Am J Physiol Regul Integr Comp Physiol 308: R379–R390, 2015. First published December 24, 2014; doi:10.1152/ajpregu.00256.2014.

Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat Fan Fan,1 Aron M. Geurts,2 Sydney R. Murphy,1 Mallikarjuna R. Pabbidi,1 Howard J. Jacob,2 and Richard J. Roman1 1

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi; and 2Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin Submitted 13 June 2014; accepted in final form 16 December 2014

Fan F, Geurts AM, Murphy SR, Pabbidi MR, Jacob HJ, Roman RJ. Impaired myogenic response and autoregulation of cerebral blood flow is rescued in CYP4A1 transgenic Dahl salt-sensitive rat. Am J Physiol Regul Integr Comp Physiol 308: R379–R390, 2015. First published December 24, 2014; doi:10.1152/ajpregu.00256.2014.—We have reported that a reduction in renal production of 20-HETE contributes to development of hypertension in Dahl salt-sensitive (SS) rats. The present study examined whether 20-HETE production is also reduced in the cerebral vasculature of SS rats and whether this impairs the myogenic response and autoregulation of cerebral blood flow (CBF). The production of 20-HETE, the myogenic response of middle cerebral arteries (MCA), and autoregulation of CBF were compared in SS, SS-5BN rats and a newly generated CYP4A1 transgenic rat. 20-HETE production was 6-fold higher in cerebral arteries of CYP4A1 and SS-5BN than in SS rats. The diameter of the MCA decreased to 70 ⫾ 3% to 65 ⫾ 6% in CYP4A1 and SS-5BN rats when pressure was increased from 40 to 140 mmHg. In contrast, the myogenic response of MCA isolated from SS rats did not constrict. Administration of a 20-HETE synthesis inhibitor, HET0016, abolished the myogenic response of MCA in CYP4A1 and SS-5BN rats but had no effect in SS rats. Autoregulation of CBF was impaired in SS rats compared with CYP4A1 and SS-5BN rats. Blood-brain barrier leakage was 5-fold higher in the brain of SS rats than in SS-5BN and SS.CYP4A1 rats. These findings indicate that a genetic deficiency in the formation of 20-HETE contributes to an impaired myogenic response in MCA and autoregulation of CBF in SS rats and this may contribute to vascular remodeling and cerebral injury following the onset of hypertension.

other hand, we have reported that a deficiency in the renal production of 20-HETE promotes sodium retention and contributes to the development of hypertension in Dahl saltsensitive (SS) rats (10, 48). Moreover, transfer of a region of chromosome 5 containing the CYP4A genes from Lewis or Brown Norway (BN) rats into the SS genetic background increases the formation of 20-HETE, promotes sodium excretion, and opposes the development of hypertension in SS-5BN consomic or congenic strains (61, 62). However, there are 1,794 genes on rat chromosome 5, so it is difficult to conclude that a genetic defect in one of the CYP4A genes on chromosome 5 is responsible for the development of hypertension in SS rats. It also remains to be determined whether there is a deficiency in the formation of 20-HETE in the vasculature that impairs the myogenic response and vascular tone in the SS rats. To test this hypothesis, the present study compared the production of 20-HETE, the myogenic response of the middle cerebral artery (MCA), autoregulation of cerebral blood flow (CBF), and the blood-brain barrier (BBB) leakage in SS and SS-5BN consomic rats. We also measured the formation of 20-HETE and characterized cerebral vascular function in a new CYP4A1 transgenic SS rat strain that we generated using a Sleeping Beauty transposon system (17, 23, 24).

CYP4A; cerebral circulation; myogenic response; middle cerebral artery; autoregulation of cerebral blood flow

General

CYTOCHROME P-450 (CYP) ENZYMES

of the 4A family, which catalyze the formation of 20-hydroxyeicosatetraenoic acid (20HETE), have been implicated in the regulation of renal function, vascular tone, and the long-term control of blood pressure (2, 10, 48, 63). CYP4A1, CYP4A2, CYP4A3, and CYP4A8 are all expressed in the kidney and the renal and cerebral vasculature and contribute to the formation of 20-HETE (8, 21). CYP4A1 has the highest catalytic activity, and its expression is induced by fibrates (31, 50, 52). 20-HETE has both prohypertensive and antihypertensive actions. It is a potent vasoconstrictor that plays an important role in the myogenic response and autoregulation of blood flow in the renal and cerebral circulations (11, 15, 20, 48, 63). In the spontaneously hypertensive rat and in androgen-dependent mouse models, the increased vascular production of 20-HETE is associated with the development of hypertension (10, 38, 51, 63, 64). On the

Address for reprint requests and other correspondence: R. J. Roman, Univ. of Mississippi, Medical Center Dept. of Pharmacology and Toxicology, 2500 N. State St., Jackson, MS 39216 (e-mail: [email protected]). http://www.ajpregu.org

MATERIALS AND METHODS

These experiments were performed using 192 SS, SS-5BN consomic and CYP4A1 transgenic SS rats that were obtained from inbred colonies maintained at the University of Mississippi Medical Center and 12 Sprague-Dawley (SD) and Lewis rats purchased from Charles River Laboratories (Wilmington, MA). The rats had free access to food and water throughout the study. All protocols involving animals received prior approval by the Institutional Animal Care and Use Committee (IACUC) of the University of Mississippi Medical Center. Generation of Rat CYP4A1 Transposon Plasmid Cloning, sequencing of rat CYP4A1 cDNA. The renal outer medulla of a Lewis rat was placed overnight in ice-cold RNAlater solution (Life Technologies, Grand Island, NY), homogenized in TRIzol solution (Life Technologies) using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA), and RNA was extracted according to the manufacturer’s instructions. CYP4A1 template was generated by adding an aliquot of the RNA (1 ␮g) to a 15-␮l reverse transcription reaction with 0.5 ␮g of a gene-specific primer (5=-GTG CAG GAC ACT GGA CAC-3=) derived from the rat CYP4A1 (NM_175837) sequence (25) using a method that we previously described (60). The primer sequences for the amplification of the full-length rat CYP4A1 cDNA were 5=-GCT GCA CCA TGA GCG TCT C-3= (sense) and 5=-GTG CAG GAC ACT GGA CAC-3= (antisense). The PCR reactions contained 25 ng of each primer, 10 ng of cDNA, 20 mmol/l

0363-6119/15 Copyright © 2015 the American Physiological Society

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Tris·HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2, 200 mol/l of each dNTP, 0.5 U Taq DNA polymerase (Qiagen, Valencia, CA). The reaction mixtures were initially denatured at 94oC for 5 min and then cycled 35 times between 94oC for 30 s, 58oC for 30 s, and 72oC for 2 min followed by extension for 10 min at 72oC. The PCR products were separated on 1% agarose gel in a Tris-borate-EDTA (TBE) buffer with 100 mg/ml of ethidium bromide (Sigma, St. Louis, MO), they were visualized, and their density was analyzed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). The final PCR product size was 2,133 bp, and it included 8 bp of the 5=-UTR and the entire 3=-UTR. The full-length CYP4A1 cDNA, purified using a PureLink PCR purification kit, was ligated into pCR4-TOPO sequencing vector (Life Technologies) and incubated at room temperature for 30 min. DH 5␣-T1 Escherichia coli-competent cells (Life Technologies) were transformed by adding 2 ␮l of the ligation reaction and incubated at 42oC for 30 s followed by placing the reactions in an ice bath for 2 min. The cells were placed in 1 ml of SOC growth medium and incubated at 37°C for 1 h. They were then placed on LB agar plates treated with ampicillin (100 ␮g/ml), and incubated at 37°C overnight. The colonies were screened by PCR for the presence of an insertion. Briefly, the colonies were picked and placed into a well of a 96-well plate preloaded with 100 ␮l of LB medium plus antibiotics and incubated at 37°C for 1 h. The samples were diluted by a factor of 25 and heated to 95°C for 10 min. The samples were screened for inserts using PCR with M13 forward and reverse primers. Positive colonies were grown in 5 ml of LB medium with 100 ␮g/ml of ampicillin overnight at 37°C. Plasmid DNA was then extracted using a PureLink Quick Plasmid Miniprep kit (Life Technologies), eluted in 50 ␮l of TE buffer, and the inserts were sequenced using M13 primers. Plasmids containing the full-length CYP4A1 cDNA without any mismatched base pairs were stored at ⫺80°C for later use. Construction of CYP4A1 transposon vector. The sequence confirmed pCR4-TOPO.CYP4A1 plasmid was cut with the restriction enzyme EcoRI (New England Biolabs, Ipswich, MA) and incubated at 37°C for 1 h. An eGFP transposon vector pT2.CAG.eGFP was also digested using EcoRI under the same conditions. The digested fragments were separated on an agarose gel. A fragment of 2772 bp from digested pCR4-TOPO.CYP4A1 and a 5757-bp fragment from pT2.CAG.eGFP were excised from the gels and purified using a

PureLink Quick Gel Extraction kit (Life Technologies). The two fragments were ligated together to obtain a CYP4A1 insertion between the CAG promoter and the right IR/DR (inverted repeat/direct repeat) in the T2 transposon backbone using T4 DNA ligase (Life Technologies) and incubated at 16°C overnight. The ligation reactions (2 ␮l) were transformed into DH 5␣-T1 competent cells, and the resulting plasmids were extracted using MiniPrep kit. The orientation of the insertions was confirmed by digestion with Pst I and by PCR using primers F1/R1 and F2/R2, as shown in Fig. 1A. The Pst I digested plasmid fragments with the correctly orientated CYP4A1 insert were 5,994 and 540 bp, respectively. Excision assay. We next performed an excision assay to ensure that the transposon construct was active. HeLa cells were cotransfected with 1.5 ␮g of pT2.CAG.CYP4A1 with a SB11 transposase plasmid (17) in a 1:1 ratio using PolyFect transfection reagent (Qiagen). Forty-eight hours after transfection, the cells were washed with CellScrub Washing Buffer (Genlantis, San Diego, CA) to remove cationic lipid/DNA complexes. Genomic DNA was extracted using genomic DNA extraction kit (Life Technologies) and was used as PCR templates (25 ng). PCR was performed using OF/OR primers followed by nested PCR using IF/IR primers, as shown in Fig. 1B. pT2.CAG.eGFP was used as a transposon-positive control, and SB11 transposase overexpression plasmid alone was used as the negative control. Western blot analysis. HeLa cells transfected with the pT2.CAG. CYP4A1 construct were incubated for 48 h at 37°C, harvested using ice-cold RIPA buffer (R0278; Sigma-Aldrich), supplemented with a protease inhibitor cocktail, and homogenized. The homogenates were centrifuged at 11,000 g at 4°C for 15 min. The supernatant was collected, and Western blots were performed using 50 ␮g of protein. The samples were separated by electrophoresis on a 10% SDSpolyacrylamide gel, transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad), and the membranes were blocked at room temperature for 1 h in a buffer containing 10% nonfat dry milk. The membranes were incubated overnight at 4°C with a mixture of 1:4,000 dilution of goat anti-CYP4A primary antibody (299230; Daiichi Pure Chemicals, Tokyo, Japan) and 1:8,000 dilution of an anti-␤ actin (ab6276; Abcam, Cambridge, MA) followed by 1:15,000 dilution of a horseradish peroxidase-coupled anti-goat antibody (sc-2020) and 1:20,000 of an anti-mouse secondary antibody (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA) for

Fig. 1. Strategy to generate the CYP4A1 transposon construct. A: transposon plasmid construction. A fulllength CYP4A1 cDNA was cut by EcoRI from TOPO TA sequencing vector pCR4-TOPO.CYP4A1 and ligated into a transposon vector pT2.CAG.eGFP in which eGFP was cut out using EcoRI. The orientation of the insertion was confirmed by digestion with PstI and PCR by using primers F1/R1 and F2/R2. B: plasmid-based excision assay. The DR of ITR (IR/DR shown in panel A) has consensus inner (Li and Ri) and outer (Lo and Ro). TA results from duplication of the original TA insertion site. OF/OR are the primers of the first A (outer) PCR and IF/IR are the primers of the nested (inner) PCR. SB transposases bind on each ITR, resulting a ⬃300-bp nested PCR product, when there is excision activity. A ⬃3,000-bp product should be produced when there is no excision.

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1 h. The blots were exposed to SuperSignal West Dura Extended Duration Substrate (34076; Thermo Scientific, Pittsburgh, PA), and the relative intensities of the bands at 50 –52 kDa for CYP4A and 42 kDa for ␤-actin were imaged using a ChemiDoc photodocumentation system (Bio-Rad, Hercules, CA). 20-HETE assay. Transfected HeLa cell homogenates (1 mg protein) were incubated in 1 ml of PSS containing (in mmol/l) 119 NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 NaH2PO4, 18 NaHCO3, 0.03 EDTA, 10 glucose, and 5 HEPES in the presence of 1 mmol/l NADPH and 40 ␮mol/l of arachidonic acid (AA) at 37°C for 30 min. The reactions were shaken under an atmosphere of 100% oxygen to maintain adequate PO2 in the incubation media (20, 22). The reactions were stopped by acidification to pH 3.5 with formic acid (1 mol/l), and the samples were extracted twice with 3 ml of ethyl acetate after the addition of 20 ng of an internal standard 20-HETE-d6 (Cayman Chemicals, Ann Arbor, MI). After centrifugation, the organic phase was dried under nitrogen. The samples were reconstituted with 50% methanol in water, and the metabolites of AA were measured using a Dionex (Sunnyvale, CA) HPLC system and an ABsciex 4000 Q trap tandem mass spectrometer with electrospray ionization (ABsciex, Foster, City, CA), as previously described (11, 61). Generation of CYP4A1 Transposon Transgenic Rats (SS.CYP4A1) in the SS Genetic Background Pronuclear injection. Full-length CYP4A1 or eGFP cDNAs in the transposon vector (3 ng/␮l) along with 10 ng/␮l of SB100 transposase mRNA in a buffer containing 5 mmol/l Tris/0.1 mmol/l EDTA, pH 7.4 were injected into the pronucleus of oocytes collected from female SS rats (17). The oocytes were incubated in EmbryoMax KSOM embryo culture medium and were changed to m-RECM2 rat 2-cell embryo culture medium with HEPE&PVA (EMD Millipore, Billerica, MA) after incubation at 37°C for 24 h. Two-cell oocytes were transferred into the oviducts of pseudo-pregnant foster SS females to generate CYP4A1 or eGFP transgenic founders. Genotyping. Tail biopsies were digested with 0.2 mg/ml proteinase K in direct PCR lysis reagents (102-T; Viagen Biotech, Los Angeles, CA) and incubated at 85°C with rotation for 45 min. The primers used to detect the CYP4A1 transgene were T2-4A1F1: 5=-TCG GGC GAT CAG ATC CAA AGG CC-3= and T2-4A1R1: 5=-GCC ATT GTG GCT GAA GGC ACA-3=, and the size of the resulting PCR product was 439 bp. The primers used to detect CAG promoter in the transgene were CAGF1: 5=-ACT GTA TCA CAA TTC CAG TGG G-3= and CAGR1: 5=-GGA AAG TCC CAT AAG GTC ATG T-3=, and the size of the product was 293 bp. Detection and mapping of the integration sites. Transposon insertion sites were detected using a ligation-mediated PCR (LM PCR) (9, 24) method, as summarized in Fig. 4. Tail DNA (1 ␮g) extracted using Genomic DNA extraction kit (Life Technologies) was digested with BfaI at 37°C overnight, following heat inactivation at 80°C for 20 min. A left side linker was prepared by annealing BfaI linker⫹ (5=-GTA ATA CGA CTC ACT ATA GGG CTC CGC TTA AGG GAC-3=) and BfaI linker ⫺ (5=-Phos-TAG TCC CTT AAG CGG AG-3= Phos) together in TE buffer and 5 M NaCl at a 1:1:2 ratio at 95°C for 5 min and then slowly cooled to room temperature. A ligation reaction was set up with 9 ␮l of BfaI-digested genomic DNA, 6 ␮l of the left side linker, and 1 ␮l of T4 ligase (Life Technologies) in a 20-␮l total volume and incubated at 14°C overnight. The outer PCR reaction was performed with 1 ␮l of the ligation reaction, 1 ␮l of each ITRL-long primer (100 ␮M; 5=-CTG GAA TTT TCC AAG CTG TTT AAA GGC ACA GTC AA-3=) and linker primer (100 ␮M; 5=-GTA ATA CGA CTC ACT ATA GGG C-3=) in a 25-␮l reaction by using 0.5 U of Taq DNA polymerase (Qiagen). The nested PCR was performed using 2 ␮l of 1:50 diluted outer PCR with 25 ␮M each of ITRL-nested (5=-GAC TTG TGT CAT GCA CAA AGT AGA TGT CC-3=) and linker-nested (5=-AGG GCT CCG CTT AAG GGA C-3=) primers in a 50-␮l reaction. The PCR products were purified and

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ligated into pCR4-TOPO sequencing vector, transformed into DH 5␣-T1 cells, and cultured on LB agar plate with antibiotics. A colony screening assay for insertions was performed by PCR using M13 primers to identify positive colonies and confirmed by sequencing. The sequenced products were then blasted through the rat genome database to identify the insertion site in the genome. Genotyping to confirm the insertion sites was performed using a tri-primer PCR strategy (TP-PCR), as shown in Fig. 5. An ITRLnested primer was used as the primer 3. Primer 1 (chromosome 3) consisted of 5=-GTC CTT GGT GTA GAT GGC TGT GTC-3= and the sequence of primer 2 (chromosome 3) was 5=-GAA CTG TTT ACT GAA CGG ACA AG-3=. The expected PCR product was 921 bp. Touchdown PCR program was performed: 95°C for 5 min, followed 5 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min, followed by a another 25 cycles using the same conditions of denaturing and extension steps but with an annealing temperature of 58°C. This was followed by another 10 cycles of PCR consisting of an annealing temperature of 56°C, and a 10-min extension at 72°C. Primer 1 used for the chromosome X insertion was 5=-GCT GTC AAT GAC ATG ATC CTT CC-3=, while primer 2 (chromosome X) was 5=-GAA TCT GGA TAC AGA GAT CTG TGG CTC-3=. The expected PCR product was 1,585 bp. The same touchdown PCR program was used except the annealing temperatures were 60°C, 55°C and 52°C, respectively. All of the PCR products obtained from the CYP4A1 insertion sites were verified by sequencing using TOPO TA cloning with M13 primer pair, as described earlier. Heterozygous founders were backcrossed to SS rats, and the progeny was brothersister mated to derive homozygous transgenic lines. A double transgenic CYP4A1 SS line homozygous for both of the insertions on chromosome 3 and the X chromosome was generated after several rounds of breeding and selection and was used for all of the experiments. Expression of CYP4A Protein and 20-HETE Production in SS.CYP4A1 Transgenic, SS-5BN, and Dahl SS Rats Preparation of renal microsomes. Nine-week-old male SS and CYP4A1 transgenic rats fed a 0.4% NaCl diet were euthanized and kidneys were collected. Microsomes were prepared from kidney, as previously described (2, 61). Briefly, 0.5 g of the renal tissue was homogenized in 3 ml of 10 mmol/l of potassium buffer (pH 7.7) containing 250 mmol/l sucrose, 1 mmol/l EDTA and 0.1 mmol/l PMSF. The homogenates were centrifuged at 11,000 g for 20 min. The supernatant was centrifuged at 100,000 g for 1 h to obtain the microsomal fraction. The pellets were resuspended in 100 mmol/l potassium buffer (pH 7.25) containing 30% glycerol, 1 mmol/l dithiothreitol, and 0.1 mmol/l PMSF, frozen in liquid nitrogen, and stored at ⫺80°C until assayed. Isolation of cerebral vessels. Cerebral vessels were isolated using the Evans blue sieving procedure, as previously described (8, 11). Briefly, the rats were anesthetized with isoflurane, and both carotid arteries were cannulated. The cerebral circulation was flushed with 10 ml of ice-cold Tyrode’s solution containing (in mmol/l): 145 NaCl, 5 KCl, 4.2 NaHCO3, 1 MgCl2, 0.05 CaCl2, 10 HEPES, and 10 glucose. Then, 5 ml of Tyrode’s solution containing 3.0% albumin stained with 1% Evans blue was injected to stain the cerebral circulation. The brain was quickly removed and pushed through a 150-␮m stainless-steel sieve with the barrel of a 30-ml glass syringe to separate the cerebral vasculature from brain parenchyma tissue. The tissue retained on the screen was repeatedly rinsed with ice-cold PSS solution. The retained vascular tissue on the top of the screen was collected, and small thick walled arterioles were microdissected from the bulk-isolated tissue using a stereomicroscope for measurement of 20-HETE production by LC/MS/MS. Western blot analysis. The expression of CYP4A protein was assessed in 50 ␮g of microsomal protein prepared from kidney of SS

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and CYP4A1 transgenic rats. Western blots were performed, as described above. 20-HETE assay. Two milligrams of freshly isolated cerebral vessels were incubated at 37°C in 2 ml PSS containing 1 mmol/l NADPH, 2 ␮mol/l indomethacin, and 40 mmol/l of AA for 1 h. 20-HETE activities were also measured in microsome (0.5 mg)prepared from kidneys of SS and CYP4A1 transgenic rats, as described above. Comparison of the Myogenic Response of MCA Isolated from SS-5BN Consomic, CYP4A1 Transgenic, and Wild-Type SS rats These experiments were performed using segments of the MCA isolated from 9 –12-wk-old rats that were killed with 4% isoflurane, as previously described (15, 41, 42). The brain was removed and placed in ice-cold PSS solution. MCAs with inner diameters ranging from 100 to 140 ␮m were dissected, mounted on glass micropipettes, and pressurized to 40 mmHg at 37°C. The vessels were bathed in PSS solution. Calcium-free PSS was prepared by replacing CaCl2 with equimolar concentration of MgCl2 and the addition of 2 mmol/l EGTA. The inflow pipette was connected to a reservoir to allow for control of intraluminal pressure that was monitored with a transducer (Cobe, Lakewood, CO). The cannulated MCAs were visualized using a videomicroscopy system consisting of a charge-coupled device camera and a stereomicroscope (model DRC; Zeiss, Oberkochen, Germany). The inner diameter of the vessels was measured using a videomicrometer (VIA-100; Boeckeler Instruments, Tucson, AZ). Magnification on the screen was approximately ⫻180, and the measurement system was calibrated with a micrometer to a diameter within ⫾2.0 ␮m. The bath solution was equilibrated with O2 (95%) and CO2 (5%) to provide adequate oxygenation and to maintain at pH 7.4. After mounting, vessels were allowed to equilibrate for 60 min and were preconditioned by pressurizing the vessel 3 times for 5 min at 5-min intervals by changing the intraluminal pressure from 40 to 140 mmHg. After preconditioning, the diameter was measured at intraluminal pressures from 40 to 140 mmHg in steps of 20 mmHg in the presence and absence of HET0016 (10 ␮mol/l). After this relationship was determined, the bath was replaced with calcium-free PSS, and the passive pressure-diameter relationship was determined. Autoregulation of CBF in SS-5BN Consomic, CYP4A1 Transgenic, and Wild-Type SS rats These experiments were performed on 9 –12-wk-old male SS-5BN consomic, CYP4A1 transgenic, and wild-type SS rats, as previously reported (41). The rats were anesthetized with ketamine (30 mg/kg im; Phoenix Pharmaceutical, St. Joseph, MO) and Inactin (50 mg/kg ip; Sigma) and placed in the supine position. A polyethylene (PE-240) cannula was placed into the trachea, and the animals were mechanically ventilated throughout the experiment to maintain PO2 and PCO2 at 100 and 35 Torr, respectively. Catheters were placed in the femoral artery for the measurement of arterial pressure and the femoral vein for an intravenous infusion of 0.9% NaCl solution at a rate of 100 ␮l/min to replace fluid losses. The rats were placed on a heating pad to maintain temperature at 37°C. The head of the rat was secured in a stereotaxic apparatus (Stoelting, Wood Dale, IL), and the scalp was removed over the parietal cranial bone. A 3 ⫻ 3 mm area of the left and right parietal bones, 2 mm distal to bregma and 7 to 8 mm from centerline was thinned using a handheld low-speed drill until superficial pial vessels became visible. CBF was monitored bilaterally with a laser-Doppler flowmeter (PF5001; Perimed, Jarfalla, Sweden) and 1-mm flow probes. The probes were lowered into position using micromanipulators, and a drop of mineral oil was applied to the probe tip to provide optical coupling. After surgery and a 30-min equilibration period, mean arterial pressure (MAP) was adjusted to 90 –100 mmHg by deepening the depth of anesthesia using a low dose of pentobarbital sodium (1–5 mg/kg iv). Baseline regional CBF was measured at this level of MAP, and pressure was then elevated in steps

of 10 –20 mmHg up to 190 mmHg by graded intravenous infusion of phenylephrine (0.5–5 ␮g/min) via the femoral vein. MAP was maintained for 3–5 min until a new steady-state level of CBF was obtained. CBF was expressed as a percentage of baseline laser-Doppler flow signal. Blood-Brain Barrier Permeability Fourteen to sixteen-week-old male SS-5BN consomic, CYP4A1 transgenic and wild-type SS rats were anesthetized with ketamine (30 mg/kg im; Phoenix Pharmaceutical) and Inactin (50 mg/kg ip; Sigma), and catheters were placed in the femoral artery and vein. Evans blue (EB) dye (3 ml/kg in 3% albumin saline) was intravenously infused, and MAP was maintained at 190 mmHg for 1 h by adjusting the infusion rate of phenylephrine to assess BBB leakage and to stain the vasculature. After this equilibration period, a 1-ml sample of blood was collected from the femoral artery, and the plasma was separated by centrifugation for 10 min at 4,000 rpm. Then, 100 ml of saline containing 10 U/ml heparin was rapidly infused via aorta to remove EB from the vascular space, and the brain was harvested. A picture of the brain was taken, and then the brain was homogenized in 1 ml of PSS solution. The homogenate was centrifuged at 10,000 rpm for 20 min, and a sample of the supernatant and plasma obtained earlier was diluted 40 and 4,000 times, respectively, with water and was loaded onto 96-well plates. Fluorescence was measured using a fluorescent microplate reader (BioTek, Winooski, VT) at excitation and emission wavelengths of 620 nm and 680 nm. Drugs HET0016 (Enzo Scientific) and AA (Sigma) were prepared in ethanol at concentrations of 10 –20 mmol/l. The drugs were used in 1:500 to 1:1,000 dilutions in PSS or assay buffer to the final concentrations used in the various experiments. Statistics Values are presented as means ⫾ SE. The significance of the differences in mean values between and within groups in the myogenic responses and autoregulation of CBF was analyzed using a two-way ANOVA for repeated-measures and Holm-Sidak test for preplanned comparisons. A P value ⬍0.05 was considered statistically significant. RESULTS

Generation and Verification of Full-Length Rat CYP4A1 Transposon Plasmid We generated the full-length CYP4A1 cDNA and subcloned it into a transposon vector under the control of a CMV early enhancer/chicken ␤-actin (CAG) promoter, which has been previously reported to drive high levels of transgene expression in the kidney and vasculature in a variety of transgenic animal models (32, 37) (Fig. 1A). The proper orientation of the insertion was confirmed by digestion with PstI and by PCR using primers F1/R1 and F2/R2. The PstI digested plasmid fragments were 5,994 and 540 bp, which were consistent with the CYP4A1 insert in the plasmid with correct orientation. The PCR products that were obtained also had the expected sizes of 325 and 292 bp using the F1/R1 and F2/R3 primers, respectively. A plasmid-based excision assay was performed (Fig. 1B) to verify the “cut-and-paste” ability and efficiency of SB transposition (30, 44) of our newly generated CYP4A1 transposon plasmid. SB transposons contain two “repeats” within each inverted terminal repeat (ITRs; IR-DR). The optimization in

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Fig. 2. Verification of functional CYP4A1 protein in transposon vector. A: Western blot to detect the expression of CYP4A protein. Samples in lanes 4 – 6 are lysate proteins from HeLa cells 48 h posttransfection with three different colonies of newly generated CYP4A1 transposon vector. Lane 1 shows an untreated HeLa cell lysate, lane 2 shows HeLa cells transfected with a pT2.CAG.eGFP vector, and lane 3 is transfected with a CYP4A1 in a lentiviral expression vector driven by the same CAG promoter. B: 20-HETE assay. Cells transfected with pT2.CAG.CYP4A1 clone 5 were picked to measure 20-HETE production after incubation with 40 ␮M AA in the presence of 1 mM NADPH. The production of 20-HETE was much higher in cells transfected with the pT2.CAG.CYP4A1 vector or a Lenti-CYP4A1 construct used as a positive control than in untransfected cells or those transfected with an eGFP construct.

specific base pairs within the DR sequences (T2), which has consensus inner (Li and Ri) and outer (Lo and Ro) enhances the efficiency of SB transposons (5, 66) comparing with original T transposon (23). We obtained the expected size of the excised bands in our newly generated pT2.CAG.CYP4A1 and eGFP transposon-positive control, but not in embryos injected with the SB11 transposase plasmid alone that served

as our negative control (data not shown). These results indicate that our newly constructed rat CYP4A1 transposon vector has the expected transposon excision capability. The ability of the vector to express functional CYP4A protein was tested following transient transfection of HeLa cells with the CYP4A1 transposon plasmid. The expression of CYP4A protein in untreated HeLa cells was very low and was markedly enhanced in cells transfected with the pT2.CAG.CYP4A1 plasmid. A CYP4A1 construct in a lentiviral expression vector driven by the same CAG promoter that we have previously described (32) was used as a positive control. A 27-kDa eGFP band was detected only in cells transfected with the pT2.CAGeGFP control plasmid, and ␤-actin was detected in all samples (Fig. 2A). Clone 5 was chosen to measure 20-HETE production vs. that seen in control HeLa cells and cells transfected with the eGFP and CYP4A1 lentiviral vectors. The results presented in Fig. 2B indicate that the production of 20-HETE was markedly elevated in cells transfected with the CYP4A1 transposon construct relative to untransfected or eGFP-transfected HeLa cells, and the levels were similar to those seen in cells transfected with the CYP4A1 lentiviral vector. Generation and Verification of CYP4A1 Transgenic SS Rats The flow scheme for the generation of CYP4A1 transposon transgenic rats is presented in Fig. 3A. The expression of the CYP4A1 transgene and CAG promoter was detected in the transgenic-positive rats but not in wild-type SS rats since one arm of the CYP4A1 primers was designed to hybridize with the portion of the sequence in transposon vector (Fig. 3B). The integration sites were identified using LM PCR (9, 24), as outlined in Fig. 4. Two integration sites were identified in our transgenic founders. One is intergenic between hypothetical protein LOC367734 and Dynlt3 on chromosome X. Another is in intron 18 of the PDE11a4 gene on chromosome 3. Each integration site was verified by TP-PCR (Fig. 5). The expression of PDE11a at mRNA and protein level was similar in Dahl SS and SS.CYP4A1 rats (data not shown). Primers 1 and 2 were designed on the flanks of the mapped insertion sites on

Fig. 3. Strategy for generation of CYP4A1 transposon transgenic rats in SS genetic background. A: flow scheme. Fertilized donor embryos from female SS rats were collected and the CYP4A1 transposon plasmid and along with SB 100 transposase mRNA, they were microinjected into the pronucleus of fertilized one-cell embryos. These embryos were incubated to two-cell stage and then transferred back into a foster mother. The founders were screened for transgenic positive rats using PCR. B: PCR genotyping. CYP4A1 transgene and CAG promoter were both detected in transgenic positive rats, whereas it was not in wild-type SS rats or no template water control.

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Fig. 4. Strategy for detection and mapping of the insertion sites using ligation-mediated PCR (LMPCR). Step 1: Digestion of genomic DNA isolated from CYP4A1 transgenic homozygous rats with BfaI. Step 2: Left side linker was prepared by annealing BfaI linker⫹ and BfaI linker⫺, and a ligation reaction was performed to create an adaptor at the left of pT2.CAG.CYP4A1 transposon integration site. Step 3: The outer PCR was performed by using ITRL-long primer and linker primer. Step 4: The nested PCR was using ITRL nested and ITRR nested primers. Step 5: The nested PCR products were ligated into pCR4-TOPO sequencing vector. Step 6: M13 forward and reverse primers were used to sequence the nested PCR products. Step 7: The sequencing products were then blasted through rat genome database.

chromosome 3 and X, respectively. In wild-type rats, the PCR products obtained with these two primers were expected to be ⬃1 kb as shown in Fig. 5, top, left, while in transgenic-positive rats with ⬃3-kb transposon elements inserted, the PCR products were expected to be ⬃4 kb (Fig. 5 top, right). ITRL-nested

primer was used as primer 3. The PCR products using primers 2 and 3 were expected to be ⬃500 bp. A single ⬃500-bp band was amplified in transgenic homozygous rats using primers 1, 2, and 3 with a short extension time (ⱕ2 min), while a single ⬃1-kb band was detected in wild-type rats, and both bands

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Fig. 5. Confirmation of genotyping at each insertion site using a Tri-Primer PCR (TPPCR) strategy. Primers 1 and 2 were designed around the mapped insertion sites on chromosome 3 and the X chromosome, respectively. A single ⬃500-bp band was amplified in transgenic homozygous rats using primers 1, 2, and 3 with a short extension time (ⱕ2 min), while a single ⬃1-kb band was detected in wild-type rats, and both bands were detected in heterozygous rats. Primer 1 (chromosome 3) consisted of 5=-GTC CTT GGT GTA GAT GGC TGT GTC- 3=, and the sequence of primer 2 (chromosome 3) was 5=-GAA CTG TTT ACT GAA CGG ACA AG-3=. Primer 1 used for the chromosome X insertion was 5=-GCT GTC AAT GAC ATG ATC CTT CC-3=, while primer 2 (chromosome X) was 5=-GAA TCT GGA TAC AGA GAT CTG TGG CTC-3=.

were detected in heterozygous rats. Heterozygous founders were backcrossed to SS rats to generate a homogeneous genetic background and the progeny was brother-sister mated to derive homozygous-transgenic lines. A double homozygous-transgenic CYP4A1 rat strain was generated after several generations. The expression of CYP4A1 protein was eight-fold higher (Fig. 6A), and the formation of 20-HETE was 4-fold higher (Fig. 6B) in the kidney of the CYP4A1 transgenic animals than in SS rats. Moreover, 20-HETE production was six-fold higher in cerebral vessels isolated from CYP4A1 transgenic and SS-5BN animals than the levels seen in SS rats (Fig. 7). Myogenic Responses in Isolated Perfused MCA The luminal diameter of the MCA decreased to 70.2 ⫾ 3% and 64.5 ⫾ 6% in CYP4A1 transgenic and SS-5BN when pressure was increased from 40 to 140 mmHg. In contrast, the myogenic response of the MCA isolated from SS rats was markedly impaired, and these vessels did not constrict when pressure was elevated over this range. Administration of the 20-HETE synthesis inhibitor HET0016 (10 ␮mol/l) (33) abolished the pressure-induced constriction of the MCA in

CYP4A1 transgenic and SS-5BN rats, but it had no effect on the myogenic response in SS rats (Fig. 8A). The passive diameter curves generated in Ca2⫹ free solution were not significantly different between the various strains (Fig. 8B). Autoregulation of CBF Baseline MAP was similar in 9 –12-wk-old male SS-5BN consomic, CYP4A1 transgenic and wild-type SS rats. MAP was significantly higher by about 10 mmHg in SS rats fed a low-salt diet than in normotensive SD rats that were used as a control strain with normal myogenic response (Fig. 9A). The relationships between MAP and CBF are presented in Fig. 9B. CBF increased by 49.1 ⫾ 6%, as MAP was increased by 60% from 100 to 160 mmHg and by 107.9 ⫾ 7% when MAP was increased to 190 mmHg in SS rats. In contrast, CBF was better autoregulated in SS-5BN strain and increased by 25.9 ⫾ 5% and 65.6 ⫾ 7% when MAP was increased over the same range. Similarly, CBF was increased by only 18.6 ⫾ 2% and 36.3 ⫾ 1% when MAP was increased from 100 to 160 and then to 190 mmHg in CYP4A transgenic SS rats and is similar to the response seen in the MCA isolated from SD rats (25.0 ⫾ 5%, 43.5 ⫾ 6%). Fig. 6. Comparison of the expression of CYP4A protein and the production of 20HETE in the kidney of SS and CYP4A1 transgenic rats. A: typical picture of Western blotting detecting CYP4A1 protein expression. Lanes 1 through 4 were loaded with microsomes (50 ␮g) prepared from the kidney of male SS rats, while lanes 5 through 9 were loaded with microsomes from CYP4A1 transgenic rats. ␤-actin was used as a loading control. The expression of CYP4A1 protein is eight-fold higher in the kidney in the transgenic animals than wildtype SS rats (A, bottom). B: formation of 20-HETE in microsomes prepared from the kidney of SS and CYP4A1 transgenic rats. The formation of 20-HETE is four-fold higher in the kidney of the transgenic animals. Mean values ⫾ SE are presented. Numbers in the bars indicate the number of animals studied in each group. *Significantly different from the corresponding value in SS rats.

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Fig. 7. Comparison of the production of 20-HETE in cerebral vessels isolated from SS, SS-5BN, and CYP4A1 transgenic SS rats. 20-HETE production was six-fold higher in cerebral vessels (0.60 ⫾ 0.1 vs. 0.12 ⫾ 0.03 pmol·mg⫺1·min⫺1) isolated from CYP4A1 transgenic than from SS rats. The production was similar to the levels seen in SS-5BN consomic rats. Mean values ⫾ SE are presented. Numbers in the bars indicate the number of animals studied in each group. *Significant difference from the corresponding value in SS rats.

a CMV early enhancer/chicken ␤ actin (CAG) promoter, which has ubiquitous high efficiency to drive transgene expression in transgenic animal models (32, 37) generated CYP4A1 transposon transgenic rats. We found that the expression of CYP4A protein was eight-fold, and the production of 20-HETE was four-fold higher in the liver (data not shown) and the kidney of CYP4A1-transgenic compared with SS rats. More importantly, the production of 20-HETE in cerebral vasculature was sixfold higher in CYP4A1 transgenic animals than in SS rats, while the levels were similar to those seen in SS-5BN consomic rats. The functional significance of the impaired production of 20-HETE in cerebral vessels of SS rats was then determined by studying the myogenic response of the MCA and autoregulation of CBF. We found that the myogenic response of MCA of SS rats was markedly impaired, and the luminal diameter of the MCA remained unaltered in SS rats, when the perfusion pressure was increased from 60 to 140 mmHg. In contrast, the

Blood-Brain Barrier Permeability Baseline MAP of the older 14 –16-wk-old Dahl SS rats in this study averaged 163 ⫾ 10 mmHg and was not significantly different than the levels measured in SS-5BN (151 ⫾ 8 mmHg) or SS.CYP4A1 rats (155 ⫾ 10 mmHg). Blood-brain barrier (BBB) leakage was clearly observed in the neocortex and hippocampus of Dahl SS rats and was less in the brain of SS-5BN and SS.CYP4A1 rats (Fig. 10A). EB concentration was about 5-fold higher in the brain of Dahl SS rats compared with the levels seen in SS-5BN and SS.CYP4A1 rats, respectively (Fig. 10B). DISCUSSION

20-HETE plays an important role in the regulation of renal function and vascular tone (2, 10, 48). It is a potent vasoconstrictor that blocks activation of KCa channel and facilitates calcium entry through the activation of PKC, tyrosine kinase, and/or the Rho kinase pathways (11, 45, 48, 55). The renal formation of 20-HETE has been reported to be reduced in SS rats, and this contributes to sodium retention and the development of hypertension when these rats are challenged with a high-salt diet (61, 62). However, it remains to be determined whether there is also a deficiency of 20-HETE production in the vasculature that impairs the myogenic response, especially in the cerebral circulation that might promote the development of vascular remodeling, stroke, and cerebral injury in SS rats. The present study examined the production of 20-HETE, the myogenic response of isolated perfused MCA, and autoregulation of CBF in SS and SS-5BN consomic rats, in which chromosome 5 from BN was transferred into the SS genetic background. We also characterized the formation of 20-HETE and cerebral vascular function in an unique strain of CYP4A1 transgenic SS rat that we newly generated using a Sleeping Beauty transposon system to determine whether the expression of CYP4A1 and the formation of 20-HETE in the cerebral vasculature was elevated and if this could rescue the impaired myogenic response and autoregulation of CBF seen in SS rats. Using an approach that Dr. Aron M. Geurts has recently described to generate transgenic rats (17), we cloned a fulllength, wild-type CYP4A1 into a transposon vector containing

Fig. 8. Comparison of the myogenic response in middle cerebral artery (MCA) isolated from SS, SS-5BN, and CYP4A1 transgenic rats. A: luminal diameter of the MCAs decreased from 100 to 70.2 ⫾ 3% in CYP4A1 transgenic rats and from 100 to 64.5 ⫾ 6% in SS-5BN rats when the perfusion pressure was increased from 40 to 140 mmHg, whereas it was not significantly altered in SS rats (from 100 to 93.1 ⫾ 5%). Administration of an inhibitor of the synthesis of 20-HETE, HET0016 (10 ␮mol/l), inhibited the myogenic response in CYP4A1, and SS-5BN rats. HET0016 had little effect on MCAs in SS rats (100 to 96.4 ⫾ 3%). B: passive pressure-diameter curves in Ca2⫹-free solution at each pressure in all strains. Values are presented as means ⫾ SE. Numbers in parentheses indicate the number of vessels studied per group. *Significant difference (P ⬍ 0.05) from the corresponding value in SS rats. #Significant difference (P ⬍ 0.05) from the corresponding values in HET0016-treated and -untreated rats.

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Fig. 9. Autoregulation of cerebral blood flow (CBF). A: baseline mean arterial pressure (MAP) of 9 –12-wk-old male SS-5BN consomic, CYP4A1 transgenic, wild-type SS and SD rats maintained on a low-salt diet containing 0.3% NaCl from weaning. B: relationship between MAP and CBF in these strains. SD served as the wild-type control strain. Values are presented as means ⫾ SE. *Significant difference (P ⬍ 0.05) from the corresponding value in SS rats. #Significant difference (P ⬍ 0.05) from the control value at 100 mmHg within a strain. Numbers in parentheses indicate numbers of animal studies per strain.

myogenic response of the MCA of CYP4A1 transgenic and SS-5BN rats were intact, and these vessels constricted by about 35% when pressure was increased over this range. Administration of HET0016, a potent synthesis inhibitor of 20-HETE, eliminated the pressure-induced vasoconstriction in CYP4A1 transgenic and SS-5BN rats, but it had no significant effect in SS rats. Autoregulation of CBF as measured by laser-Doppler flowmetry was also impaired in SS rats. CBF increased by 49.1 ⫾ 6% when pressure was increased from 100 to 160 mmHg in SS rats vs. only 18.6 ⫾ 2% and 25.9 ⫾ 5% in CYP4A1 transgenic and SS-5BN rats, respectively. These studies are the first to document that the myogenic response and autoregulation of CBF is impaired in SS rats prior to the development of hypertension and that it is associated with a genetic deficiency in the formation of 20-HETE in the cerebral vasculature. The present results are consistent with previous reports that the myogenic response of cerebral arteries and autoregulation of CBF is impaired following the development of hypertension in Dahl SS rats fed a high-salt diet, and this contributes to BBB leakage (43, 54). This pathway may play a more generalized role in cerebral vascular dysfunction since Toth et al. (58) recently reported that downregulation of the formation of 20-HETE plays an important role in the impaired autoregulation of CBF in aging mice subjected to ANG II hypertension.

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Overall, the present findings that endogenously formed 20HETE plays a critical role in autoregulation of CBF are consistent with previous reports from our laboratory and the others that 1) elevations in transmural pressure increase the synthesis and release of 20-HETE in cerebral arteries, 2) 20-HETE inhibitors block the pressure-induced vasoconstriction of rat MCA in vitro, and 3) attenuates autoregulation of CBF in vivo (15, 18). The mechanism by which 20-HETE enhances the myogenic response in cerebral arteries remains to be fully determined. It activates PKC (27, 36), mitogen-activated protein kinases (MAPK) (34), tyrosine kinase (55), and the Rho kinase pathway (45) to promote Ca2⫹ entry through depolarization of vascular smooth muscle cells secondary to blockade of the large-conductance, calcium-sensitive K⫹ (BK) channel (20, 27). 20-HETE also increases the conductance of L-type Ca2⫹ channels through activation of PKC (16). More recently, 20HETE has been reported to enhance the activation of inward nonselective cation currents through transient receptor potential canonical 6 (TRPC6) channels (3), which are implicated in the myogenic response (4). Moreover, Toth et al. (57) reported that 20-HETE contributes to human and rat cerebral arteries constriction via TP receptors and involves enhanced production of ROS, COX activity. On the other hand, the present findings indicating that a deficiency in the formation of 20-HETE contributes to an impaired myogenic response in SS relative to SS-5BN and SD rats differ with a recent report by Lukaszewicz et al. (28, 29) that the expression of CYP4A protein is higher in SS than in SS-5BN rats (28, 29). These authors suggested that upregulation of the production of 20-HETE following exposure to a highsalt diet impairs endothelial function and the vasodilatory response to ACh in the cerebral circulation of SS rats. The reason for the differences remains to be determined. It may have something to do with the diet, as the rats in the previous studies were fed low- and high-salt AIN76 diets, whereas the rats in the present study were fed a grain-based diet containing 0.4% NaCl. It should also be noted that the previous study did not measure vascular 20-HETE levels but only inferred that there may be differences based on the expression of CYP4A protein and the response to a 20-HETE inhibitor. Finally, it should be noted that the antibodies used in the two studies are different. We used a well-documented CYP4A antibody (299230; Daiichi Pure Chemicals, Tokyo, Japan) known to cross react with the CYP4A1, CYP4A2, CYP4A3, and CYP4A8 that all produce 20-HETE, whereas the previous study used a mouse monoclonal antibody (sc-53247; Santa Cruz Biotechnology, Santa Cruz, CA) specifically raised against rat liver CYP4A2. The later observation may be important because a previous study by Dunn et al. (8) indicated that CYP4A3 rather than CYP4A2 isoform may be the primary isoform expressed and responsible for the formation of 20HETE in the middle cerebral artery of rats. Autoregulation of CBF is one of the major mechanisms to protect the brain from elevations in perfusion pressure that promote vascular leakage and swelling of the brain (18, 19). Chronic hypertension promotes alterations in the cerebral vasculature, including enhanced formation of atherosclerotic plaques (6), inward vascular remodeling and stiffening, vascular leakage, and fibrinoid necrosis of penetrating arteries and arterioles supplying the white matter; it also causes lacunar

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Fig. 10. Blood-brain barrier permeability. A: representative images of the extravasation of Evans blue in the brains of Dahl SS vs. SS-5BN and SS.CYP4A1 rats after mean blood pressure (MAP) was elevated to 190 mmHg for 1 h. B: tissue concentration of Evans blue measured in the brains of these strains. Values are presented as means ⫾ SE. *Significantly different (P ⬍ 0.05) from the corresponding value in SS rats.

infarcts and diffuse white matter damage (12, 19, 26). Impaired autoregulation of CBF in hypertensive patients has been suggested to accelerate these changes and promote the development of a cognitive decline (12, 26). Amyloid plaques and neurofibrillary tangles are also increased in the brain of patients with a history of hypertension, suggesting that hypertension may contribute to the development of vascular dementia and Alzheimer’s disease (12, 19, 53). Hypertension is also the most powerful risk factor for stroke (35, 59). Disruption of BBB occurs in many pathological conditions and leads to ischemic foci and triggers the inflammation that promotes glial activation and neurodegeneration (53, 56). Our finding that there is far more BBB leakage in the neocortex and hippocampus, which are essential for the control of learning and memory, in Dahl SS rats compared with SS-5BN and SS CYP4A1 rats suggests that the deficiency of 20-HETE in cerebral arterioles may contribute to the development of vascular cognitive impairments in Dahl SS rats with aging and following the development of hypertension.

Perspectives and Significance Several genome-wide association studies have linked the sequence variants of human CYP4A11, the homologs of rat CYP4A1 and CYP4A2, to the development of hypertension, stroke, and cerebral vascular disorders in human population studies (7, 10, 13, 14, 63), but the mechanisms remain to be determined. The present finding that the myogenic response in the cerebral circulation and autoregulation of CBF is impaired in SS rats due to a genetic deficiency in the formation of 20-HETE and is restored in our new CYP4A1 transgenic SS rats now fills the knowledge gap and provides investigators an important new model to study the mechanism by which genetic defects in the myogenic response can contribute to the development of small vessel disease and brain injury. 20-HETE has also been reported to play a critical role in angiogenesis (65), restenosis (40), acute renal injury (10, 46, 49), chronic kidney disease (10), cancer (1), hypertension (10, 48, 63), and ischemic and hemorrhagic stroke (8, 39, 47). The availability of the Dahl SS rat model deficient in the formation

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of 20-HETE and along with our new rescued CYP4A1 transgenic SS rat model also provides investigators with a unique opportunity to study the mechanisms by which 20-HETE contributes to the pathophysiology of these diseases.

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ACKNOWLEDGMENTS

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We thank Dr. Rodney Baker and Mrs. Chris Purser in HPLC/Mass Spectrometry Analytical Core in the Department of Pharmacology and Toxicology at the University of Mississippi Medical Center for their supports to analysis of 20-HETE production.

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GRANTS This work was funded in part by National Institutes of Health (NIH) R01-HL36279 and DK-104184 (to R. J. Roman), GO grant HL-101681 (to H. Jacob), new innovator award OD-8396 (to A. Geurts) and NIH 1 PO1-GM-104357 (Core B, C); postdoctoral fellowship 11POST7520052 (to S. Murphy), scientist development grants 14SDG20160020 (to S. Murphy) and 13SDG14000006 (to M. Pabbidi) from the American Heart Association. DISCLOSURES

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No conflicts of interest, financial or otherwise, are declared by the authors.

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AUTHOR CONTRIBUTIONS Author contributions: F.F., A.M.G., H.J.J., and R.J.R. conception and design of research; F.F., A.M.G., S.R.M., M.R.P., and R.J.R. performed experiments; F.F., A.M.G., S.R.M., M.R.P., and R.J.R. analyzed data; F.F., A.M.G., S.R.M., and R.J.R. interpreted results of experiments; F.F. and R.J.R. prepared figures; F.F. and R.J.R. drafted manuscript; F.F. and R.J.R. edited and revised manuscript; F.F. and R.J.R. approved final version of manuscript.

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