(Received for publication, January 16, 1997, and in revised form, June 5, 1997)
From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0682
The molecular and cellular mechanisms by which hypertension enhances atherosclerosis are poorly understood. Angiotensin II (Ang II) has been implicated in the regulation of cellular lipoxygenases (LO), which are thought to play a role in atherogenesis by inducing oxidative modification of low density lipoprotein (LDL). We sought to test the hypothesis that Ang II would stimulate murine macrophage LO activity (which has both 12- and 15-LO activity). Competitive binding studies revealed the presence of Ang II AT1 receptors on mouse peritoneal macrophages (MPM) and J-774 cells, but not on the RAW cell line. Valsartan, a specific AT1 receptor antagonist inhibited Ang II binding, whereas PD 123319, an AT2 receptor antagonist did not. Incubation of MPM or J-774 cells with Ang II (10 pM to 1 µM) for 24 h led to a 2.5-3.5-fold increase in LO activity, measured as generated 13-HODE or 12(S)-HETE. This stimulation was inhibited by valsartan, but not by PD 123319. In contrast, Ang II did not stimulate LO activity in RAW macrophages. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 2-3-fold increase in LO mRNA in MPM, but not in RAW cells after treatment with Ang II. Ang II also induced an increase in 12-LO protein. In addition, pretreatment of J-774 cells with Ang II increased in a dose-dependent manner the ability of the cells to modify LDL, resulting in greater chemotactic activity for monocytes, typical of minimally modified LDL. This stimulation was inhibited by AT1 receptor blockade.
In summary, these data suggest that Ang II increases macrophage LO activity via AT1 receptor-mediated mechanisms and this further increases the ability of the cells to generate minimally oxidized LDL. These studies provide a link between hypertension and the associated increased atherosclerosis observed in hypertensive patients.
The importance of high blood pressure as a risk factor for developing cardiovascular disease is well established. However, it has been found that drug therapies that successfully reduce blood pressure and the incidence of stroke do not necessarily reduce the morbidity and mortality associated with coronary artery disease (1). These findings appear to indicate a multifactorial relationship between hypertension and atherosclerosis, suggesting that additional mechanisms, other than increased systemic blood pressure, contribute to the accelerated atherosclerosis associated with hypertension. Increased plasma or tissue levels of vasoactive hormones, such as Ang II, may play a role in such mechanisms, by inducing vascular cell growth in the vessel wall (2-4), stimulation of proto-oncogene expression (5, 6), and modulation of myocardial hypertrophy and fibrosis (7, 8).
For many years our laboratory has been interested in the "oxidation hypothesis" of atherosclerosis (9). Oxidation of LDL1 converts it into an atherogenic form contributing to the development of the atherosclerotic lesion. Foam cells, one of the hallmarks of atherosclerosis, develop when monocyte-derived macrophages or smooth muscle cells within the artery wall take up oxidized LDL via scavenger receptors. There is increasing evidence to suggest that certain cellular lipoxygenase (LO) enzymes are involved in this process by inducing oxidation of LDL (10, 11). Evidence supporting a causal role of human 15-LO in LDL modification during atherosclerosis includes co-localization of 15-LO protein and mRNA with epitopes of oxidized LDL in macrophage-rich areas of atherosclerotic lesions (12, 13), and the presence of stereospecific products of 15-LO activity in lesions, but not in normal arteries (14, 15). Additionally, fibroblasts transfected with human 15-LO cDNA showed an enhanced ability to seed hydroperoxides into LDL incubated with such cells (16, 17) and transfer of the 15-LO cDNA into arteries in vivo led to the appearance of oxidation-specific products that co-localized with the transduced 15-LO (18).
Ang II has been shown to up-regulate both lipoxygenase activity and
expression in human smooth muscle cells (19). Murine macrophages and
murine macrophage cell lines contain a "macrophage-type" lipoxygenase which possesses both 12- and 15-LO activity (20), which is
highly homologous to human and rabbit 15-LO, the enzyme believed to
play a role in the maturation of red blood cells and the oxidative
modification of LDL (21). Accordingly, due to their dual specificity,
these enzymes have been referred to as 12/15-LO (20). In contrast, not
much is known about the role of vasoactive agents, such as Ang II, on
macrophage function and metabolism. Although Ang II mediates some
immunological responses, such as increased interferon- production
and increased macrophage phagocytosis, the receptor subtype involved
has not been defined (22-25).
The present studies test the hypothesis that Ang II increases the oxidative modification of LDL by stimulating the activity of macrophage 12/15-LO through an Ang II-receptor-mediated pathway. We show that Ang II, through the Ang II AT1 receptor subtype, stimulates expression of 12/15-LO mRNA, protein, and activity in MPM and the J-774 cell line. In turn, this results in increased modification of LDL incubated with these cells, rendering it more chemotactic for human monocytes.
Ang II (human), FMLP, indomethacin, Tyrode's salts, and glutaraldehyde were purchased from Sigma. [Sar1,Ile8]Ang II was from Bachem Fine Chemicals (Torrance, CA). Valsartan was a generous gift from Dr. M. de Gasparo (Novartis, Basel, Switzerland) and PD 123319 and PD 146176 were from Parke-Davis (Ann Arbor, MI). [14C]Linoleic acid (53.0 mCi/mmol), [125I-Sar1,Ile8]Ang II (0.2 nM, specific activity, 2200 Ci/mmol) and [3H]thymidine (35 Ci/mmol) were from NEN Life Science Products (Boston, MA). 13(S)-Hydroxyoctadecadienoic acid (13-HODE) and eicosatetraynoic acid (ETYA) were purchased from Cayman Chemical Co. (Ann Arbor, MI). HPLC grade water and methanol were obtained from Fischer (Fair Lawn, NJ). SDS gels were obtained from Novex (San Diego, CA), the enhanced chemiluminescence reagents were from Amersham, and the polyvinylidene fluoride blotting membranes were from Millipore (Bedford, MA). The peroxidase-conjugated rabbit anti-sheep was purchased from Cappel (Aurora, OH). The sheep antibody against rabbit reticulocyte 15-LO and authentic 15-LO protein were a generous gift from Dr. J. Cornicelli.
Measurement of 15-LO ActivityResident mouse peritoneal macrophages were harvested from C57BL/6J (Jackson Laboratory, Bar Harbor, ME) by lavage with ice-cold phosphate-buffered saline. Cells were plated in 6-well plates (Costar, Cambridge, MA) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicilline, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells of the murine macrophage cell lines J-774 A.1 and RAW 264.7 (noted as J-774 and RAW, respectively) were kept in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 50 µg/ml gentamycin, and 2 mM L-glutamine. The activity of 15-LO was determined as the rate of formation of 14C-labeled 13-HODE measured by reverse-phase high performance chromatography (HPLC) from [14C]linoleic acid (16, 26). If not otherwise mentioned, cells were treated with Ang II for 24 h prior to incubation with radiolabeled linoleic acid. To determine which receptor subtype mediated the effects of Ang II, cells were first treated with Ang II receptor antagonists for 6 h before incubating them with Ang II. Protein content was determined after lysing the cells with 0.2 N NaOH using the bicinchoninic acid assay reagent (Pierce).
Measurements of 12-LO ProductsThese assays were performed according to previously published methods (27). Briefly, J-774 cells were treated for various times with Ang II and 12(S)-HETE was extracted from cell supernatants on C18 mini columns (Varian, Harbor City, CA) and measured by radioimmunoassay (Perceptive Diagnostics, Cambridge, MA). The 12-HETE antibody used for radioimmunoassay specifically recognizes 12(S)-HETE. Results were corrected for extraction recovery and data expressed as picogram/mg of cell protein.
125I-Angiotensin II Binding[125I-Sar1,Ile8]Ang II binding and competition assays were performed on adherent cells as described previously with minor modifications (28). 1.5 × 106 MPM, J-774, or RAW cells were preincubated in phosphate-buffered saline containing 1 g/liter glucose and 1% BSA for 15 min at room temperature. It was established that binding equilibrium occurred after a 60-min incubation. Therefore, the cells were incubated with increasing concentrations of [125I-Sar1,Ile8]Ang II for 60 min at room temperature. The reaction was stopped by aspirating the supernatant and washing the cells with ice-cold phosphate-buffered saline. Total cell-bound radioactivity was measured after lysing the cells with 0.2 N NaOH. Nonspecific binding was determined in the presence of 1 µM cold [Sar1,Ile8]Ang II. Specific binding was calculated by substracting nonspecific from total binding. To determine the extent of binding via specific Ang II receptor subtypes, competition curves were performed in the presence of subtype-specific antagonists. Dissociation constant (Kd) and maximum binding (Bmax) were determined using non-linear least regression curve analysis according to the equation: b = c × Bmax/(c + Kd), where b is bound radioligand, c is the radioligand concentration, Bmax is maximum binding, and Kd is the dissociation constant.
Chemotaxis AssayThe chemotactic response of human monocytes to FMLP, Ang II-, and/or LDL-containing media conditioned by exposure to cells was assessed in a 48-well chemotactic chamber (Neuroprobe, Cabin John, MD). Human mononuclear cells were isolated by Ficoll-Paque density centrifugation (29). LDL (d = 1.019-1.063 g/ml) was isolated by density gradient ultracentrifugation from pooled healthy human plasma (30). J-774, MPM, or RAW cells were plated in a 96-well plate. After incubation in the absence or presence of Ang II for 24 h, cells were carefully washed and incubated in the absence or presence of 250 µg/ml LDL in Ham's F-10 for another 24 h. The supernatants of the treated cells, in the absence or presence of LDL, with and without pretreatment with Ang II, as well as native LDL alone, were placed in the lower compartment of a chemotactic chamber which was separated from the upper compartment by a 5-µm pore-sized polycarbonate membrane (Poretics, Livermore, CA). 5 × 106 monocytes resuspended in 0.1% BSA/Tyrode per chamber were loaded in the upper wells. The cells were then incubated for 90 min at 37 °C (5% CO2, 100% humidity). The filters were washed and cells remaining on the upper surface of the filters were removed mechanically. After fixation in 1% glutaraldehyde and staining of the filters in 0.1% crystal violet, the number of migrated cells was counted microscopically. Each experimental condition was carried out in quadruplicate, and 6 fields were examined for each well encompassing 50-1000 cells. In additional experiments Ang II antagonists, lipoxygenase and cyclooxygenase antagonists were incubated with macrophages for 6 h prior to addition of Ang II. FMLP (10 nM) in 0.1% BSA/Tyrode's salt buffer served as positive and 0.1% BSA/Tyrode's as negative control.
The role of secreted low-molecular weight proteins into the medium (e.g. MCP-1) induced by minimally modified LDL (31) was assessed after filtration of the cell supernatants. Cell supernatants were filtered through a 25,000 Mr cut-off filter cone (Amicon, Beverly, MA) at 1000 × g for 30 min. The 25,000 molecular mass cut-off was chosen to exclude low molecular mass compounds, such as MCP-1 which has a mass of 14 kDa (32). The LDL fraction was diluted to the original LDL protein concentration. The filtrate and the remainder in the cone were assessed for chemotactic activity and compared with an aliquot of the supernatant prior to filtration.
Measurements of LDL ModificationThe mobility of LDL was determined by agarose gel electrophoresis (33). The degree of LDL modification was also determined by the amount of thiobarbituric acid-reactive substances (TBARS) (34).
RT-PCR of 12-LO mRNATotal RNA was extracted from
cultured J-774, RAW, and freshly isolated MPM treated with Ang II
(RNAzol B, Tel-Test Inc., Friendswood, TX). 2.0-3.0 µg of RNA were
reverse transcribed with PCR buffer (50 mM Tris-HCl, pH
8.2, 6 mM MgCl2, 10 mM
dithiothreitol, 100 mM NaCl), 2.5 mM of dNTP,
10 mg/ml BSA, 30 µM RNase inhibitor (RNasin), 3 µM oligo(dT)15 and Moloney murine leukemia
virus reverse transcriptase (Supercript II 200 U, Life Technologies,
Inc.) in a final volume of 50 µl. The samples were reverse
transcribed in a thermal cycler at 72 °C for 4 min, 42 °C for 60 min, and 94 °C for 10 min. Murine 12-LO cDNA amplification was
carried out by mixing 0.16 µg of cDNA in a 25-µl volume with
0.24 µM each of the 5- and 3
-primer for 12-LO
(5
-GTTTGGCTCCTGGGCAGACG-3
and 5
-TTTCTACCAGGCTGGGCCGC-3
) and 0.75 units of Taq polymerase (Boehringer Mannheim). The primers
were chosen based on the published sequence of the murine leukocyte
12-LO (35). They were encoded across several exons to exclude the
possibility of DNA contamination. The resulting PCR product for 12-LO
resulted in a single band of the predicted size (195 kilobases). As
internal standard, GAPDH mRNA was coamplified by the addition of
primers for murine GAPDH. To ensure that GAPDH amplification would not
reach the plateau phase earlier than the target gene, addition of GAPDH
primers was delayed, as described previously (36). GAPDH primers (0.24 µM each of 5
- and 3
-primer;
5
-CTGCCATTTGCAGTGGCAAAGTGG-3
and 5
-TTGTCATGGATGACCTTGGCCAGG-3
) were
added after completion of the fifth extension phase. PCR was carried
out with a denaturing step at 94 °C for 1 min, annealing at 66 °C
for 1 min, and extension at 72 °C for 1 min for 30-35 cycles.
Similar PCR conditions were used for the quantitative determination of
GAPDH expression by competitive PCR, which confirmed that GAPDH
expression in cells was constant and not affected by Ang II. The
competitor cDNA was identical to the GAPDH PCR product, except for
a 102-base pair deletion (kindly supplied by Dr. H. Lukhaup,
Heidelberg). Six increasing concentrations of competitor (3-30
pg/µl) were used and the amount of PCR product formed was determined
relative to the expression of GAPDH. Blank reactions with no RNA or
cDNA template were carried out through the RT and PCR steps to test
for possible contamination. The PCR products were electrophoresed on a
3% agarose gel (NuSieve, Rockland, ME) containing ethidium bromide
and the intensity analyzed using Optimas 4.0 imaging software (Bioscan) and compared with GAPDH as internal control (37). Each image analysis
was performed twice by the same observer. Gene expression was
determined in triplicate. The identity of the amplified product from
MPM and J-774 was confirmed by sequence analysis of the 12-LO product
purified from agarose gels (data not shown).
Cultured MPM were washed with phosphate-buffered saline and lysed in lysis buffer. Lysates were subjected to SDS-PAGE on 8% gels and separated proteins were transferred to polyvinylidene fluoride membranes. Blots were blocked with 5% dry milk, incubated with sheep anti-15-LO antibody, and then incubated with peroxidase-conjugated rabbit anti-sheep antibody. Immunopositive bands were visualized by enhanced chemiluminescence. Purified reticulocyte 15-LO was included as positive control; non-immune sheep IgG was used as negative control. This antibody has previously been shown to cross-react with both human and mouse 12/15-LO (38).
Statistical AnalysisData are analyzed using analysis of variance (ANOVA). Student's two-tailed, unpaired t test was used to identify the groups between which the differences were significant. Data are represented as mean ± S.E., and p < 0.05 was considered statistically significant.
The presence of Ang II receptors
and/or its receptor subtype have not been formally established on MPM
or on macrophage cell lines. Therefore, we determined the parameters of
Ang II binding to MPM, J-774, and RAW cell lines. Specific binding of
[125I-Sar1,Ile8]Ang II to intact
MPM or J-774 cells was saturable. Fig.
1A shows representative
saturation isotherms in MPM and in J-774 yielding an apparent
Kd value of 0.18 nM and a
Bmax of 0.80 fmol/mg protein in MPM and a
Kd of 0.50 nM and
Bmax 1.20 fmol/mg protein in J-774,
respectively. No specific binding could be found on RAW cells (data not
shown). Ang II-specific binding on J-774 and on MPM could be
dose-dependently competed by valsartan, an AT1
receptor antagonist with an IC50 of 1 nM,
whereas PD 123319, an AT2 receptor blocker was not able to
inhibit [125I-Sar1,Ile8]Ang II
binding even at millimolar concentrations (Fig. 1B).
Effect of Ang II on 15/12-LO Activity
Incubation of MPM or
J-774 cell lines with Ang II for 24 h induced a
dose-dependent increase in 15-LO activity, as measured by
the generation of 13-HODE from linoleic acid. An EC50 was
apparent at 10 pM with a maximum response with 100 pM. Higher concentrations did not increase 15-LO activity
any further (Fig. 2). In contrast, Ang II
did not augment 15-LO activity in RAW cells (data not shown). The Ang
II-induced increase was inhibited by 76% by 2 nM
concentration of the Ang II AT1 antagonist valsartan (Table
I), similarly to its inhibition of Ang II
binding (Fig. 1B). In contrast, the AT2 receptor
antagonists PD 123319 did not have any inhibitory effect (Table I). The
antagonists alone had no effect on basal 15-LO activity and were not
different from control (data not shown). None of the antagonists were
toxic to cells in the concentration used, as evaluated by cell protein
determination and trypan blue exclusion. Similarly, Ang II increased
macrophage 12-LO activity. Treatment with Ang II at concentrations of
1011 and 10
7 M for 1 h
significantly stimulated the release of 12(S)-HETE, as
measured by radioimmunoassay in the J-774 cell line, in a
dose-dependent manner (control, 262 ± 31; Ang II, 10 pM 488 ± 85; Ang II, 100 nM 955 ± 26 pg/mg protein, respectively).
|
To determine if Ang II treatment of macrophages altered the
level of 12/15-LO mRNA in MPM and in the J-774 macrophage cell line, semiquantitative RT-PCR was used to amplify the murine
"macrophage-type" 12/15-LO (20). In the absence of Ang II, both
murine macrophage cell lines, J-774 and RAW, as well as MPM contained
the predicted 195-base pair sized product, consistent with the
macrophage-type of 12/15-LO. The amplified product was confirmed to be
of murine 12-LO origin by sequence analysis (data not shown). In
response to incubation with Ang II over a range of 10 pM to
1 µM, expression of murine GAPDH remained constant in MPM
and J-774 yielding a regression line of the ratio of GAPDH expression
to exogenous GAPDH standard of r 0.995 for J-774 and
0.985 for MPM, respectively. Since GAPDH remained constant, the
increase in 12/15-LO mRNA was expressed as a function of GAPDH.
Ang II (1 nM) increased 12-LO mRNA expression in MPM
starting at the 2-h incubation time, with a maximum increase of
2.8-fold after a 24-h incubation (Fig.
3). In contrast, Ang II did not have any
effect on 12-LO mRNA expression in RAW cells (data not shown).
Effect of Ang II on 12-LO Protein in MPM
To determine whether
Ang II increased 12/15-LO activity in MPM through up-regulation of
12-LO protein, cells were treated for increasing periods of time with
Ang II. Cell lysates were then prepared for SDS-PAGE, electroblotted
onto polyvinylidene fluoride membranes, and subjected to Western blot
analysis using an antibody to rabbit reticulocyte LO, which was
previously shown to cross-react with 12-LO in MPM (38). Incubation of
MPM with Ang II (1 nM) increased LO protein expression
starting at 4 h, with a maximum seen after 24 h of
incubation. Incubation with Ang II for 48 h, however, did not
further increase LO protein levels (data not shown) (Fig.
4).
Effect of Ang II on LDL Modification by Macrophages
We have previously shown that fibroblasts transfected with human 15-LO had an increased ability to modify LDL (16). Accordingly, we determined the Ang II-induced effect on macrophage-mediated LDL modification by two different parameters. One type involves physical properties of the conditioned LDL and the other involves altered biological behavior. For the physical properties we determined the appearance of lipid peroxidation decomposition products as measured by the TBARS assay and the effect on LDL mobility in agarose gel.
LDL incubated with J-774 showed an increase in TBARS compared with non-cell control or native LDL, but no significant difference in TBARS from LDL exposed to J-774 alone or compared with cells that had been pretreated with Ang II could be found (Table II). Of all the lipoxygenase and cyclooxygenase inhibitors used, only ETYA, an unspecific LO inhibitor (39), and PD 146167, a lipoxygenase inhibitor lacking significant antioxidant properties (40), inhibited the cell-mediated increase in TBARS, whereas the cyclooxygenase inhibitor indomethacin had no effect (Table II).
|
LDL incubated with J-774 showed greater mobility than LDL incubated in the absence of cells or native LDL (data not shown). However, LDL incubated with Ang II-pretreated cells did not show any greater increase in mobility than J-774-conditioned LDL (data not shown).
Ang II-mediated Increase in ChemotaxisIt is well established
that although minimally modified forms of LDL have few discernible
changes in physical properties, they have altered biological
characteristics (41). Therefore, we assayed for the chemotactic
activity of conditioned LDL. Native LDL or LDL conditioned with and
without cells for 24 h, elicited a small chemotactic response.
However, pretreatment of J-774 and MPM with Ang II (10 pM
to 100 nM) prior to the incubation with LDL, increased in a
dose-dependent manner the chemotactic ability of the
conditioned LDL. The EC50 was approximately 10 pM (Fig. 5A). In
contrast, LDL from media conditioned by Ang II-treated RAW cells, did
not elicit an increased chemotactic response (data not
shown).
Because LDL was not reisolated in these experiments, it was possible that some or all of the chemotactic activity was due to components (e.g. low molecular weight proteins, such as MCP-1) in the conditioned media not associated with LDL. Therefore, the conditioned supernatants were filtered through 25,000 Mr cut-off filters and the filtrate and the LDL fraction assessed for chemotactic activity. 85 ± 1.0% of the recovered chemotactic activity was associated with the LDL fraction, whereas 15 ± 1.1% was found in the filtrate (n = 3, p = 0.0009). Since the filtrate was only marginally chemotactic compared with the LDL in the filter or unfiltered supernatants, it is not likely that low molecular factors contributed markedly to the chemotactic activity.
The Ang II-induced increase in chemotactic response to LDL conditioned
by J-774 and MPM could be inhibited up to 77% by valsartan, the Ang II
AT1 receptor antagonist, whereas the AT2
antagonist PD 123319, even at the highest dose, had little effect (Fig.
5B). The IC50 value for valsartan was shown to
be 10 nM. To further show that the Ang II-induced increase
in chemotaxis of LDL-conditioned media was mediated through 12/15-LO
activity, we incubated J-774 cells with Ang II in the presence of ETYA
or PD 146176, two lipoxygenase inhibitors, or in the presence of
indomethacin, a cyclooxygenase inhibitor. Both lipoxygenase inhibitors
reduced the cell modification of LDL, as measured by the chemotactic
response, whereas indomethacin had little effect. The IC50
of ETYA and PD 146176 were approximately 10-100 µM each
(Fig. 6).
These studies show that Ang II stimulates macrophage-mediated
modification of LDL, both in MPM and in the murine macrophage cell line
J-774 through up-regulation of 12-LO mRNA, LO protein, and
enzymatic activity. Both cells showed the presence of AT1 receptors in binding studies. This ability of Ang II to increase 12/15-LO activity was shown to be mediated primarily through the AT1 receptor, as valsartan, an AT1 receptor
antagonist substantially inhibited the increase in 12/15-LO activity
and inhibited the Ang II-induced effect on LDL modification, as
measured by enhanced monocyte chemotaxis, whereas PD 123319, an
AT2 antagonist, did not. Further evidence that Ang II was
specifically acting through Ang II receptors to stimulate 12/15-LO
activity and subsequent cell-mediated modification of LDL was provided
by studies with RAW cells. These cells, which failed to show specific
Ang II binding, also failed to have an increase in 15-LO activity or to
modify LDL, when preincubated with Ang II, despite expressing basal
levels of 12/15-LO mRNA. The concentrations of Ang II used in our
studies (1011 to 10
7 M) are
comparable to physiological plasma levels of Ang II in humans, which
lie in the pico- to nanomolar range (42).
It has previously been shown that Ang II stimulates 12-LO activity, mRNA expression, and protein levels in porcine and human smooth muscle cells (19, 43). A recent study provided evidence that Ang II stimulated the ability of macrophages to oxidatively modify LDL, as measured by enhanced degrees of lipid peroxidation in the LDL (44). Since phenidone, presumably a 15-LO inhibitor, inhibited the Ang II-mediated stimulation of LDL-lipid peroxidation, it was concluded that 15-LO may be involved. However, this study did not demonstrate specific binding sites on macrophages for Ang II, nor did it demonstrate a direct action of Ang II on 12/15-LO enzyme activity, on mRNA expression or on 12/15-LO protein induction in macrophages. Additionally, very high concentrations of Ang II were used (100 nM) to stimulate the macrophages and high concentration of saralasin (100 µM), a nonspecific Ang II receptor antagonist, were required to test receptor specificity of the Ang II-induced macrophage-mediated oxidative modification of LDL (44). Our data demonstrate a dose-dependent increase in 12/15-LO activity with Ang II concentrations in the physiological range and the increase required binding of Ang II to the AT1 receptors on macrophages, as only valsartan significantly inhibited this increased activity. RT-PCR analysis demonstrated that Ang II stimulated an increase in 12-LO mRNA, consistent with regulation at the transcriptional level, although additional post-transcriptional regulation cannot be ruled out as well. Furthermore, we show that Ang II induces LO protein in MPM. In our studies we did not find enhanced levels of lipid peroxidation in the LDL incubated with macrophages preincubated with Ang II, in contrast to the report of Keidar et al. (45). This difference could be due to the fact that we used doses of Ang II in the physiological range. Alternatively, our studies also differ in that in the studies of Keidar et al. (44) Ang II and LDL were coincubated with the macrophages, a potentially confounding variable, since the same authors have shown that Ang II-LDL complexes are more readily bound by macrophages (45). Despite these differences, our finding that Ang II, by binding to the AT1 receptor, increases macrophage-mediated modification of LDL via a 12/15-LO-dependent pathway, extends the findings of Keidar et al. (44) and proves a novel role for Ang II on macrophage metabolism.
The recent discovery of selective, high affinity nonpeptide antagonists has resulted in the identification of at least two major Ang II receptor subtypes with differential tissue distribution and functional responses, namely the AT1 and the AT2 receptor (46, 47). While most of the known physiological and pathophysiological actions of Ang II (hemodynamics, aldosterone release, growth of vascular cells) have been attributed to the AT1 receptor, the role of the AT2 subtype is less clear (48). We document the presence of Ang II AT1 receptors on MPM and the J-774 cell line with an apparent Kd of 0.18 and 0.50 nM, respectively, which is in agreement with studies on macrophages isolated from murine liver granulomas following Schistosoma mansoni infection (49). The fact that only an AT1 receptor antagonist was able to compete for the Ang II binding on MPM and J-774, suggests that AT1-binding sites are present and that binding of Ang II to its receptor is required for the Ang II-induced effects. Additional support comes from the finding that RAW cells lacking the AT1 receptor, did not respond to Ang II treatment in any of the parameters measured. Because Ang II has the ability to affect many metabolic and cellular processes, these data suggest that Ang II may have an important effect on other macrophage functions as well.
To further assess whether the Ang II-induced effect on macrophages chemotaxis was due to a more extensively oxidized LDL particle or a minimally modified LDL, we used both physicochemical and biological parameters. The increase of TBARS and the electrophoretic mobility were not enhanced and suggest that the effect of Ang II on LO only induced "minimal" further modification of LDL (41) than was already induced by the macrophages themselves. The finding that the Ang II-induced stimulation of 12/15-LO resulted in no further measures of oxidative modification of the LDL, as measured by TBARS and agarose gel electrophoresis, yet resulted in LDL with enhanced chemotactic properties, is similar to recent studies from this laboratory, showing that fibroblasts overexpressing human 15-LO induce only minimal changes in lipid peroxidation in LDL, yet these LDL are bioactive (50). Further evidence that the Ang II-induced macrophage-mediated modification of LDL was due to stimulation of 12/15-LO was provided by the experiments using PD 146176, ETYA, and indomethacin. This suggests that the chemoattractant activity is mediated via a lipoxygenase-dependent pathway, rather than cyclooxygenase-dependent pathway, since only PD 146176, supposedly a specific 12/15-LO inhibitor (40) and ETYA were able to reduce the Ang II-induced ability of macrophages to modify LDL chemoattractant activity. The fact that Ang II stimulation of RAW cells also failed to increase LDL's chemotactic activity further supports this hypothesis.
The precise mechanisms linking the Ang II-induced stimulation of 12/15-LO activity to the increased modification of LDL, rendering it more chemotactic for human monocytes, has not been established in these studies. One possibility is that macrophage 12/15-LO acts on endogenous lipids in the cell resulting in fatty acid hydroperoxides which are subsequently transferred to extracellular LDL. In turn, the LDL seeded with hydroperoxides may undergo subtle degrees of lipid peroxidation, giving rise to products that have chemotactic activity (16, 17, 41). Another possibility is that 12/15-LO, despite being an intracellular enzyme, acts directly on LDL making contact with the cell's surface or that its activation is linked with the increased generation of reactive oxygen species, as suggested by Cathcart and co-workers (51). Additionally, minimally modified LDL has been shown to induce secretion of MCP-1 (31). Although the majority of the chemotactic activity was associated with the LDL fraction, it cannot be excluded that chemotactic proteins, such as MCP-1, could be bound to LDL. This possibility, however, is unlikely as MCP-1 is very hydrophilic and thus not likely to associate with LDL. Whatever the exact mechanism, it appears that activation of the 12/15-LO pathway increases LDL's chemotactic properties out of proportion to the extent of lipid peroxidation, at least as measured by the TBARS assay.
Despite the large body of literature accumulated around the hypothesis that lipoxygenases are involved in oxidative modification of LDL (10-13, 16, 17), the role of these enzymes in atherosclerosis in vivo are less clear. Sendobry et al. (40) have reported that administration of PD 141176 to cholesterol-fed rabbits significantly inhibited the progression of atherosclerosis. On the other hand, a recently published report suggests that macrophage-specific overexpression of human 15-LO in rabbits does not increase atherosclerosis, and possibly even protects from lipid deposition in the arterial wall (52). Nevertheless, the finding that Ang II stimulates macrophage-mediated LDL modification in vitro via a 12/15-LO pathway and consequently enhances monocyte chemotaxis, suggests a novel role for Ang II on macrophage metabolism and an important link between its known role in hypertension and atherosclerosis. Obviously more studies in this area will be needed.
Although very little is known about local concentrations of Ang II within the vascular wall, it has been suggested that locally generated Ang II, whose levels may exceed the circulating levels, may exert important autocrine and paracrine functions. Consequently, it has been proposed that local synthesis of Ang II in cardiovascular organs, such as the vasculature may play an important role in the development of hypertension and cardiovascular diseases. Thus, increased local Ang II concentrations in the artery wall may not only promote hypertension (53), but also promote atherosclerosis by stimulation of macrophage lipoxygenase activity and subsequent enhanced oxidative modification of LDL, rendering it potentially more atherogenic and resulting in enhanced monocyte recruitment into the arterial wall.
We thank Dr. J. Cornicelli (Parke-Davis) for the generous supply of PD 146176 and 15-LO antibody as well as authentic 15-LO protein, and Dr. M. de Gasparo (Novartis) for the gift of valsartan and helpful advice.