Published online before print October 2, 2008, doi: 10.1161/ATVBAHA.108.177113
© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Hypercholesterolemia Enhances 15-Lipoxygenase–Mediated Vasorelaxation and Acetylcholine-Induced Hypotension
From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee.
Correspondence to William B. Campbell, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail wbcamp{at}mcw.edu
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Abstract |
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Objective— Arachidonic acid (AA) metabolites from 15-lipoxygenase-1 (15-LO-1), trihydroxyeicosatrienoic acid (THETA), and hydroxyepoxyeicosatrienoic acid (HEETA) relax arteries. We studied 15-LO-1 expression, THETA and HEETA synthesis, and their effect on arterial relaxations and blood pressure in hypercholesterolemic nonatherosclerotic rabbits.
Methods and Results— Immunoblots, RTPCR analysis, and 14C-AA metabolism revealed that hypercholesterolemia increased 15-LO-1 expression in the endothelium and THETA and HEETA synthesis in the arteries. Isometric tension recording, in presence of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors, showed greater relaxations to acetylcholine (ACH) and AA (max 76.0±4.6% and 79.5±2.4%, respectively) in aortas from hypercholesterolemic rabbits compared with normal rabbits (max 39.1±2.8% and 39.9±2.2%, respectively). AA induced greater hyperpolarization in the smooth muscle cells of hypercholesterolemic aortas (–45.85±3.0 mV) compared with normal aortas (–31.45±1.9 mV). The ACH- and AA-relaxations were inhibited by 15-LO-1 inhibitors. ACH induced hypotensive responses were greater in hypercholesterolemic rabbits in absence (–54.9±3.3%) or presence (–48.5±3.2%) of NOS and COX-inhibitors compared with control rabbits (–31.6±3.3% and –24.3±1.6%, respectively). BW755C reduced these responses in hypercholesterolemic rabbits to –29.3±2.3%.
Conclusion— Hypercholesterolemia increases endothelial 15-LO-1 expression, THETA and HEETA synthesis and enhances vasorelaxation.
Key Words: hypercholesterolemia • 15-lipoxygenase • endothelium-derived hyperpolarizing factors • blood pressure • arachidonic acid
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Introduction |
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In presence of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibitors, agonist-induced relaxations are attributed to endothelium-derived hyperpolarizing factors (EDHFs) that regulate vascular tone.1 Arachidonic acid (AA) metabolites from 15-lipoxygenase-1 (15-LO-1), 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA), and 11,12,15-trihydroxyeicosatrienoic acid (THETA) are EDHFs in rabbit arteries.2–4 Other LO metabolites of AA, 15-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 15-hydroperoxyeicosatetraenoic acid (HpETE), are inactive in rabbit arteries.5 15-LO-1 is important and sufficient to increase ACH- or AA-relaxations in rabbit arteries.6,7 15-LO-1 expression increases in early atherosclerotic lesions in rabbit aorta.8 However, progression of atherosclerosis begins with increased total plasma cholesterol levels. Hypercholesterolemia without appearance of any fatty streak lesion in the arteries alter gene expression9 including intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and P-selectin. Therefore, we hypothesized that hypercholesterolemia without atherosclerosis would effect 15-LO-1 expression. In the aortas from rabbits that were fed a 2% cholesterol diet for 2 weeks, ACH-relaxations sensitive to a LO inhibitor nordihydroguaiaretic acid (NDGA), and synthesis of AA-metabolites increased in the arteries.10 NO- and prostaglandin (PG)-independent ACH-relaxations were also greater in the renal arteries from hypercholesterolemic rabbits.11 ACH-induced relaxations in the rabbit arteries are mediated by THETA and HEETA that open small conductance calcium-dependent potassium (SKCa) channels and K+ that is released from intermediate conductance, calcium-dependent potassium (IKCa) channels.4
Therefore, the present study was designed to determine the effect of hypercholesterolemia on the expression of 15-LO-1, synthesis of THETA and HEETA, SKCa or IKCa channel–mediated ACH relaxations, AA-induced relaxations, and contribution of THETA and HEETA to these relaxations. We also measured mean arterial pressure (MAP), heart rate (HR), and ACH-induced fall in MAP in vivo.
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Methods |
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Rabbits, Arterial Tissue, and Total Plasma Cholesterol Levels
Animal protocols were approved by the institutional animal care and use committee of the Medical College of Wisconsin, and procedures were performed in accordance with the National institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Six-week-old male New Zealand White rabbits (Kuiper Rabbit Ranch, Ind) were either maintained on normal rabbit chow or were given a cholesterol enriched rabbit chow for a period of 2 weeks. The cholesterol enriched diet consisted of 2% cholesterol in cholesterol-free vegetable oil mixed with normal chow.10 After 2 weeks rabbits were either used for the in vivo experiments or euthanized with a pentobarbital overdose. Various arteries were removed and maintained at 4°C in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (mM): 10 HEPES, 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 6 glucose, pH 7.4. Two ml blood was drawn through an intracardiac injection into a tube containing 0.5% heparin (Sigma). Heparinized blood was centrifuged to separate plasma and stored. Total cholesterol level in the plasma was measured by a cholesterol kit (Sigma Diagnostic).
Immunoblotting, Immunohistochemistry, Metabolism of 14C-AA, Measurement of Membrane Potential, and Isometric Tension
The methods for immunoblotting, immunohistochemistry, 14C-AA metabolism studies, measurement of membrane potential and isometric tension recording were carried out as previously published.3,6 For details, see the methods section of the online supplement at http://atvb.ahajournals.org.
Quantitative RT-PCR
Total nucleotides were isolated from rabbit arteries using Trizol reagent, cDNA was synthesized from total RNA and amplified for 15-LO-1 and GAPDH in 25x10–6 L reaction mixtures using BIO-Rad iCycler. The reaction mixture contained 2x10–7 g cDNA, 2x10–7 mol/L primers and RT2 SYBR Green/Fluorescein qPCR Master Mix (Super Array Bioscience Technologies) and the program for the iCycler was 94°C for 30 s, 58°C for 1 minute, and 72°C for 1.5 minutes, repeated 40 times followed by final extension at 72°C for 7 minutes. The amplified PCR products were separated by 1% agarose gel electrophoresis and visualized. For details, see the methods section of the online supplement at http://atvb.ahajournals.org.
Mean Arterial Pressure (MAP) and Measurement of ACH Responses
Age matched (8-week-old) hypercholesterolemic or normal rabbits were anesthetized with 20 mg/kg pentobarbital intravenously (IV), mechanically ventilated via an endotracheal cannula, and MAP was measured through the right carotid artery as previously published.6 Three groups of rabbits were treated with vehicle (0.9% wt/vol NaCl), INDO (6 mg/kg, IP) and L-nitroarginine methyl ester (LNAME; 20 mg/kg IV and 5 mg/kg/h, IV), or INDO, LNAME, and BW755C (20 mg/kg) IV, stabilized for 45 minutes and change in MAP and HR to ACH doses (0.4 to 4000 ng/kg, IV) were recorded. For details, see the methods section of the online supplement at http://atvb.ahajournals.org.
Statistical Analysis
The experimental data were expressed as means±SEM. Student t test was performed to compare immunoblots, metabolite synthesis, and membrane potential. A repeated measure 2-way ANOVA followed by Bonferronni post test was performed to analyze responses to each ACH dose in vitro or in vivo and effect of inhibitors on these responses. Values were considered significant at P<0.05 or smaller.
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Results |
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Arterial Histology and Cholesterol Concentrations
Gross examination of the lumen did not reveal fatty streaks in the mesenteric and femoral arteries or aortas from hypercholesterolemic rabbits. Total plasma cholesterol increased from 53±6 mg/dL in normal rabbits to 965±253 mg/dL in the hypercholesterolemic rabbits.
15-LO-1 Protein and mRNA Expression
Expression of 15-LO-1 was analyzed by Western blots in arteries from normal and hypercholesterolemic rabbits using an antirabbit 15-LO-1 antibody (Figure 1A). Band density of a 75-kDa protein was increased in the arterial lysates from hypercholesterolemic rabbits compared with normal rabbits indicating an increase in 15-LO-1 expression. 15-LO-1 band densities, normalized to β-actin, were increased in thoracic aorta, abdominal aorta, and renal and carotid arteries from cholesterol-fed rabbits compared with the aortas or arteries from normal rabbits (Figure 1B).
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15-LO-1 mRNA was quantified in the arteries from hypercholesterolemic or normal rabbits by qRT-PCR. Cycle threshold (Ct) of 15-LO-1 cDNA was normalized to the Ct of GAPDH in the same sample to obtain 
Ct. 15-LO-1 mRNA expression increased with cholesterol feeding in all the arteries with the greatest increase in aortas (Figure 1C). Separation of the amplified products from the qRTPCR reactions revealed a single band for 15-LO-1 (326 bp) or GAPDH (170 bp), whereas no band was observed in absence of reverse transcriptase or cDNA in the reaction mixture (Figure 1D).
15-LO-1 Expression by Immunohistochemistry
Immunoflourescence was performed to determine the expression pattern of 15-LO-1 in thoracic aorta and mesenteric arteries (supplemental Figure I). Fluorescence signal from arteries treated with vehicle or only secondary antibodies was negligible (panel A, E, I, and M). Presence of intact endothelium was confirmed by staining with the endothelial marker protein PECAM (panels B, F, J, and N). Fluorescence signal for 15-LO-1 was weaker in the endothelium of normal rabbits aortas (panel C) compared with the signal in the endothelium of hypercholesterolemic rabbits aortas (panels G and C). Similarly, the fluorescence signal for 15-LO-1, limited to the endothelium, in the mesenteric arteries of hypercholesterolemic rabbits was greater than normal arteries (panels K and O). When stained for 15-LO-1, no inflammatory cells were observed in the vessel wall of the arteries or aortas from either normal or hypercholesterolemic rabbits. Adjacent layers of cells were shown by nuclear staining with DAPI (panel D, H, L, and P) did not express 15-LO-1.
Metabolism of 14C-AA
Enzymatic activity of the 15-LO-1 in aorta of normal or hypercholesterolemic rabbits was determined by analyzing 14C-AA metabolism (Figure 2). In the presence of INDO, aortas from normal (Figure 2A) and hypercholesterolemic (Figure 2B) rabbits metabolized 14C-AA to 14C-THETA, 14C-HEETA, and 14C-15-HETE. Synthesis of 14C-THETA increased 4.7-fold, 14C-HEETA increased 3.18-fold, and 14C-HETEs increased 3.7-fold in aortas from hypercholesterolemic rabbits (Figure 2D). The metabolism of 14C-AA to the products was reduced in aortas from hypercholesterolemic rabbits preincubated with INDO and the LO inhibitor, BW755C (Figure 2C). In the absence of the endothelium, the metabolism of 14C-AA was abolished in aorta from both hypercholesterolemic or normal rabbits (data not shown).
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ACH- or AA-Induced Relaxations
Relaxations to ACH were greater in aortas from hypercholesterolemic (EC50=1.2x10–8 M) rabbits than normal (EC50=8x10–8 mol/L) rabbits (supplemental Figure II). PG and NO-independent relaxations to ACH or AA were also measured in aortas from hypercholesterolemic and normal rabbits. L-Nitroarginine (LNA; 3x10–5 M or 3x10–4 mol/L) inhibited ACH relaxations in the rabbit aortas to the same extent (supplemental Figure IIB). Thus 3x10–5 mol/L LNA was used in these studies. ACH and AA caused concentration-dependent relaxation in the aortas (Figure 3). In the presence of INDO and LNA, aortas from hypercholesterolemic rabbits had greater ACH-relaxations (max 76.0±4.6%) compared with normal rabbits (max 39.1±2.8%) (Figure 3A). INDO and LNA-resistant relaxation to AA in aortas from hypercholesterolemic rabbits were also greater (max 79.5±2.4%) compared with normal (max 39.9±2.2%) at 3x10–4 mol/L AA (Figure 3B). In contrast, the maximum relaxation to dipropylenetriamine (DPTA) NONOate, an NO donor, and P1075, a KATP channel opener, were the same (supplemental Figure IIC and IID). Maximum contractions to 44 mmol/L KCl were 2.97±0.34 gm and 2.97±0.09 gm, and to cumulative concentrations of PNE were 4.2±0.3 gm and 4.2±0.2 gm, in aortas from normal and hypercholesterolemic rabbits, respectively.
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Contribution of AA metabolites from 15-LO-1 to ACH and AA relaxations were determined in presence of INDO, LNA and various 15-LO inhibitors. In aortas from hypercholesterolemic rabbits, the maximum NO- and PG-independent ACH relaxations (69.3±3.0%) were reduced by BW755C to 22.8±2.3%, by ebselen to 27.4±4.3%, and by cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC) to 13.9±1.4% (Figure 3C). In presence of INDO, maximum AA relaxations were reduced by BW755C from 81.4±2.4% to 4.3±2.2% (Figure 3D). Similarly, in aortas from normal rabbits, NO- and PG-independent maximum ACH relaxations (39.1±2.8%) were reduced by BW755C to 24.1±4%, by ebselen to 26.1±5%, and by CDC to 7.1±0.7% (supplemental Figure IIIA). AA-relaxations in aortas from normal rabbits were also reduced by BW755C from 41.7±2.2% to 0.5±2.5% (supplemental Figure IIIB).
ACH relaxes rabbit arteries because of a synergistic action of SKCa channels and IKCa channels. THETA and HEETA activate SKCa channels but not IKCa channels in rabbit mesenteric arteries.4 Therefore, ACH relaxations were investigated in presence of IKCa channel inhibitor and THETA and HEETA synthesis inhibitor (supplemental Figure IV). In the aortas from hypercholesterolemic rabbits, IKCa channel inhibitors charybdotoxin (CTX) or TRAM-34 in combination with BW755C reduced the maximal relaxations to 20.7±2.9% or 19.9±4.4%, respectively (supplemental Figure IVA). These reductions of relaxation were greater when compared with the reduction by BW755C alone.
The effect of hypercholesterolemia on the ACH-induced relaxation mediated by IKCa channels alone was investigated in the aortas (supplemental Figure IV). In presence of INDO and LNA, in the aortas from hypercholesterolemic rabbits, CTX reduced maximal ACH relaxations from 73.1±2.2% to 28.6±4.5% whereas TRAM-34 reduced these relaxations from a maximum of 68.0±2.7% to 16.5±2.9% (supplemental Figure IVB). Similarly, in the aortas from normal rabbits, CTX and TRAM-34 reduced the NO- and PG-independent maximal ACH relaxations from 39.7±2% to 23.4±1.1% and 26.3±5%, respectively (supplemental Figure IVC). The extent of the reductions by CTX or TRAM alone is not different in aortas from normal or hypercholesterolemic rabbits. The effect of hypercholesterolemia on IKCa channels was further confirmed by measuring relaxations to an IKCa channel opener, 1-ethyl-2-benzimidazolinone (1-EBIO), in presence of INDO and LNA (supplemental Figure V). Maximum 1-EBIO relaxations were not different in aortas from hypercholesterolemic (86.6±2%) or normal rabbits (80.6±6%) (supplemental Figure VA). Finally, effects of BW755C or ebselen on IKCa channels were investigated by measuring the effect on 1-EBIO relaxations. 1-EBIO-relaxations in aorta from hypercholesterolemic rabbits were reduced to 57.8± 2.7% by CTX, but not by BW755C (87.1±1.7%) or ebselen (80.5±2.7%; supplemental Figure VB).
Membrane Potential of Smooth Muscle Cells in Aortas
The resting Em in the smooth muscle cells (SMCs) from the normal rabbits was –61.66±7.6 mV and from the hypercholesterolemic rabbits was –55.41±5.5 mV (Figure 4). INDO did not alter the resting Em, but phenylephrine (PNE) depolarized the Em in SMCs from normal and hypercholesterolemic aortas. Addition of AA to the PNE-treated SMCs repolarized the Em in aortas from normal rabbits to a lesser extent than in the aortas from hypercholesterolemic rabbits (P<0.013). The AA-induced hyperpolarization was inhibited by BW755C. BW755C alone did not alter the Em in INDO treated aortas.
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Basal Hemodynamics and ACH Responses In Vivo
Basal MAP was elevated in the hypercholesterolemic rabbits compared with normal rabbits, whereas HR did not change (Table). ACH induced a dose-related decrease in MAP (Figure 5). HR with 4000 ng/kg of ACH did not change from the basal values in either normal or hypercholesterolemic rabbits. The maximal MAP decrease to ACH was –31±3% in normal rabbits and –58±3% in hypercholesterolemic rabbits (Figure 5A). With INDO and LNAME treatment, MAP did not differ from the basal values in either hypercholesterolemic or normal rabbits (Table). The maximum MAP decrease in INDO- and LNAME-treated normal rabbits was –24±1% but was –48±3% in hypercholesterolemic rabbits (Figure 5B). In INDO- and LNAME-treated hypercholesterolemic rabbits, BW755C reduced the ACH-induced decrease in MAP to –29±2% (Figure 5C). With BW755C treatment, MAP and HR remained unchanged compared with INDO- and LNAME-treated rabbits (Table). None of the treatments altered HR in either normal or hypercholesterolemic rabbits.
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Discussion |
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We determined whether hypercholesterolemia without atherosclerosis alters 15-LO-1 expression, THETA and HEETA synthesis, and vasorelaxation. Feeding rabbits a 2% cholesterol enriched diet for 2 weeks raised the total plasma cholesterol concentration without inducing lesion formation.10 15-LO-1 mRNA and protein expression was increased in arteries from these hypercholesterolemic rabbits. Immunoflourescence revealed that this increased 15-LO-1 expression was limited to the endothelium. The mechanism of induction of 15-LO-1 protein expression in arteries by elevated cholesterol is not known. However, hypercholesterolemia without atherosclerotic lesions causes aberrant DNA-methylation patterns including both hypo- and hypermethylation.13 Conflicting reports have indicated that hyper- or hypomethylation of the CpG island in the promoter region of the 15-LO-1 gene alters its expression.14,15 This is further substantiated by the reported genomic hypomethylation in atherosclerotic rabbit aorta,16 and increased 15-LO-1 expression in these atherosclerotic lesions.8 As hypercholesterolemia progresses to atherosclerosis, it is conceivable that 15-LO-1 expression will continue to increase in the endothelium and eventually be restricted to the lesions as reported.
Increased 15-LO-1 expression increases THETA and HEETA synthesis in rabbit arteries and endothelial cells (ECs).6 THETA and HEETA synthesis increased in arteries of hypercholesterolemic rabbits. Endothelial denudation inhibited this synthesis, confirming that induction of 15-LO-1 expression was limited to the endothelium and the endothelium was necessary for THETA and HEETA synthesis. Additionally, a LO inhibitor BW755C inhibited THETA and HEETA synthesis, confirming the role of 15-LO-1. 15-LO-1 expression, and increase in THETA and HEETA synthesis, is sufficient to increase ACH- and AA-relaxations in rabbit arteries.6 We determined the NO- and PG-independent ACH- and AA-relaxations in the thoracic aortas as the increase in the 15-LO-1 expression with hypercholesterolemia was greatest in aortas compared to other arteries. AA-induced relaxation increased in aortas from hypercholesterolemic rabbits compared to aortas from normal rabbits, suggesting a contribution of the AA metabolites. Furthermore, NO- and PG-independent ACH-relaxations were increased in the aortas from hypercholesterolemic rabbits. Similar results were obtained in the mesenteric arteries. A similar increase in NO- and PG-independent EDHF-mediated ACH relaxation was reported in renal arteries from rabbits fed a 0.5% cholesterol chow for 5 weeks17 and in thoracic aortas from rats fed a 1% cholesterol chow for 8 weeks.18 On the contrary, ACH-induced relaxations were decreased in thoracic aorta and femoral arteries from rabbits fed with 0.5% cholesterol chow for 20 weeks.19 Thus the influence of hypercholesterolemia on ACH relaxations depends on the duration of cholesterol feeding. Longer feeding times may induce lesion formation in the arteries that is associated with endothelium damage. In the present study, the NO- and PG- independent ACH relaxations were increased by hypercholesterolemia. ACH relaxations, without NOS and COX inhibition, in the aortas from hypercholesterolemic rabbits were also enhanced. Additionally, the endothelium independent relaxations determined by measuring relaxations to an NO donor and to an ATP-sensitive K channel (KATP) channel opener were not different. This suggests that only the endothelium-dependent relaxations were enhanced. Contractions to KCl and PNE were not changed by hypercholesterolemia. The NO- and PG-independent relaxations to ACH were inhibited by 3 different LO inhibitors, BW755C, ebselen, and CDC, confirming the contribution of 15-LO-1 metabolites. None of the LO inhibitors altered the basal tone of the aortas from either normal or hypercholesterolemic rabbits. Additionally, the inhibitors reduced the NO- and PG-independent ACH-relaxations to a greater extent in hypercholesterolemic rabbit aortas compared to normal rabbit aortas. This is consistent with higher expression of 15-LO-1 and greater synthesis of THETA and HEETA in the aortas from hypercholesterolemic rabbits. This confirmed that enhanced expression of 15-LO-1 in hypercholesterolemic rabbits is sufficient to increase ACH or AA relaxations.
ACH-induced relaxations in rabbit arteries are mediated by synergistic action of SKCa channels in SMCs and IKCa channels in ECs.4 THETAs and HEETAs open the apamin-sensitive SKCa-channels, whereas CTX-sensitive IKCa-channels are involved in K+ efflux. Both the mechanisms act together to hyperpolarize smooth muscle cells and relax rabbit arteries.4 Therefore, ACH relaxations were further reduced when IKCa channels inhibitors CTX and TRAM-34 were used in combination with BW755C. However, it is not known whether hypercholesterolemia influences the activity of IKCa channels. To test this possibility, NO- and PG-independent ACH relaxations were measured in presence of IKCa channel inhibitors, CTX and TRAM-34 alone. Both CTX or TRAM-34 reduced the ACH relaxations, but the reduction in aortas from normal and hypercholesterolemic rabbits did not differ. This suggests that the sensitivity of the IKCa channel pathway did not change with hypercholesterolemia. Additionally, the IKCa channel opener 1-EBIO relaxed the aortas from normal or hypercholesterolemic rabbits similarly, confirming that IKCa channel–mediated relaxations did not change with hypercholesterolemia. Specificity of BW755C and ebselen to SKCa channels was also determined. BW755C or ebselen did not inhibit the 1-EBIO relaxations suggesting that these inhibitors do not block the IKCa channels and their effect is attributable to the inhibition of 15-LO–mediated activation of SKCa channels. Overall, the relaxation studies suggested that with hypercholesterolemia, only SKCa channel mediated ACH relaxations were enhanced because of enhanced synthesis of THETA and HEETA, and the IKCa channel–mediated relaxation did not change.
The effect of EDHFs varies with type and size of arteries.12 Therefore, we measured the MAP, HR, and ACH responses in rabbits to evaluate the contribution of 15-LO-1 in regulating blood pressure. MAP of hypercholesterolemic rabbits was slightly elevated compared to the MAP of normal rabbits. This pattern was the same in the presence and absence of NO- and PG-inhibitors. Therefore, it appears that the increased synthesis of THETA and HEETA does not contribute to the basal MAP in hypercholesterolemic rabbits. We have observed similar results in the rabbits infected with an adenovirus containing 15-LO-1 cDNA to overexpress 15-LO-1 in arteries.6 Presumably, compensatory mechanisms negate the effect of elevated THETA and HEETA synthesis. Furthermore, with INDO and LNAME treatment, we observed only a transient increase in MAP that returned to the basal values in 6 to 10 minutes. Rajapakse et al20 also observed no differences in the basal MAP levels with vehicle or nitro-L-arginine treatment, and Oliver et al21 reported that COX inhibition by ibuprofen increased the MAP only transiently for 10 minutes. This confirms our previous results6 and suggests a compensatory mechanism returns the raised MAP to basal values after COX and endothelial NOS inhibition in rabbits.
We measured ACH-induced hypotensive responses in normal or INDO and LNAME treated rabbits. Similar doses of ACH lowers MAP in rabbits.22 ACH caused a transient fall in MAP and was used to assess endothelium-dependent vasodilation.22 We and others have reported that the ACH-induced hypotensive responses are not related to the basal MAP.6,22 ACH decreased MAP to a greater extent in hypercholesterolemic rabbits when compared with the normal rabbits. When responses to ACH were measured in INDO and LNAME treated rabbits, MAP decreased to a greater extent in hypercholesterolemic rabbits compared with the normal rabbits indicating greater amount of EDHFs released. This finding is consistent with the enhanced ACH-induced relaxation in isolated aortas. BW755C reduced the ACH-induced decrease in MAP in the INDO- and LNAME-treated rabbits, confirming the role of 15-LO-1 metabolites in the hypotension.
In summary, hypercholesterolemia increases the expression of 15-LO-1 in rabbit arterial endothelium, increasing the synthesis of THETAs and HEETAs. The increased THETA and HEETA synthesis enhances ACH- or AA-relaxations in the isolated aortas and ACH-induced decrease in MAP in hypercholesterolemic rabbits. The roles of these 15-LO-1 metabolites in other changes in the vascular wall with hypercholesterolemia await further study.
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Acknowledgments |
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The authors thank Dr Yuttanna Chawengsub for his helpful discussions, Daniel Goldman for his technical assistance, and Gretchen Barg for her secretarial assistance.
Sources of Funding
The studies were supported by a grant from the National Heart, Lung, and Blood Institute (HL-37981) and a predoctoral fellowship to N.T. Aggarwal from the American Heart Association, Greater Midwest Affiliate.
Disclosures
None.
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Footnotes |
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Original received May 20, 2008; final version accepted September 20, 2008.
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