer. By using the antisense primer at the end of CYP4A1 as backward primer, a 2.4-kb fragment was amplified. However, no PCR product was amplified when the sense primer of CYP4A1 was used as backward primer with the primer in the SM22 promoter (Figure 1), indicating that CYP4A1 cDNA was cloned with the correct orientation.

View larger version (43K): [in this window] [in a new window]   Figure 1. PCR analysis of the Ad-SM22-4A1-pCA3 shuttle vector. PCR analysis was performed to verify positive clones and insert orientation using CYP4A1-specific and full-length primers as well as a SM22 primer, as described in Materials and Methods.

Ad-SM22-4A1–Directed Smooth Muscle Cell–Specific Expression of CYP4A1
Cell-specific expression was examined by infecting endothelial, smooth muscle, and fibroblast cells with Ad-SM22-nLacZ. As seen in Figure 2, the expression of ß-gal was only detected in A7r5 smooth muscle cells, indicating that the SM22 promoter specifically drove gene expression to smooth muscle cells. The transduction efficiency was 95% to 100%. Functional expression of CYP4A1 was additionally examined in A7r5 and PAC1 cells transduced with Ad-SM22-4A1 by reverse transcriptase–PCR using CYP4A1 full-length primers. As seen in Figure 3A, the 2.1-kb band was only amplified in cells transduced with Ad-SM22-4A1, indicating that Ad-SM22-4A1 expressed CYP4A1 mRNA. CYP4A1 immunoreactive protein was detected with antibody against CYP4A1 only in cells transduced with Ad-SM22-4A1 (Figure 3B). Cells transduced with 10 µL of 1x10¹² pfu/mL of virus showed the highest expression, whereas increasing the amount of virus to 50 µL decreased the expression, presumably because of virus cytotoxicity.

View larger version (102K): [in this window] [in a new window]   Figure 2. Cell-specific expression of ß-gal after transduction with Ad-SM22-nLacZ. A7r5, 3T3, and RLMV cells were transfected with Ad-SM22-nLacZ, and expression of LacZ was detected by ß-gal staining, as described in Materials and Methods. Right panel,x20; left panel, x10.

View larger version (65K): [in this window] [in a new window]   Figure 3. CYP4A1 expression in cells transduced with Ad-SM22-4A1. A, RT-PCR was performed using total RNA from PAC1 and A7r5 cells transduced with Ad-SM22-nLacZ (-) or Ad-SM22-nLacZ (+) and CYP4A1 full-length primers. Amplification of CYP4A1 plasmid was used as a positive control. B, Representative immunoblot of CYP4A in cells transduced with Ad-SM22-4A1. A7r5 cells were transduced with Ad-SM22-nLacZ (-) or with 1, 10, or 50 µL of 1x1012 pfu/mL Ad-SM22-4A1 (+). Western blot analysis (n=3) was performed using cell homogenates and antibody against CYP4A1 protein, as described in Materials and Methods. Recombinant CYP4A1 and CYP4A2 proteins were used as standards (St).

To determine whether transduction of cells with Ad-SM22-4A1 yielded an active CYP4A1 enzyme, the preferred substrate lauric acid was added to transduced cells. As seen in Figure 4, A7r5 cells transduced with Ad-SM22-4A1 metabolized lauric acid to its -hydroxylated metabolite, 12-hydroxy lauric acid, at a rate 5 times greater (P<0.05) than that of cells transduced with Ad-SM22-nLacZ (4.79±1.77 versus 0.97±0.57 nmol 12-hydroxy lauric acid/10⁶ cells per hour, n=4), indicating an increase in CYP4A1 function. Moreover, addition of the CYP4A inhibitor, HET0016, blocked lauric acid -hydroxylation (Figure 4C), additionally demonstrating that the transduced CYP4A1 is a functional enzyme.

View larger version (38K): [in this window] [in a new window]   Figure 4. Lauric acid -hydroxylation in Ad-SM22-4A1–transduced A7r5 cells. Representative HPLC elution profiles of radiolabeled metabolites formed in cells incubated with [1-14C] lauric acid. A, Ad-SM22-nLacZ–transduced cells. B, Ad-SM22-4A1–transduced cells. C, Ad-SM22-4A1–transduced cells preincubated with HET0016 (1 µmol/L).

Smooth Muscle Expression of CYP4A1 in Renal Arteries Stimulates Angiogenic Activity
Smooth muscle–specific expression driven by the SM22 promoter was additionally assessed in isolated renal microvessels. Renal interlobar arteries were transduced with Ad-SM22-nLacZ virus, as described in Materials and Methods, and sections of these vessels were stained for ß-gal and with nuclear fast red for background staining. As shown in Figure 5A, many of the smooth muscle cells in the media were stained as blue, indicating that the SM22 promoter specifically drove gene expression to smooth muscle cells. To additionally determine whether transduction of renal interlobar arteries with Ad-SM22-4A1 increases CYP4A activity, 20-HETE levels were measured. As seen in Figure 5B, the levels of 20-HETE in the culture medium from Ad-SM22-4A1–transduced arteries were 3-fold higher than those in Ad-SM22-nLacZ–transduced arteries. Addition of HET0016 reduced the levels of 20-HETE by 60% and 70% in Ad-SM22-4A1–transduced and Ad-SM22-nLacZ–transduced arteries, respectively, indicating that transduction with Ad-SM22-4A1 increased CYP4A1 enzymatic activity (Figure 5B).

View larger version (50K): [in this window] [in a new window]   Figure 5. A, Cell-specific expression of ß-gal in interlobar arteries after transduction with Ad-SM22-nLacZ. A, B, C, and D, Control untransduced vessel counterstained with nuclear fast red, Ad-SM22-nLacZ–transduced vessel counterstained with nuclear fast red, Ad-SM22-nLacZ–transduced vessel (x10), and Ad-SM22-nLacZ–transduced vessel (x40), respectively; arrows indicate smooth muscle staining. B, 20-HETE levels in culture medium of transduced interlobar arteries. Microdissected arteries were transduced with Ad-SM22-nLacZ or Ad-SM22-4A1 and placed in culture for 72 hours in the presence and absence of HET0016 (10 µmol/L). The culture medium was collected, and 20-HETE was extracted with ethyl acetate, separated by HPLC, and subjected to GC/MS analysis, as described in Materials and Methods. Results are mean±SE, n=4. P<0.01 from LacZ-transduced vessels; *P<0.01 from vessels not treated with HET0016.

A recent study by Amaral et al²⁰ demonstrated that treatment of rats with HET0016 blocked skeletal muscle angiogenesis elicited by electrical stimulation. Interestingly, we found that 20-HETE, the arachidonate metabolite of CYP4A1, is a potent angiogenic stimulus in cultured endothelial cells. As seen in Figure 6, addition of 20-HETE or 20-HETE analogues at concentrations of 1 nmol/L resulted in a marked stimulation of capillary-like tube formation in endothelial cells grown on Matrigel compared with the vehicle control, and this effect was comparable to that of 1 nmol/L of VEGF. In contrast, the structurally similar eicosanoid, 19-HETE, had no significant effect on the angiogenic response of endothelial cells (Figure 6B). Based on this finding and the observation made by Amaral et al,²⁰ we argued that increased expression of CYP4A1 protein might result in increased angiogenic response of renal arteries. We transduced renal microvessels with Ad-SM22-4A1 or Ad-SM22-nLacZ and examined their capacity to produce capillary-like network when grown on Matrigel. Figure 7 shows representative photographs of the angiogenic response in these cultures. Vessels that were transduced with Ad-SM22-nLacZ exhibited only rare (1 out of 5 preparations) sprouts (Figure 7A), whereas vessels transduced with Ad-SM22-4A1 showed a prominent angiogenic response in all preparations, as evidenced by a marked increase in microvessel sprouting and branching (Figures 7B and 7C). The origin of the cells sprouting from the arterial explants was examined using the lectin Ulex europeus, which specifically binds to endothelial cells. As seen in Figures 7D and 7E, most (75±20%, n=10) cells forming capillary tube-like network were stained with Ulex europeus, indicating that the sprouting cells were endothelial cells.

View larger version (61K): [in this window] [in a new window]   Figure 6. Capillary tube–like formation in microvessel endothelial cells. RLMV endothelial cells grown on Matrigel were incubated for 24 hours with 1 nmol/L of either VEGF, 19-HETE, 20-HETE, or 20-HETE analogues [UMK-IV-101-30, N-(20-hydroxy-eicosa-5(Z),14(Z)-dienoyl)benzenesulfonamide; UMK-V-26-36, N-(20-hydroxy-eicosa-5(Z),14(Z)-dienoyl)-4-iodobenzenesulfonamide]. The vehicle (ethanol) was used as a negative control. A, Representative photographs depicting the angiogenic response of 20-HETE and its analogues. B, Quantitative analysis of capillary length (µm); mean±SE, n=3. *P<0.01 from vehicle control.

View larger version (71K): [in this window] [in a new window]   Figure 7. Sprouting of neovessels in renal interlobar arteries transduced with Ad-SM22-4A1. Phase-contrast photographs of arteries transduced with Ad-SM22-nLacZ (A) and Ad-SM22-4A1 (B, x4; C, x10) were taken 7 days after transduction. Sprouting of neovessels is seen only in arteries transduced with Ad-SM22-4A1; arrows indicate branching points. D and E, Representative phase-contrast and fluorescence photographs of sprouting neovessels treated with the lectin Ulex europeus (x10).

Quantitative analysis of the angiogenic response is shown in Figure 8. Endothelial sprouting, as measured by total length and number of branching points, increased by 7-fold in vessels transduced with Ad-SM22-4A1 compared with vessels transduced with Ad-SM22-nLacZ. Vessels that were not transduced with virus showed similar angiogenic activity as Ad-SM22-nLacZ–transduced vessels (1.58±1.35 versus 1.79±0.89 mm, n=5). That the increased expression of CYP4A1 contributed to the angiogenic activity was additionally confirmed using a CYP4A-selective inhibitor. Addition of HET0016 (10 µmol/L), but not the vehicle control (ethanol), to the incubation medium of Ad-SM22-4A1–transduced arteries blocked the angiogenic activity (Figure 8), suggesting that CYP4A1 activity underlies the increased angiogenic activity. The effect of HET0016 was dose-dependent, with 20%, 50%, and 90% inhibition at 1, 5, and 10 µmol/L HET0016, respectively (n=5). More importantly, the inhibitory effect of HET0016 was reversed by the addition of the 20-HETE analogue, UMK-IV-101-30, suggesting the involvement of 20-HETE in mediating the angiogenic response displayed by Ad-SM22-4A1–transduced arteries.

View larger version (27K): [in this window] [in a new window]   Figure 8. Angiogenic activity of renal interlobar arteries. Angiogenic activity of arteries transduced with Ad-SM22-nLacZ and Ad-SM22-4A1 was measured as the total length of the capillary-like tube network (A) and the number of branching points (B) in cultures treated with and without HET0016 (10 µmol/L) in the presence and absence of the 20-HETE analogue (20-HETEag, 1 µmol/L) UMK-IV-101-30. Results are mean±SE, n=5. *P<0.05 from the corresponding vehicle (ethanol) control; P<0.05 from vehicle-treated Ad-SM22-nLacZ–transduced arteries.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
CYP-derived eicosanoids are recognized as important autocrine and paracrine mediators of cell function. They have been implicated in the regulation of vascular tone, ion transport mechanisms, inflammation, cell proliferation and differentiation, renal hemodynamics, and salt and water reabsorption and secretion.²⁴ This diversity of biological activities stems not only from the array of structurally different metabolites but also from the fact that there are numerous CYP isoforms that exhibit cell-specific localization and regulation and have the ability to catalyze arachidonic acid oxygenation. Such is the case of 20-HETE, the -hydroxylation product of arachidonic acid. In the kidney, it is formed in epithelial cells along the nephron and in smooth muscle cells of the kidney vasculature by numerous CYP isoforms. 20-HETE in vitro is a potent vasoconstrictor of renal arterioles and an inhibitor of renal tubular transport and K⁺ channel activity. In vivo, 20-HETE affects renal vascular resistance, autoregulation of renal blood flow, and tubuloglomerular feedback. To discern the biological significance of 20-HETE, it is necessary to establish preparations that target its synthesizing system in a cell-specific manner.

The present study describes the construction of an adenoviral vector that specifically transduces CYP4A1 expression to smooth muscle cells in vitro in cultured cells and ex vivo within the blood vessel wall. CYP4A1 is the most efficient arachidonate -hydroxylase in vitro, producing 20-HETE at a rate that is 20 to 40 times greater than CYP4A2/4A3.³ This feature is useful when designing a vector to increase CYP4A expression and activity. Because the smooth muscle composes the layer within the renal vasculature from which 20-HETE is believed to be originated, targeting CYP4A1 expression to these cells should provide a valuable approach to studying its relevance to vascular function. We used the smooth muscle promoter, SM22,²⁶ to drive cell-specific expression of CYP4A1.

Smooth muscle–specific functional expression of CYP4A1 was established in cultured cells and in isolated renal microvessels. We used several cell lines, including the rat aortic-derived A7r5 and PAC1 cells, which exhibit characteristics of smooth muscle cells. Transduction of A7r5 or PAC1 cells, but not endothelial cells or fibroblast cells, with Ad-SM22-nLacZ resulted in expression of ß-gal. Likewise, only A7r5 or PAC1 cells showed increased CYP4A1 mRNA and immunoreactive protein 48 hours after transduction with Ad-SM22-4A1. The increased expression of CYP4A1 protein in A7r5 cells was associated with increased CYP4A enzymatic activity, as measured by -hydroxylation of lauric acid, the preferred substrate of CYP4A enzymes, indicating that transduction of CYP4A1 protein via Ad-SM22-4A1 yielded an active CYP4A1 enzyme. Smooth muscle–specific expression was also evident in isolated renal interlobar arteries. Thus, transduction of renal microvessels placed in culture with Ad-SM22-nLacZ displayed considerable ß-gal staining only in the smooth muscle layer. Although expression of ß-gal was significant, it was not robust. This may be attributable to the presence of permeability barriers limiting adenoviral particles from reaching smooth muscle cells, as suggested by others.^33,34 This level of expression, together with the lack of good-quality specific CYP4A1 antibody, limited our ability to detect by immunohistochemistry an increase in CYP4A1 protein. This notwithstanding, transduction with Ad-SM22-4A1 yielded an increase in a functional CYP4A1 protein, as evidenced by a 3-fold increase in 20-HETE levels in the culture medium of vessels transduced with Ad-SM22-4A1 that was inhibited by the addition of the CYP4A-selective inhibitor HET0016.

We additionally examined whether smooth muscle–specific transduction of CYP4A1 alters vascular functions that may be attributed to its activity, such as angiogenesis.²⁰ Coincidentally, we found that 20-HETE and 20-HETE analogues at concentrations as low as 1 nmol/L stimulated capillary-like tube formation of microvessel endothelial cells grown on Matrigel by 10- to 20-fold compared with the vehicle control. This finding suggests that the endothelium is a target for 20-HETE, thus raising the possibility that 20-HETE generated in the smooth muscle cells affects endothelial cell function by stimulating an angiogenic response. Indeed, renal arterial vessels placed on Matrigel showed a marked angiogenic response after transduction with Ad-SM22-4A1 but not with Ad-SM22-nLacZ. This angiogenic activity, measured as the length of sprouting neovessels that were composed of endothelial cells, was evident as early as 4 days. Moreover, it was fully blocked when HET0016 was added to the medium, suggesting that the increased expression of CYP4A1 in the media was responsible for the observed angiogenic activity.

The demonstration that addition of 20-HETE analogues, which exhibit angiogenic potency like that of 20-HETE, reversed the inhibitory effect of HET0016 on angiogenesis in Ad-SM22-4A1–transduced vessels additionally supports the concept that the high angiogenic activity exhibited by these vessels is linked to 20-HETE. This conclusion is strengthened by our results that 20-HETE and its analogues are potent angiogenic factors for endothelial cells in vitro and that vessels transduced with Ad-SM22-4A1 produced more 20-HETE and by the findings of Amaral et al.²⁰ Additional studies using specific 20-HETE antagonists may substantiate this claim. Nonetheless, the fact that increased smooth muscle CYP4A1 expression yields endothelial response in renal microvessels is novel and intriguing. The role of angiogenesis in the maintenance of the renal microvasculature is unclear. However, numerous studies demonstrated decreased glomerular and interstitial capillary density in progressive renal diseases.^35,36 Aging, diabetes, and hypertension are a few conditions in which renal interstitium capillary density and angiogenic activity are reduced. Thus, maintenance of endothelial function, including its angiogenic capability, may be important for sustaining normal kidney function. Our observation that CYP4A1 transduction induced angiogenic activity of the renal microvasculature suggests that CYP4A1 expression or activity may be an important component for maintaining angiogenic capability of the endothelium. To this end, studies have shown that renal CYP4A1 expression is decreased with age, as is the ability of the kidney to produce 20-HETE.^37,38

	Acknowledgments

 
This study was supported by NIH Grants PO1 HL34300, HL62572, and DK-38226 and by the Robert A. Welch Foundation.

	Footnotes

 
Original received July 11, 2003; resubmission received October 17, 2003; accepted November 14, 2003.

	References

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