Published online before print April 21, 2003, doi: 10.1161/01.HYP.0000069011.18333.08
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(Hypertension. 2003;41:1294.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Reduced Growth Factor Responses in Vascular Smooth Muscle Cells Derived from 12/15-Lipoxygenase–Deficient Mice
From the Department of Diabetes, Beckman Research Institute of the City of Hope, Duarte, Calif.
Correspondence to Rama Natarajan, PhD, Department of Diabetes, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010. E-mail rnatarajan{at}coh.org
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Abstract |
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Biochemical and genetic evidence support the involvement of leukocyte-type 12/15-lipoxygenase enzyme and its products in the atherogenic process. We recently showed that products of the 12/15-lipoxygenase pathway play an important role in mediating hypertrophy, matrix protein production, and inflammatory gene expression in vascular smooth muscle cells (VSMC) through activation of mitogen activated protein kinases and key transcription factors. The current study is aimed at establishing the in vivo role of 12/15-lipoxygenase in VSMC by comparing growth factor–induced responses in VSMC derived from 12/15-lipoxygenase knockout mice versus genetic control wild-type mice. In the lipoxygenase knockout cells, 12/15-lipoxygenase protein was not expressed, and levels of its product, 12(S)-hydroxyeicosatetraenoic acid, were reduced (51% of wild type). Knockout cells exhibited significantly lower rates of growth factor–induced migration, fibronectin production, and incorporation of 3H-thymidine and 3H-leucine (54%, 55%, 61%, and 57% of wild type, respectively). Growth factor–induced superoxide production and p38 mitogen–activated protein kinase activation were also reduced in knockout cells. Serum-stimulated AP-1 transcription factor activation was markedly reduced (50% of wild type), whereas cAMP response element binding protein activation was abrogated in knockout cells. Furthermore, growth factor–induced mRNA expression of immediate early genes and fibronectin were also greatly reduced. These results suggest that the modulation of specific signaling pathways and growth-responsive genes may be responsible for the altered growth factor responses in the lipoxygenase knockout cells. They also demonstrate the important in vivo role of vascular 12/15-lipoxygenase in VSMC growth, migration, and matrix responses associated with hypertension, atherosclerosis, and restenosis.
Key Words: lipoxygenase • muscle, smooth, vascular • signal transduction • angiotensin II • platelet-derived growth factor • gene regulation
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Introduction |
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The lipoxygenase (LO) pathway has been implicated in the pathogenesis of atherogenesis and hypertension.1,2 LOs metabolize arachidonic acid to oxidized lipids and are classified as 5-, 8-, 12-, and 15-LOs, based on the ability to insert molecular oxygen at the corresponding carbon atom of arachidonic acid. Three major functionally distinct isoforms of 12-LO, namely, platelet-type, leukocyte-type, and epidermal-type 12-LO, have been cloned.3–5 Human and rabbit 15-LOs as well as the leukocyte-type 12-LO have high homology and are classified as 12/15-LOs because they can form both 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE)] and 15(S)-HETE from arachidonic acid via their peroxy precursors, and mainly 13(S)-hydroperoxy-octadecadienoic acid from linoleic acid.3–5 Leukocyte-type 12-LO has a wide tissue distribution, including vascular smooth muscle cells (VSMC).6,7 In mice, both leukocyte 12/15-LO and platelet 12-LO are expressed.8
Studies indicate that LO inhibition reduced blood pressure in renovascular hypertensive rats.9 Furthermore, 12(S)-HETE levels were elevated in spontaneously hypertensive rats compared with age-matched Wistar-Kyoto rats, and 12(S)-HETE directly regulated calcium signals in VSMC.10,11 Several lines of evidence implicate 12/15-LO in the development of atherosclerosis. 12/15-LO can mediate the oxidation of LDL,12 and the enzyme and its products have been detected in atherosclerotic lesions.13,14 Convincing evidence comes from recent data showing that disruption of 12/15-LO in apoE-/- or LDL-R-/- mice significantly reduced atherosclerosis in these mice models.15,16
Additionally, growth factors (GFs) such as angiotensin II (Ang II) and platelet-derived growth factor (PDGF) and cytokines such as IL-1, IL-4, and IL-8 could induce 12/15-LO activity and expression in VSMC.7,17–19 Furthermore, the 12-LO product 12(S)-HETE could induce VSMC migration and extracellular matrix production.20,21 Ang II–induced hypertrophy as well as PDGF-induced chemotactic effects were significantly blocked by pharmacological LO inhibitors and a molecular inhibitor, 12-LO ribozyme.17,21–23 Expression of 12/15-LO was also increased in balloon-injured arteries, whereas 12-LO ribozymes and pharmacological inhibitors reduced the extent of neointimal thickening in these injured rat carotid arteries.22,24 Recently, we showed that 12-LO products directly stimulated VSMC hypertrophy, activation of p38 mitogen–activated protein kinase (p38 MAPK), transcription factors NF-kappa B (NF-
B), and cAMP response element binding protein (CREB) and induced transcription from the fibronectin and VCAM-1 promoters.25,26 Furthermore, stable 12/15-LO overexpression in VSMC increased MAPK activity and induced hypertrophy.25 Thus, 12/15-LO activation in VSMC may play a key role in vascular growth and injury responses. The aim of the current study was to determine the in vivo pathologic role of 12/15-LO in VSMC by comparing basal and GF-mediated responses of VSMC derived from 12/15-LO knockout mice (LOKO)27 with those isolated from genetic control (WT) mice. Our new results indicate that cellular growth, matrix production, and migration are greatly attenuated in VSMC from the LOKO mice relative to WT mice.
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Methods |
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All animal experiments were conducted in accordance with National Institutes of Health guidelines with an approved protocol from the Research Animal Care Committee of the City of Hope.
Isolation and Culture of MVSMC
Control (WT, C57BL/6) and leukocyte 12/15-LO knockout mice (LOKO) on a C57BL/6 background (strain name: B6.129S2-Alox15tm1Fun; stock No: 002778) were obtained from Jackson Laboratories. VSMC from aortas of 7- to 9-week-old mice were isolated by enzymatic digestion as described earlier.28 These primary cultures of mouse VSMC (MVSMC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% FCS, glutamine (2 mmol/L), streptomycin (100 µg/mL), penicillin (100 U/mL), and amphotericin B (25 µg/mL) as fungizone. The SMC identity of the cells was confirmed by staining with SMC-specific 
-actin monoclonal antibody (clone 1A4, Sigma Chemicals Inc). In all experiments, MVSMC (passages 4 to 8) were serum-depleted for 48 hours in medium containing 0.2% BSA and treated with GFs for the indicated time intervals.
12(S)-HETE Assay
MVSMC were serum-depleted for 48 hours and basal 12(S)-HETE levels (without any added arachidonic acid) in the cell supernatants were quantified with the use of a specific radioimmunoassay (RIA) as described earlier.7,29 This RIA is specific for 12(S)-HETE and does not cross-react with 12(R)-HETE.
[3H]-Leucine and [3H]-thymidine incorporation were determined as described earlier.18,21
Fibronectin Assay
Cells were serum-starved for 48 hours, replaced with fresh serum-free medium, and incubated for another 24 hours. The supernatant conditioned medium was then assayed for fibronectin by a specific sandwich ELISA as described earlier.21
Intracellular Superoxide Production
Intracellular superoxide production was evaluated with the use of a superoxide probe dihydroethidium (Molecular Probes Inc) as described.30 MVSMC cultured on chamber slides were treated with PBS or the indicated GFs for 30 minutes, washed, and incubated with dihydroethidium (10 µmol/L) for 15 minutes. Cells were then washed to remove extracellular dye, and the red fluorescence in cells, which is an indicator of intracellular superoxide production, was detected by a confocal microscope. Cells were also stained with Hoechst dye (Molecular Probes) to detect nuclei.
Immunoblotting and Gel Shift Assays
These were performed as described previously.25
RNA Isolation and RT-PCR
After stimulation with agonists, total RNA was extracted, and gene-specific relative multiplex RT-PCRs, with 18S RNA used as internal control, were performed as described earlier.31 After normalization to 18S RNA, results were expressed as fold expression over unstimulated cells. Mouse fibronectin mRNA was amplified using primers 5'-GCACAACAGACCACCAAACTCG-3' (forward) and 5'-CTGAAGTCACTTCTCGGGGTGC-3' (reverse). Primers for c-fos, c-jun, and 18S RNA were from Ambion Inc.
Migration Assay
VSMC migration assay was performed with the use of a modified 48-well Boyden’s microchemotaxis chamber as described earlier.17 Briefly, VSMC were placed in the upper chamber and PDGF (0.1 nmol/L) in the lower chamber. The number of cells migrated to the lower side of filter were counted after 4-hour incubation.
Data Analyses
Data are expressed as mean±SEM of multiple experiments. Paired Student t tests were used to compare 2 groups, or ANOVA with the Dunnett posttest for multiple groups using Prism software (Graph Pad). Statistical significance was detected at the 0.05 level.
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Results |
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Characterization of MVSMC From WT and LOKO Mice
To examine the in vivo role of 12/15-LO signaling in VSMC and in GF responses, MVSMC were isolated from WT or LOKO mice. Both WT and LOKO cells stained positive with SMC-specific
-actin and did not show significant differences in morphology. Immunoblotting of whole-cell lysates with 12/15-LO–specific antibody17 showed that in contrast to WT cells, LOKO cells did not have a 72-kDa protein (Figure 1A). Lysates from porcine VSMC, which we have shown to express leukocyte-type 12-LO,7 was included for comparison (lane PV). Furthermore, basal levels of 12(S)-HETE (Figure 1B), an arachidonic acid metabolite of 12/15-LO, were significantly lower in MVSMC from LOKO mice (65±7 pg/106 cells in WT versus 33.5±4 pg/106 cells in LOKO cells).
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Protein and DNA Syntheses Are Decreased in MVSMC From LOKO Mice
Next, we examined the effect of 12/15-LO deficiency on cellular growth–promoting effects by incubating MVSMC in the presence of 3H-Leucine or 3H-thymidine to determine the rates of protein and DNA syntheses, respectively. As shown in Figure 2, rates of both 3H-Leucine (A) or 3H–thymidine (B) incorporation were significantly reduced in LOKO cells compared with WT cells (61% and 57% of WT, respectively, P<0.01). These results suggest that LO activation plays an important role in MVSMC hypertrophy and growth.
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GF-Stimulated Superoxide Production Is Reduced in MVSMC Derived From LOKO Mice
Since oxidant stress is essential for GF signaling and LO activation has been associated with increased oxidant stress,32,33 we next compared superoxide production in WT versus LOKO cells. Quiescent MVSMC from WT and LOKO were stimulated with FBS (10%) or Ang II (0.1 µmol/L) or PDGF-BB (0.1 nmol/L) for 30 minutes. Intracellular superoxide production was then monitored by red fluorescence resulting from oxidation of intracellular probe DHE. As seen in Figure 3, on stimulation with GFs (FBS, Ang II, or PDGF), WT cells produced markedly greater amounts of superoxide compared with LOKO cells. Thus, reduced superoxide production may be one of the mechanisms by which GF-induced responses are inhibited in 12-LOKO cells.
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Reduced Activation of p38 MAPK in LOKO MVSMC
Earlier studies from our laboratory demonstrated that p38 MAPK plays a key role in hypertrophy as well as inflammatory gene induction by 12-LO products.25,26 Furthermore, p38 MAPK activity was augmented in rat VSMC and cardiac fibroblasts overexpressing 12-LO cells.25,34 Hence, we hypothesized that p38 MAPK activation may be reciprocally attenuated in LOKO VSMC. MVSMC were stimulated with serum (10% FBS) and cell lysates immunoblotted with phosphospecific-p38 MAPK antibody. Results showed that serum-activated p-p38 MAPK was attenuated in LOKO cells compared with WT cells (Figure 4A, top panel, and Figure 4B). Levels of total p38 MAPK were unchanged (Figure 4A, middle panel). In contrast, serum-stimulated p44/42 MAPK activation was similar in the 2 cell types (Figure 4A, lower panel). Reduced p38 MAPK activation associated with 12/15-LO deficiency is consistent with our previous in vitro observations.
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Comparison of AP1 and CREB DNA Binding Activities
Since serum-induced MAPK activation was reduced in MVSMC, we evaluated whether the activation of key downstream MAPK target transcription factors such as AP1 and CREB are altered. AP1 and CREB are induced by GFs and induce expression of genes required for cell growth and matrix protein production.35,36 Nuclear extracts prepared from FBS-stimulated (10%) MVSMC were analyzed by gel shift assays, using 32P-labeled oligonucleotides containing consensus DNA-binding sequences for AP1, CREB, and a constitutively active transcription factor SP1. Results in Figure 5A show that both basal (3851 versus 11 960 cpm phosphorimager counts) as well as serum-induced (6933 versus 45 020 cpm) AP1 DNA-binding activities were greatly reduced in LOKO relative to WT cells. Basal CREB DNA binding (30 506 versus 42 247 cpm) was also reduced, and serum failed to increase CREB DNA-binding activity in LOKO cells (31 556 cpm compared with 92 123 in WT cells). However, SP-1 DNA-binding activity (50 000 to 60 000 cpm/lane) was similar in these nuclear extracts, indicating equal loading of nuclear proteins and specificity of effects for AP1 and CREB.
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Expression of Immediate Early Response Genes
Activation of MAPK cascade by GF stimulation leads to the expression of immediate early response (IE) genes such as c-fos and c-jun, which are involved in cellular growth, migration, and differentiation. We therefore compared their expression in the 2 cell types. MVSMC were stimulated with FBS (10%) for 15 minutes to 4 hours, and c-fos and c-jun mRNA expression was determined by relative RT-PCR. Results showed that the expression of c-fos (Figure 5B) and c-jun (Figure 5C) mRNAs were stimulated by serum in both WT and LOKO cells with similar time course, that is, peak at 30 minutes and returning to basal after 2 hours. However, the fold induction of both genes was greatly reduced in LOKO compared with WT. Thus, reduced expression of IE genes may be one of the mechanisms by which LO-deficient cells exhibit reduced rates of migration and hypertrophy.
Reduced Fibronectin Expression in LOKO Cells
GFs induce the transcription of the key extracellular matrix (ECM) protein fibronectin through activation of CREB. Hence, we compared fibronectin mRNA expression by relative RT-PCR after stimulation with Ang II (0.1 µmol/L) for 4 hours. Basal levels were attenuated and Ang II–induced fibronectin mRNA levels were greatly reduced in LOKO cells relative to WT (Figures 6A and 6B). In addition, Figure 6C shows that basal fibronectin protein levels (measured by ELISA in culture supernates) were significantly lower in LOKO compared with WT cells (55% of WT, P<0.001). Thus, 12-LO may play an important role in GF-induced matrix production in VSMC.
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GF-Induced Migration Is Reduced in LO-Deficient MVSMC
Using pharmacological inhibitors and 12-LO–specific ribozymes, we previously demonstrated that LO plays an important role in GF-induced migration in VSMC. To further determine the in vivo functional significance of 12/15-LO in VSMC migration, we compared rates of PDGF-induced migration in MVSMC from LOKO mice versus WT mice. Figure 7 shows that PDGF-induced migration was significantly attenuated in LOKO compared with WT cells (54% of WT, P<0.001). These results clearly demonstrate an important role for the 12/15-LO pathway in GF-induced migration.
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Discussion |
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In this study, we examined the functional role of mouse 12/15-LO expression in VSMC by evaluating basal and GF-stimulated oxidant stress, growth, migration, MAPK activation, and IE gene and fibronectin expression in MVSMC derived from WT versus LOKO mice. The rationale partly stems from earlier observations that 12-LO activity and expression were increased in porcine VSMC in response to GFs and cytokines.7,17–19 Furthermore, the 12-LO pathway was demonstrated to mediate Ang II–induced hypertrophic responses and PDGF-induced chemotactic responses in PVSMC.17,21 Additionally, 12-LO products such as 12(S)-HETE could directly induce VSMC hypertrophy and fibronectin expression in VSMC through activation of p38 MAPK and CREB activation.25
MVSMC from LOKO mice showed absence of the leukocyte-type 12/15-LO protein and reduced levels of 12(S)-HETE. Funk and coworkers8 showed that mice express both platelet 12-LO and leukocyte-type 12/15-LO. Since LOKO mice in which leukocyte 12/15-LO was disrupted still express platelet 12-LO,27 this could be responsible for the residual 12(S)-HETE seen in the LOKO VSMC. There is no specific report of a platelet-type 12-LO in murine VSMC. However, since mice express both types of 12-LO8,27 and there is one report of a novel platelet 12-LO in human VSMC,37 it is possible that murine VSMC express both isoforms or another novel as yet unidentified 12-LO isoform. Both platelet and vascular 12(S)-HETE have been implicated in hypertension,9–11,38–40 whereas macrophage and VSMC 12/15-LO have been implicated in atherosclerosis.7,13–16,41 Activation of LO is associated with oxidant stress and superoxide production.12,32,33 Our present observations of decreased GF-induced superoxide production in the LOKO cells are consistent with the pro-oxidative role of LO. We also observed reduced rates of 3H-thymidine and 3H-leucine incorporation in the MVSMC from LOKO mice relative to WT.
12/15-LO products such as 12(S)-HETE and 13(S)-HPODE have been shown to induce cellular effects through activation of MAPKs such as ERK1/2 and p38 in VSMC.25,26,42 We observed that serum-induced p38 MAPK is attenuated in the LOKO cells relative to WT. Furthermore, DNA binding activities of key MAPK target transcription factors AP-1 and CREB were attenuated in the LOKO MVSMC. These effects were functionally relevant, since serum-induced expression of the target immediate early genes c-fos and c-jun (components of AP-1) were greatly reduced in LOKO cells. Furthermore, the expression of the key ECM protein, fibronectin, which is regulated by CREB, was also significantly reduced in the LOKO cells. Our present observations in vivo are supportive of the recent in vitro data that 12(S)-HETE leads to the transcription of fibronectin via CREB DNA binding and transactivation of the fibronectin promoter in PVSMC.25 They are also in agreement with observations that overexpression of mouse 12/15-LO in VSMC or fibroblasts leads to increased hypertrophy and p38 MAPK activity.25,34
Evidence suggests that 12(S)-HETE can directly induce VSMC migration20 and 12-LO can mediate PDGF-induced chemotactic effects in VSMC.17,22,23 In the current study, we observed that PDGF-induced migration was significantly attenuated in the LOKO cells relative to WT, thus providing in vivo relevance to the in vitro observations.
Animal models have now demonstrated the key role of the LO pathway in the development of hypertension, restenosis, and atherosclerosis.1,9,15,16,22,24 Pharmacological 12/15-LO inhibitors as well as a rat 12/15-LO ribozyme could significantly reduce neointimal thickening in injured rat arteries.22,24 A 15-LO inhibitor could block diet-induced atherosclerosis in rabbits.43 Increased 12/15-LO expression has been reported in a swine model of atherosclerosis.41 Recent observations that cross-breeding LOKO mice with the apoE-/- or LDL-R-/- mice could greatly reduce atherosclerosis development in the latter 2 mice models provide the first clear demonstration of the significant role played by leukocyte 12/15-LO in atherosclerosis.15,16 In these in vivo models, the decrease in atherosclerosis was attributed to reduced LDL oxidation caused by the absence of macrophage 12/15-LO.15,16 Very recently, a novel observation was made that the macrophages from 12/15-LO–deficient mice had a selective defect in LPS-induced interleukin-12 synthesis.44 Our present observations of reduced growth and matrix responses in VSMC from LOKO mice suggest that these could be additional mechanisms for the atheroprotective role conferred by ablation of 12/15-LO. Since LO activation can lead to the consumption of nitric oxide,45 this could be another key route by which vascular LO mediates the pathogenesis of atherosclerosis and hypertension. Patricia et al46 showed that 12(S)-HETE treatment of endothelial cells could lead to increased monocyte binding and that the 12/15-LO ribozyme blocked high glucose-induced binding of monocytes to endothelial cells.23 Thus, lack of 12/15-LO in endothelial cells of the LOKO mice may also contribute to the atheroprotective effects of these mice. A recent genetic study suggesting that 5-LO may be an important proatherogenic gene is interesting,47 and it is not yet clear how it relates to the data on 12/15-LO. Furthermore, the relevance of LO to human hypertension and atherosclerosis needs to be established. Taken together, our studies show for the first time that vascular 12/15-LO may play an important role in VSMC growth, migration, oxidant stress, and ECM production under pathological conditions such as hypertension, restenosis, and atherosclerosis.
Perspectives
LO enzymes in vascular, inflammatory, renal, and other cells can form products that have several physiological and pathological effects. These include potent growth-promoting, chemotactic, adhesive, and inflammatory effects, which therefore implicate them in the pathogenesis of diseases such as atherosclerosis, hypertension, and diabetic complications. The in vivo role of LO in these diseases is further supported by recent data with mouse models of 12/15-LO deficiency, including our present studies. These data therefore make 12/15-LO an attractive target for drug design. There are currently no clinically available safe and selective pharmacological inhibitors of the LO enzymes. One approach would be to use genetic approaches to block the enzyme. The ribozyme targeted to 12/15-LO has been effective in vitro in VSMC and endothelial cells and in vivo in animal models of restenosis. Hence, therapeutic approaches to block this pathway may provide new ways to combat cardiovascular diseases.
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Acknowledgments |
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These studies were supported by grants from the National Institutes of Health (PO1-HL55798 and RO1-58191).
Received January 30, 2003; first decision February 28, 2003; accepted March 1, 2003.
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References |
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