Growth factors induce monocyte binding to vascular smooth muscle cells: implications for monocyte retention in atherosclerosis

Qiangjun Cai, Linda Lanting, and Rama Natarajan

Gonda Diabetes Center, Beckman Research Institute of City of Hope, Duarte, California 91010

Submitted 1 April 2004 ; accepted in final form 4 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte {beta}1- and {beta}2-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

angiotensin II; platelet-derived growth factor-BB; cell interaction; CD36


MONOCYTES-MACROPHAGES play a key role in all stages of atherosclerosis (28). In response to atherogenic stimuli, monocytes in circulation adhere and migrate across the endothelium. These initial processes may be reversible, whereas the subsequent prolonged intimal retention of monocytes-macrophages is a central pathogenic process in atherogenesis (20). However, the mechanisms by which subendothelial retention of monocytes occurs and the role of vascular smooth muscle cells (VSMC) in this process are unclear.

Migration and proliferation of VSMC are also well-documented events in atherosclerosis (30). Increasing evidence suggests that adhesive interactions between migrated monocytes and VSMC may contribute to monocyte-macrophage retention within the vasculature. The potential of VSMC to interact with monocytes is suggested by the fact that VSMC express adhesion molecules within atherosclerotic lesions but not in normal vessels (2). In addition, a highly significant association was found between VSMC vascular cell adhesion molecule (VCAM)-1 expression and intimal macrophage content (17, 25). Furthermore, a strong focal expression of intercellular adhesion molecule (ICAM)-1 on VSMC in atherosclerosis-prone regions was observed preceding mononuclear cell infiltration in man, suggesting a causative role in lesion development (7).

Direct cell-to-cell interactions between monocytes and VSMC enhance monocyte procoagulant activity and increase production in both cell types of atherosclerosis-related factors such as metalloproteinase-1 (40). These data suggest that VSMC and monocytes-macrophages are not merely innocent coexistent neighbors in the vessel but that VSMC-monocyte interactions are additional regulatory signals in the pathogenesis of atherosclerosis. However, the cellular, molecular, and signal transduction mechanisms are not clear.

Angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB play pivotal roles in vascular remodeling and atherosclerosis (5, 28, 39). They potently induce VSMC migration, hypertrophy, and proliferation (1, 38, 39). However, the role of these growth factors in VSMC-monocyte interactions and macrophage accumulation in the vessel wall is not known and is the focus of this study. We also evaluated VSMC adhesion molecules and secreted factors, as well as the role of monocyte integrin counterreceptors in mediating these interactions. In addition, because the cellular actions of ANG II and PDGF-BB can be mediated by the activation of phospholipases and by arachidonic metabolism via the lipoxygenase (LO) and cyclooxygenase (COX) pathways (1, 13, 22, 23, 38) as well as key signal transduction events, we evaluated the role of these factors in mediating monocyte binding to VSMC. We also examined the functional impact and pathological consequences of VSMC-monocyte interactions induced by these factors by examining monocyte expression of CD36, a key scavenger receptor involved in oxidized low-density lipoprotein (ox-LDL) uptake and foam cell formation (16).


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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All procedures were performed in conformance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Cell culture. Human aortic smooth muscle cells (HVSMC; Clonetics, San Diego, CA) were cultured in SMC basal medium according to the supplier's instructions. Primary cultures of porcine VSMC (PVSMC) were obtained and cultured as described previously (23). Mouse aortic SMC (MVSMC) from macrophage-type 12/15-LO knockout (LOKO) mice (35) and wild-type (WT) mice on a C57BL/6 background were isolated and cultured as described previously (27) in accordance with a protocol approved by the Research Animal Care Committee. The human monocytic cell line THP-1 (American Type Culture Collection, Manassas, VA) was cultured in RPMI 1640 medium with 10% heat-inactivated FBS and 5 x 10–5 M {beta}-mercaptoethanol. The mouse monocytic cell line WEHI78/24 was grown in DMEM and 10% FBS. Human peripheral blood monocytes (PBMC) were obtained from the blood of normal healthy volunteers with informed consents approved by the Institutional Review Board. Monocyte fractions were isolated as described previously (31).

Reagents. The LO inhibitor cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate (CDC), the LO products 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], 15(S)-HETE, 5(S)-HETE, the isomer 12(R)-HETE, and the COX-2 product prostaglandin E2 (PGE2) were all purchased from Biomol (Plymouth Meeting, PA). Inhibitors of p38 MAPK (SB-202190), MEK (PD-98059), phosphoinositide 3-kinase (PI3K; LY-294002), and Src kinase (PP2) were purchased from Calbiochem (San Diego, CA). The ANG II type 2 (AT2) receptor antagonist PD-123319, human ANG II, human {beta}-actin antibody, and the fluorescent label 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). The ANG II type 1 (AT1) receptor antagonist Irbesartan was purchased from Merck Pharmaceuticals (Whitehouse Station, NJ). The COX-2-selective inhibitor NS-398 was obtained from Cayman Chemical (Ann Arbor, MI). Recombinant human PDGF-BB was purchased from Invitrogen Life Technologies (Carlsbad, CA). Mouse anti-human integrin {beta}1 (MAB1987Z) and {beta}2 (MAB1962) antibodies were obtained from Chemicon (Temecula, CA). Rabbit anti-murine COX-2 polyclonal antibody was obtained from Cayman Chemical.

Binding assay with human PBMC. HVSMC plated in 48-well culture plates were serum starved for 48 h after reaching confluence and were washed twice, and then 1 x 105 PBMC was added to each well. After 30-min incubation at 37°C, unbound cells were washed off, and bound cells were fixed with 1% glutaraldehyde and counted using phase-contrast microscopy. A minimum of five fields was counted per well.

Binding assay with monocytic cells. THP-1 cells or WEHI78/24 cells were preincubated in RPMI 1640 or DMEM containing 2% FBS and 5 µg/ml fluorescent BCECF-AM at 37°C for 30 min in foil-covered tubes. Fluorescently labeled cells were washed twice to remove unincorporated dye and were then resuspended in DMEM containing 0.2% BSA. Loaded monocytic cells (5 x 104) were added to each well of VSMC and incubated at 37°C. After 30 min, unbound monocytes were withdrawn and VSMC layers with attached monocytes were gently washed twice with DMEM and lysed with 0.2 ml of 0.1% Triton X-100 in 0.1 M Tris per well. Fluorescence (excitation 485 nm, emission 535 nm) was measured using an fMax microplate reader (Molecular Devices, Sunnyvale, CA). Data were analyzed with SoftMax Pro. The number of adherent cells per well was expressed as the percent fluorescence of control.

Blocking antibody studies. Normal human PBMC were preincubated with monoclonal antibodies to human integrins {beta}1 or {beta}2 (20 µg/ml) for 30 min at 4°C and then added to HVSMC monolayers for binding assays. A nonspecific mouse immunoglobulin (IgG) was used as negative control.

Transient overexpression experiments. Fifty percent confluent VSMC in 48-well plates were transfected with expression vectors for mouse leukocyte-type 12/15-LO (pcDNA-m12-LO) or human COX-2 (pcDNA-COX-2) or with an empty vector, using Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's protocols. After overnight recovery, cells were serum depleted for 24 h and then used in binding assays.

Western blot analysis. After transfection, PVSMC were lysed and protein concentrations were determined with a Bio-Rad kit (Bio-Rad Laboratories, Hercules, CA). Aliquots of 30 µg of protein were treated with Laemmli sample buffer, heated at 100°C for 5 min, and electrophoresed at 30 µg/lane in a 10% acrylamide denaturing SDS-polyacrylamide gel. Proteins were transferred to Hybond-ECL membrane (Amersham Life Science, Arlington Heights, IL) using a Hoeffer semidry blotting apparatus (Hoeffer Instruments, San Francisco, CA). The membrane was incubated in blocking buffer (5% nonfat milk) for 1 h at room temperature and then incubated overnight at 4°C with a polyclonal antibody to leukocyte 12/15-LO, COX-2, or {beta}-actin. Membrane was washed and developed with chemiluminescent agent (ECL; Amersham Life Science). Immunoblots were scanned using a GS-800 densitometer.

Flow cytometry analysis. To examine the effects of VSMC-monocyte interactions on monocyte cell surface CD36 expression, 5 x 106 THP-1 cells were added to confluent monolayers of HVSMC. After 24 h, unbound cells were collected by gentle aspiration, and bound cells were collected separately by shaking the plates with or without gentle pipetting. The latter process is gentle enough that the HVSMC layer is undisturbed as observed under the microscope. Cell surface CD36 expression on these two cell populations was measured by flow cytometry with a R-phycoerythrin-conjugated mouse anti-human CD36 monoclonal antibody (Pharmingen, San Diego, CA). For this determination, 1 x 106 THP-1 cells were washed twice with PBS containing 5% heat-inactivated FBS, resuspended in 100 µl of medium containing 20 µl of CD36 antibody or isotype control, and then incubated at 4°C for 30 min, washed, and analyzed on a MoFlo MLS flow cytometer (Dako-Cytomation, Fort Collins, CO). Results are expressed as the percentage of cells positive for CD36.

Statistical analysis. Results are expressed as means ± SE from multiple experiments. Data were analyzed by ANOVA followed by Tukey's test, or by Student's t-test for two groups. P < 0.05 was considered statistically significant.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased binding of THP-1 cells to HVSMC pretreated with ANG II or PDGF-BB. We first examined whether treatment of key VSMC growth factors can induce monocytes to bind to them relative to untreated VSMC. HVSMC were pretreated for various time periods with increasing doses of ANG II or PDGF-BB and then subjected to the binding assays with THP-1 cells. Figure 1, A and B, shows that ANG II-induced binding of THP-1 cells to HVSMC was significant at 1 x 10–8 M and peaked at 1 x 10–7 M, whereas PDGF-BB effects were significant at 2 x 10–10 M and continued to increase up to 8 x 10–10 M. A significant increase in binding appeared by 30 min or 1 h with ANG II (1 x 10–7 M) or PDGF-BB (8 x 10–10 M), respectively, and peaked at 6 h (Fig. 1, C and D). ANG II effects were mediated by the AT1 receptor, since they were completely blocked by the AT1 blocker irbesartan but not by the AT2 blocker PD-123319 (data not shown).



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Fig. 1. Increased binding of THP-1 cells to human aortic vascular smooth muscle cells (HVSMC) treated with angiotensin (ANG) II or platelet-derived growth factor (PDGF)-BB. HVSMC were treated for 6 h with increasing concentrations of ANG II (A) or PDGF-BB (B) or for increasing time periods with 1 x 10–7 M ANG II (C) or 8 x 10–10 M PDGF-BB (D) before binding assays were performed with THP-1 cells. *P < 0.01, {dagger}P < 0.001 vs. basal binding (n = 6).

 
Involvement of monocyte integrin receptors in growth factor-induced monocyte binding. Using THP-1 monocytic cells enabled us to avoid interexperimental and interindividual variability that could arise with PBMC. However, because THP-1 cells may not fully represent the phenotype of PBMC, we next examined whether ANG II and PDGF-BB could also induce the binding of HVSMC to PBMC isolated from normal healthy adults. Figure 2 shows that, comparable to the results with THP-1 cells, preincubation of HVSMC with ANG II (1 x 10–7 M) or PDGF-BB (8 x 10–10 M) for 6 h also increased PBMC binding by 162.5 and 175.0% (P < 0.001), respectively. VSMC-monocyte binding may be mediated by interactions of specific adhesion molecules (VCAM-1 and ICAM-1) on VSMC with their respective integrin ({beta}1 and {beta}2) counterreceptors on monocytes. Monoclonal antibodies to human integrin {beta}1 or {beta}2 were used to determine their functional role. As shown in Fig. 2, both {beta}1- and {beta}2-integrin interactions mediated ANG II- and PDGF-BB-induced binding. In contrast, a nonspecific control IgG had no effect.



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Fig. 2. Blocking of monocyte integrins reduces the binding of peripheral blood monocytes (PBMC) to HVSMC. Human PBMC were incubated with blocking antibodies to {beta}1-({beta}1 Ab) or {beta}2-integrin ({beta}2 Ab) or with control (Ctrl) IgG (20 µg/ml, 30 min) before coculture with unstimulated HVSMC or with HVSMC pretreated with ANG II or PDGF-BB. Bound cells were fixed and counted. Data represent the mean (±SE) monocyte number bound per field. *P < 0.001, {dagger}P < 0.01 vs. basal binding. {ddagger}P < 0.001 vs. ANG II or PDGF-BB (n = 3).

 
Soluble factors are responsible for ANG II- and PDGF-BB-induced HVSMC-THP-1 cell binding. Increased binding in response to ANG II or PDGF-BB may be regulated by 1) increased HVSMC surface expression of VCAM-1 and ICAM-1 and/or 2) THP-1 cell integrin activation. Our above-described experiments indicated the involvement of monocyte integrins. Our following experiments clearly showed that soluble factors released from HVSMC are also responsible. First, we found no significant change in HVSMC surface expression of VCAM-1 and ICAM-1 at up to 6 h of ANG II or PDGF-BB treatment (by flow cytometry; data not shown). Second, HVSMC fixed with 2% paraformaldehyde after stimulation with ANG II or PDGF-BB (to prevent HVSMC from releasing soluble factors) failed to induce THP-1 cell binding (Fig. 3A). In contrast, THP-1 cells stimulated with conditioned medium (CM) from ANG II- or PDGF-BB-treated HVSMC showed significantly increased binding to HVSMC (by 92.8 and 100.6%, respectively; P < 0.001) (Fig. 3B).



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Fig. 3. Role of soluble factors in ANG II- and PDGF-BB-induced HVSMC-THP-1 cell binding. A: after stimulation with ANG II or PDGF-BB for 6 h, HVSMC were either left unfixed or were fixed with paraformaldehyde to prevent release of soluble factors and were then used in binding assays. *P < 0.001 vs. basal binding (n = 6). B: after stimulation with ANG II or PDGF-BB for 6 h, HVSMC-conditioned media (CM) were collected. THP-1 cells stimulated with CM were left to bind to unstimulated HVSMC. *P < 0.001 vs. control CM (n = 6).

 
Arachidonic acid metabolism and HVSMC-THP-1 binding: Effects of inhibiting or overexpressing 12/15-LO and COX-2. Specific inhibitors were first employed to investigate the role of 12/15-LO and COX-2, two major arachidonic acid (AA)-metabolizing enzymes, in ANG II- and PDGF-BB-induced monocyte binding. As shown in Fig. 4A, pretreatment of HVSMC with an inhibitor of 12/15-LO (1 x 10–5 M CDC) or of COX-2 (2 x 10–5 M NS-398) significantly attenuated binding induced by ANG II and PDGF-BB. These results suggest that LO and COX-2 pathways could be involved, at least in part, in ANG II- and PDGF-induced binding.



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Fig. 4. Arachidonic acid (AA) metabolism and HVSMC-THP-1 cell binding. A: before stimulation with ANG II or PDGF-BB and subsequent binding assay, HVSMC were pretreated with the lipoxygenase (LO) inhibitor cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate (CDC; 1 x 10–5 M) or the cyclooxygenase (COX)-2 inhibitor NS-398 (2 x 10–5 M) for 30 min. *P < 0.01 vs. ANG II or PDGF-BB stimulation without inhibitors. B: HVSMC were treated for 6 h with AA. *P < 0.01, {dagger}P < 0.001 vs. basal binding. C: before stimulation with AA and subsequent binding assay, HVSMC were pretreated with CDC (1 x 10–5 M) or NS-398 (2 x 10–5 M), or both, for 30 min. *P < 0.001 vs. basal binding. {dagger}P < 0.01, {ddagger}P < 0.001 vs. AA without inhibitors. D: HVSMC were treated for 6 h with 1 x 10–7 M 12(S)-hydroxyeicosatetraenoic acid (HETE), 12(R)-HETE, 5(S)-HETE, or 15(S)-HETE. *P < 0.01, {dagger}P < 0.05 vs. basal binding. E: direct stimulation of THP-1 cells for 30 min with 1 x 10–4 M AA or with 1 x 10–7 M 12(S)-HETE, 15(S)-HETE, 12(R)-HETE, or 5(S)-HETE before binding assay. *P < 0.01 vs. basal binding. F: direct stimulation of THP-1 cells for 30 min with PGE2. *P < 0.01 vs. basal binding (n = 6).

 
Direct stimulation of HVSMC with AA also dose-dependently increased THP-1 cell binding (Fig. 4B). The effects of AA were significantly inhibited by LO or COX-2 inhibitor (CDC or NS-398, respectively) and almost completely blocked by a combination of both (Fig. 4C). These results further implicated LO and COX products produced by HVSMC in the adhesion process. We then examined whether LO and COX-2 products could directly induce binding. Direct stimulation of HVSMC with 12(S)- or 15(S)-HETE, two major 12/15-LO products, also significantly increased binding by 60.3% (P < 0.01) and 46.3% (P < 0.05), respectively, whereas 5(S)-HETE, a 5-LO metabolite, was only moderately effective (P > 0.05) (Fig. 4D). In contrast, 12(R)-HETE, an enantiomer of 12(S)-HETE and not a LO product, was ineffective (Fig. 4D). Interestingly, direct stimulation of THP-1 cells with HETEs (but not with AA) for 30 min also yielded similar results (Fig. 4E). This 30-min time period was chosen to mimic the contact time period of THP-1 cells with HVSMC during the adhesion assay, the only time during which THP-1 cells could be exposed to AA. Preincubation of THP-1 cells with PGE2, a COX-2 metabolite of AA, also dose-dependently increased binding (Fig. 4F).

We next hypothesized that if 12/15-LO products played a role in monocytes binding to VSMC, then VSMC derived from LOKO mice (MVSMC) would show less binding than genetic control mice. MVSMC from LOKO mice showed a 47.5% decrease in binding to WEHI78/24, a mouse monocytic cell line that has been successfully used to show binding to mouse endothelial cells (12), compared with VSMC from WT mice (P < 0.001) (Fig. 5A). This finding is consistent with data showing that LOKO VSMC produce significantly lower amounts of 12(S)-HETE relative to VSMC from WT mice (27).



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Fig. 5. Further evidence for the role of 12/15-LO and COX-2 in binding. A: VSMC from 12/15-LO knockout (LOKO) mice or WT were used in binding assays with mouse monocytic WEHI cells. *P < 0.001 vs. WT. B: porcine VSMC (PVSMC) were transfected with mouse 12/15-LO cDNA (pcDNA-m12-LO) or an empty vector (pcDNA3.1) before binding with THP-1 cells. *P < 0.001 vs. vector. C: PVSMC were transfected with human COX-2 cDNA or empty vector before binding with THP-1 cells. *P < 0.001 vs. vector. Data represent means ± SE of 6 experiments. Insets: overexpression of 12/15-LO and COX-2 detected by Western blotting.

 
Because 12/15-LO or COX-2 blockade could attenuate binding, we next examined whether gain-of-function by overexpression of these genes in PVSMC can reciprocally increase binding. Our unpublished results showed that PVSMC behave much like HVSMC with respect to binding to THP-1 cells. PVSMC transfected with mouse 12/15-LO cDNA or human COX-2 cDNA displayed significantly increased binding to THP-1 cells [69.9% (P < 0.01) and 75.7% (P < 0.001), respectively] relative to pcDNA empty vector (Fig. 5, B and C, respectively). Increased 12/15-LO and COX-2 protein expression by these transfections is documented in Fig. 5, B and C, insets.

Signal transduction pathways involved in HVSMC-THP-1 cell binding. Specific signal transduction inhibitors were used to explore potential signaling pathways involved in growth factor-induced monocyte binding to VSMC. ANG II-induced binding was significantly attenuated by inhibitors of PI3K, Src kinase, ERK1/2, and p38 MAPK, whereas the effects of PDGF-BB were blocked by inhibitors of PI3K and Src kinase. These inhibitors did not affect THP-1 binding to untreated control VSMC (Fig. 6).



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Fig. 6. Signal transduction pathways involved in HVSMC-THP-1 cell binding. HVSMC were preincubated for 30 min with or without inhibitors of phosphoinositide 3-kinase (PI3K; LY-294002, 2 x 10–5 M), Src (PP2, 1 x 10–5 M), ERK1/2 (PD-98059, 5 x 10–5 M), or p38 MAPK (SB-202190, 5 x 10–6 M), followed by stimulation with ANG II or PDGF-BB for 6 h. Binding assay was performed with THP-1 cells. *P < 0.001; {dagger}P < 0.01 vs. ANG II or PDGF-BB without inhibitors. Data represent means ± SE of 6 experiments.

 
Induction of THP-1 cell surface CD36 expression upon interaction with HVSMC. To determine the potential pathological consequences, we examined whether THP-1 cell CD36 expression could be upregulated upon binding to VSMC. As illustrated in Fig. 7A, in the basal state, only ~5.0% of THP-1 cells showed surface expression of CD36 compared with isotype control cells (b vs. a). After 24 h of coculture, cells bound to HVSMC were 12.5% positive for CD36 (P < 0.001) (c). Unbound THP-1 cells, i.e., cells remaining in suspension above the VSMC, also showed a similar increase in surface CD36 expression (14.5%, P < 0.001) (e). CD36 mRNA levels also were elevated in bound and unbound THP-1 cells after coculture (data not shown). The percentage of CD36-positive cells was further increased by 6-h preincubation of HVSMC with PDGF-BB (22.0 and 21.7% for bound and unbound THP-1 cells, respectively) (d and f). Both direct cell-cell contact and soluble factors released from HVSMC appeared responsible, because THP-1 cells either incubated with HVSMC-conditioned medium (g and h, with and without PDGF, respectively) or bound to HVSMC fixed with paraformaldehyde (i) showed increased CD36 expression. Figure 7B shows a bar graph representing the flow cytometry data from multiple experiments.



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Fig. 7. Induction of monocyte CD36 expression upon coincubation with HVSMC. A: representative THP-1 cell flow cytometry data. After coculture with HVSMC for 24 h, bound and unbound THP-1 cells were collected separately, followed by flow cytometry analysis of surface CD36 expression: a, isotype control; b, basal THP-1 cells; c, THP-1 cells bound to HVSMC; d, THP-1 cells bound to PDGF-stimulated HVSMC (8 x 10–10 M, 6 h); e, unbound THP-1 cells after coculture; f, unbound THP-1 cells after coculture with PDGF-stimulated HVSMC; g, THP-1 cells incubated in HVSMC CM; h, THP-1 cells incubated with CM from PDGF-stimulated HVSMC; i, THP-1 cells bound to HVSMC fixed with paraformaldehyde. PE, phycoerythrin. B: quantitation of THP-1 cell flow cytometry data (n = 3). *P < 0.001 vs. basal. {dagger}P < 0.001, PDGF vs. control. C: PBMC flow cytometry data (n = 3). PBMC were cocultured with HVSMC for 24 h, and surface CD36 expression of bound PBMC was analyzed by flow cytometry. *P < 0.001 vs. basal.

 
To confirm that similar results are obtained with isolated primary human monocytes, we repeated the experiments using PBMC from normal human volunteers. Figure 7C shows that there is a similar, significant increase in CD36 expression in human PBMC when they bind to HVSMC.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mechanisms responsible for the intimal persistence of monocytes-macrophages in the pathogenesis of atherosclerosis are not very clear. Our studies indicate for the first time that ANG II and PDGF-BB, apart from their other well-known vascular effects, can also induce the binding of monocytes to HVSMC monolayers. These growth factors may therefore facilitate the progression of atherosclerosis by augmenting VSMC-monocyte adhesive interactions.

Increased binding did not appear to be due to elevated surface adhesion molecule expression on VSMC, at least not in the short incubation time period of our present experiments. However, VCAM-1 and ICAM-1 may still be involved during longer cellular contact and under in vivo conditions, because both ANG II and PDGF-BB have been shown to induce their expression in VSMC in vitro (3, 21, 37). Furthermore, in our present study, VSMC were 100% confluent before stimulation by ANG II or PDGF-BB. Evidence shows that PDGF-BB stimulates ICAM-1 expression in subconfluent but not confluent HVSMC (21), suggesting that cell confluence is an important factor determining adhesion molecule expression. Our current data indicate that soluble factors released from VSMC by ANG II or PDGF-BB are mainly responsible for enhanced VSMC-monocyte binding under our experimental conditions. In the vessel wall in vivo, both adhesion molecules and soluble factors may mediate VSMC-monocyte interactions.

VSMC can release a variety of soluble factors upon stimulation with ANG II and PDGF-BB. Among the potential mediators, we focused on AA and its metabolites because they are released by growth factors via phospholipase actions and can regulate cardiovascular functions. AA is further metabolized by LO to HETEs or by COX pathways to prostaglandins such as PGE2 (33). Rat, murine, and porcine leukocytes and VSMC express a leukocyte-type 12/15-LO (10, 23). These LOs incorporate molecular oxygen into AA to form 12(S)- and 15(S)-HETE. Considerable evidence now supports a role for 12/15-LO in promoting atherosclerosis and restenosis (4, 10, 24). Both ANG II and PDGF-BB significantly increase 12/15-LO activity and expression in VSMC (22, 23). Furthermore, the growth-promoting effects of ANG II and the chemotactic effects of PDGF-BB are mediated, at least in part, by 12/15-LO (22, 23). On the other hand, COX-2 and its products have inflammatory properties. Increased COX-2 expression also has been demonstrated in atherosclerotic plaques (29). Furthermore, ANG II and PDGF-BB can induce COX-2 expression and PGE2 production in VSMC (8, 13, 19).

Our following observations clearly demonstrate the involvement of AA and its metabolites for the first time in ANG II- and PDGF-BB-induced VSMC-monocyte interactions. 1) Inhibition of 12/15-LO or COX-2 activity in VSMC significantly reduced ANG II- and PDGF-induced binding of THP-1 cells to HVSMC, whereas overexpression of 12/15-LO or COX-2 in VSMC was able to reciprocally increase binding. 2) MVSMC derived from 12/15-LO knockout mice showed decreased binding to mouse monocytes compared with those derived from WT mice. This is further supported by our unpublished data showing that rat VSMC transfected with expression plasmids for short interfering RNAs (siRNAs) directed against rat 12/15-LO showed decreased monocyte binding compared with mock-transfected cells. 3) Direct stimulation of either HVSMC or THP-1 cells with 12(S)- or 15(S)-HETE mimicked the effects of ANG II and PDGF-BB; 5(S)-HETE had no effect, although leukotrienes, which are also 5-LO products, were not tested. 4) Stimulation of THP-1 cells with the COX-2 inflammatory metabolite PGE2 also induced binding. 5) Direct stimulation of HVSMC with AA also increased binding. This effect was blocked by 12/15-LO or COX-2 inhibitors, indicating that AA acts via its metabolites. Interestingly, in contrast to its effects on VSMC, AA had no direct effect on THP-1 cells, suggesting that it is not AA itself but its metabolism in VSMC to LO and COX products that is responsible for enhanced binding.

The precise mechanisms by which 12(S)- and 15(S)-HETE and PGE2 increase VSMC-monocyte binding are unclear. Evidence shows that 12(S)-HETE can activate protein kinase C and several MAPKs (24). HETEs can also be incorporated into membrane phospholipids, which then generate HETE-containing diacylglycerol species to activate protein kinase C (18). 12(S)-HETE can also induce monocyte binding to endothelial cells via activation of {beta}1-integrin and CS-1 fibronectin (26). Similar mechanisms may be operative in VSMC. Studies in HeLa cells indicate that LO and COX enzymes mediate cell adhesion to extracellular matrix via cytoskeletal reorganization (34). The acute effects of ANG II and PDGF-BB most likely occur via phospholipase activation leading to increased substrate AA and metabolite availability, whereas chronic effects may occur via an increase in the expression of LO and COX-2 enzymes. It is also well known that ANG II is a potent inducer of NADPH oxidase and associated oxidant stress, which has been attributed to several of the pathological effects of ANG II (11). In unpublished studies, we have shown that a general antioxidant, pyrrolidine dithiocarbamate, can attenuate ANG II-induced binding. However, two NAPDH oxidase inhibitors, diphenylene iodonium and apocynin, had no significant effect on ANG II-induced monocyte binding. Hence, although oxidant stress may be involved, NADPH oxidase may not be a direct mediator. Additional studies are needed in this area.

It is also possible that monocyte chemotactic protein-1 (MCP-1) could be another potential soluble mediator of the growth factor-induced VSMC-monocyte interactions, because our unpublished data show that a MCP-1 antibody can partially block ANG II- or PDGF-BB-induced binding. Furthermore, our recent report (6) showed that LO products can directly increase MCP-1 expression in VSMC.

In our evaluation of the potential signal transduction mechanisms, both ANG II- and PDGF-BB-induced binding seemed to involve Src and PI3K activation. It has been demonstrated that inhibition of PI3K abolishes ANG II-induced cytosolic phospholipase A2 (cPLA2) phosphorylation and AA release (32). In our study, ANG II-induced VSMC-monocyte interactions also appeared to be mediated by MAPKs. This observation is in accordance with a recent report that activation of both ERK1/2 and p38 MAPK is required for the full induction of COX-2 expression by ANG II (13). Our studies also implicate Src activation for the first time in VSMC-monocyte interactions. Further studies are needed to examine the full significance of Src activation in these responses.

Monocyte-macrophage differentiation is accompanied by increased expression of scavenger receptors such as CD36 that are involved in lipid uptake and foam cell formation. CD36 has been documented to play a key role in the pathogenesis of atherosclerosis (9, 16), although it is not the only ox-LDL receptor. We noted for the first time that THP-1 cell expression of CD36 was significantly increased after coincubation with VSMC. Interestingly, these effects were further augmented by PDGF-BB. Both direct cell-cell contact and soluble factors derived from VSMC seemed to be involved in CD36 upregulation. The precise underlying molecular mechanisms are not yet clear. However, unpublished data from our laboratory show that {beta}1-integrin activation can induce CD36 expression in THP-1 cells. Adhesion-modulated CD36 mRNA expression in monocytes also occurs upon their interaction with tumor necrosis factor-{alpha}-treated endothelium (15). Furthermore, recent studies have shown that MCP-1 can induce CD36 expression and that other soluble factors, such as unsaturated fatty acids, including 12/15-LO products, can promote monocyte CD36 expression and act as seeding molecules in LDL via peroxisome proliferator-activated receptor-{gamma}-related mechanisms (14, 36). Collectively, our new results show for the first time that VSMC-monocyte interactions may be regulatory signals in the phenotypic transformation to macrophages. This could be one of the mechanisms for the dramatically accelerated atherosclerosis observed in ANG II-infused rodent models (5).

In summary, as shown in Fig. 8, when growth factors bind to their VSMC surface receptors, signals such as Src and PI3K (for ANG II and PDGF-BB) and MAPK (for ANG II) are activated and lead to phospholipase-mediated release of AA and its metabolites 12(S)- and 15(S)-HETE and PGE2. Once released, these soluble factors activate monocyte integrins and stimulate VSMC-monocyte binding interactions, leading to monocyte CD36 expression. This can induce ox-LDL internalization, monocyte-macrophage differentiation, and foam cell formation. Overall, these new observations demonstrate that, in addition to inducing VSMC growth and migration, another role for ANG II or PDGF-BB in atherosclerosis is to facilitate monocyte retention and foam cell formation.



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Fig. 8. Schematic diagram showing how growth factors can induce VSMC-monocyte binding interactions, monocyte CD36 expression, and foam cell formation. EC, endothelial cell; ox-LDL, oxidized low-density lipoprotein.

 

    GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported by National Institutes of Health Grants R01 DK-065073 and P01 HL-55798, Juvenile Diabetes Foundation International Grant 1-2001-108, and, in part, by General Clinical Research Center Grant M01 RR-00043 (from National Center for Research Resources to City of Hope Medical Center).


    ACKNOWLEDGMENTS
 
We thank Dr. J. Berliner (University of California, Los Angeles) for WEHI78/24 cells, Dr. T. Hla (University of Connecticut) for the COX-2 expression vector, Dr. C. Funk (University of Pennsylvania) for the mouse 12/15-LO expression vector, and L. Brown and C. Spalla for help with flow cytometry.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Natarajan, Gonda Diabetes Center, Beckman Research Institute of City of Hope, 1500 East Duarte Rd., Duarte, CA 91010 (E-mail: rnatarajan{at}coh.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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