Human Macrophage-Induced Vascular Smooth Muscle Cell Apoptosis Requires NO Enhancement of Fas/Fas-L Interactions
Objective— We have previously shown that macrophages induce vascular smooth muscle cell (VSMC) apoptosis in vitro by cell-cell proximity and Fas-L/Fas interactions. Because NO is a short-range mediator, we tested whether NO mediates macrophage-induced VSMC apoptosis.
Methods and Results— NO synthase (NOS) inhibitors markedly inhibited macrophage-induced apoptosis of carotid plaque VSMCs (apoptotic indices, 81±2.9% for control and 28.2±3.9% for NG-nitro-l-arginine methyl ester [L-NAME] treatment) and coronary medial VSMCs (apoptotic indices, 76±5.5% for control and 3.5±0.8% for L-NAME treatment). Inactive enantiomers were without effect (P>0.05). Cultured macrophages, but not VSMCs, expressed inducible NOS (but not neuronal NOS or endothelial NOS) concomitant with activation and secreted 1.51±0.3 fmol nitrite per cell, which was blocked by L-NAME (100 μmol/L). Diethylene triamine nitric oxide (DETA/NO) and sodium nitroprusside (NO donors) induced VSMC cell-surface Fas and enhanced plaque VSMC apoptosis induced by agonistic anti-Fas antibody (apoptotic indices, 6.6±1.8% for control, 6.3±1.5% for DETA/NO, 26±1.8% for Fas, and 44±6.9% for Fas+DETA/NO). In isolated macrophages, NOS inhibitors reduced and NO donors increased surface Fas-L, indicating an NO-dependent autocrine enhancement of macrophage surface Fas-L.
Conclusions— Together, these data indicate that macrophage-derived NO is required for macrophage-induced VSMC apoptosis and that it acts by enhancing Fas-L/Fas interactions.
The rupture of human atherosclerotic plaques causes myocardial and cerebral infarction.1 Atherosclerotic plaque rupture is associated with infiltration of the fibrous cap by macrophages and T lymphocytes and with reduced numbers of vascular smooth muscle cells (VSMCs).1,2⇓ An important mode of loss of plaque in VSMCs is through apoptosis,3 which is increased in plaque compared with normal vessels and is higher in ruptured than in stable plaque.4,5⇓ Because VSMCs are the only cells within plaque that are capable of synthesizing structurally important collagen isoforms, VSMC apoptosis may therefore promote plaque rupture.6
We have recently shown that human blood–derived macrophages induce human VSMC apoptosis in coculture via cell-cell proximity and Fas-L/Fas signaling.7 Macrophage-derived NO is cytotoxic to tumor cells8,9⇓ by an uncertain mechanism and has a short half-life and path length in physiological solution.10 Therefore, we wondered whether NO contributes to proximity-dependent macrophage-induced VSMC apoptosis.
Macrophages synthesize the unstable free radical NO.11 There are 3 isoenzymes of NO synthase (NOS): NOS I, NOS II, and NOS III.11 NOS I (endothelial NOS [eNOS]) is expressed by endothelial cells, and NOS III (neuronal NOS [nNOS]) is expressed by neurons.11 NOS I and NOS III are low-output pathways.11 The inducible NOS (iNOS) pathway is a high-output pathway that may liberate NO in a sufficient concentration to damage by direct free radical modification.11 Macrophages may express iNOS, indicating that iNOS is the likeliest isoform for mediating macrophage-induced VSMC apoptosis.11
Although rodent macrophages secrete high concentrations of NO,11 it is unclear whether human macrophages liberate sufficient concentrations of NO to be directly toxic.11 Some reports suggest that human macrophages do not liberate NO,12 whereas others have recorded small amounts of NO from stimulated human macrophages.10,13⇓ Even if human macrophages do not liberate sufficient NO to directly induce apoptosis, NO could contribute to macrophage-induced VSMC apoptosis by indirect mechanisms. Recent reports indicate that NO may stimulate fibroblast apoptosis by inducing the proapoptotic gene p53.14 We have shown that p53 induces surface Fas expression in VSMCs via translocation of a preformed pool from the Golgi apparatus.15 If NO induces p53, then it is possible that NO may induce the translocation of Fas to the cell surface. Conversely, leukocyte Fas-L may be activated by trafficking from the cytoplasm to the cell surface in calcium ionophore–activated T lymphocytes.16 In neutrophils, similar trafficking (so-called degranulation) may be stimulated by cGMP, a second messenger of NO.17
We tested the hypothesis that NO contributes to human macrophage–induced VSMC apoptosis. Our data indicate that NO contributes to macrophage-induced VSMC apoptosis by upregulating VSMC surface expression of the death receptor, Fas, and macrophage expression of the death ligand, Fas-L.
Culture of Macrophages and VSMCs.
Human peripheral blood–derived macrophages were prepared, as described previously,7,18,19⇓⇓ by differential centrifugation and adherence (for details, please see expanded Methods, available online at http://atvb.ahajournals.org). Human aortic VSMCs and plaque VSMCs were isolated and cultured as before (for details, please see online Methods).7,20⇓
Detection of Apoptosis in Macrophage/VSMC Cocultures
Macrophages and VSMCs were cocultured as described previously.7 VSMC apoptosis was assessed as described before by differential staining of live and apoptotic cells with DNA fluorochromes and differential staining of VSMCs and macrophages with a fluorescent macrophage marker (for details, please see online Methods).7,18⇓ To examine the contribution of NOS to apoptosis in cocultures, apoptosis in cocultures was assessed in the presence or absence of accepted NOS inhibitors and enantiomeric controls.
Western Blotting, Flow Cytometry, and Immunohistochemistry
Flow cytometry confirmed that we were culturing 99% pure blood–derived macrophages.7 Western analysis of macrophage and VSMC protein lysates for iNOS, flow cytometric analysis of expression of iNOS in VSMCs and macrophages, and immunohistochemistry of autopsy-ruptured plaques were performed by an optimization of previous methods7 (please see online Methods).
Analysis of NO-Derived Nitrite
Statistical analysis was carried out with SigmaStat on a PC. Binomially distributed data were analyzed by ANOVA, and other data were analyzed by the Dunn test, a nonparametric ANOVA. Significance was adjusted for multiple simultaneous comparisons.
Effects of NOS Inhibitors on Macrophage-Induced Plaque and HCMED1-E6 VSMC Apoptosis
To examine the mechanism of macrophage-induced VSMC apoptosis, macrophages were cultured from human peripheral blood, and VSMCs were cultured from human carotid plaques. Macrophages and VSMCs were cocultured for 8 days, and VSMC apoptosis was assessed by using DNA-binding dyes and the macrophage-marker CD14-FITC. The apoptotic index in CD-14–negative cells was assessed (Figure 1A). The data are expressed as apoptotic index (mean±SEM, n=20 per data point): control VSMCs 5.2±0.9%, macrophage coculture 81±2.9%, and 100 μmol/L NG-monomethyl-l-arginine (L-NMMA) 39.5±5.1% (P<0.00001 by ANOVA). NG-Nitro-l-arginine methyl ester (L-NAME, 3 to 300 μmol/L) reduced macrophage-induced apoptosis in a dose-related manner, with half-maximal inhibition of ≈5 μmol/L and a maximum of ≈60% inhibition at 100 μmol/L (apoptotic index 28.2±3.9%; Figure 1A and online Figure I, which is available at http://atvb.ahajournals.org; P<0.00001 by ANOVA). Matched concentrations (100 μmol/L) of the (control) inactive enantiomers d-NAME and d-NMMA were without effect (d-NAME 71.5±4% [n=20, P>0.05] and d-NMMA 70±7.3%, P>0.05; Figure 1A). Thus, the NOS inhibitors L-NAME and L-NMMA (100 μmol/L) inhibited macrophage-induced VSMC apoptosis, confirming a role for NO.
HCMED1-E6 VSMCs are representative of aortic VSMCs and plaque-derived VSMCs in studies of macrophage-induced apoptosis7 but are more easily studied7 (see Methods). Therefore, we assessed the dependence of macrophage-induced HCMED1-E6 VSMC apoptosis on NO (Figure 1B). Data are expressed as mean±SEM apoptotic index (n=28 per data point): basal HCMED1-E6 VSMC apoptosis 7.6±2.9%, macrophage coculture 76±5.5%, L-NAME 3.5%±0.8% (P<0.00001 by Dunn test), L-NMMA 16±6.7% (P<0.00001 by Dunn test), d-NAME 76±11% (P>0.05), and d-NMMA 75±4% (P>0.05). Thus, HCMED1-E6 VSMCs show similar levels of basal and macrophage-induced apoptosis to plaque-derived VSMCs.
An iNOS selective inhibitor, l-N6-(1-iminoethyl)lysine (L-NIL), has become available. L-NIL reduced macrophage-induced apoptosis in a concentration-dependent manner, with a concentration for half-maximal effect of <0.3 μmol/L. This was more potent than L-NAME and is in keeping with published estimations of the EC50 of L-NIL for the iNOS isoform. This indicates that NO derived from macrophages is required for macrophage-induced VSMC apoptosis and implicates the iNOS isoform.
Cultured Macrophages Express iNOS
Flow cytometry was used to assess iNOS expression by fresh peripheral blood monocytes before purification by double staining for iNOS and the monocyte marker CD14. Freshly isolated monocytes were negative for iNOS (Figure 2A). In contrast, macrophages in early culture (culture day 1) and macrophages in later culture (culture day 8) strongly expressed intracellular iNOS, consistent with the Western blot data. This indicated that the adherence separation and 1 day of culture were sufficient to induce iNOS. Cultured macrophages did not express eNOS or nNOS by flow cytometry (Figure 2A). In contrast, eNOS and nNOS were identified in peripheral blood neutrophils, which are known to express eNOS and nNOS (not shown).
Macrophages in early culture (culture day 1) and late culture (culture day 8) expressed the cell-surface activation markers CD16 (FcγRIII) and HLA-DR. This indicated that iNOS upregulation in cultured macrophages is part of a more generalized activation that occurs after 1 day of culture.
Western analysis confirmed iNOS at similar levels in macrophages at culture day 1 through culture day 8 but did not detect iNOS in VSMCs (Figure 2B).
Cultured Macrophages, but Not VSMCs, Synthesize NO
Macrophage nitrite production over an 8-day period was 1.51±0.3 (range 0.3 to 5.4) fmol per cell (mean±SEM, n=16 donors). Griess reactivity was blocked with 100 μmol/L L-NAME, the dose used to block functional responses, indicating that the nitrite detected reflected NO generated by NOS (n=3 donors, not shown). By use of this assay, confluent VSMCs showed no detectable nitrite efflux over a culture period of 1 week (not shown).
NO Donors SNP and DETA/NO Upregulate Cell-Surface Fas on VSMCs
For macrophages to induce VSMC apoptosis via cell-cell contact and Fas/Fas-L, macrophages must express Fas-L on the cell surface, and VSMCs must express Fas on the cell surface.7 We assessed VSMC surface Fas expression by flow cytometry in the presence or absence of DETA/NO.
Diethylene triamine nitric oxide (DETA/NO) (1 mmol/L), but not DETA (1 mmol/L), induced cell-surface expression of Fas in HCMED1-E6 VSMCs (n=3, Figure 3A). DETA/NO (1 mmol/L) also upregulated surface Fas on aortic VSMCs (n=3 experiments), indicating that this was probably a more general property of human VSMCs (Figure 3B). Sodium nitroprusside (SNP, 1 mmol/L) increased HCMED1-E6 VSMC surface Fas expression from 30% Fas-positive cells to 60% Fas-positive cells (Figure 3C). SNP induced cell-surface receptors (at 5 to 10 hours) more rapidly than DETA/NO (at 24 hours), consistent with its more rapid release of NO. Thus, NO donors upregulated surface Fas on VSMCs.
NO-Induced Upregulation of Surface Fas on VSMCs Is by Translocation From the Golgi Apparatus
To assess surface and intracellular expression of Fas, we compared flow cytometric analysis of permeabilized or nonpermeabilized VSMCs (please see online Figure IIA, which is available at http://atvb.ahajournals.org). There was some surface expression of Fas in aortic VSMCs, but expression was increased in permeabilized cells, indicating that Fas was predominantly intracellular (n=5 donors). Fas expression in HCMED1-E6 VSMCs was entirely intracellular.
Immunofluorescence microscopy showed that Fas colocalized with the 68-kDa Golgi marker in a perinuclear distribution consistent with the Golgi apparatus (please see online Figure IIB). Therefore, we assessed the effects of brefeldin-A, an agent that disrupts the Golgi apparatus15 and prevents p53-induced Fas trafficking,15 on DETA/NO-induced surface Fas expression (please see online Figure III, available at http://atvb.ahajournals.org). Brefeldin-A (5 μg/mL) blocked DETA/NO-induced surface Fas translocation. The protein synthesis inhibitor cycloheximide (10 μg/mL) did not prevent DETA/NO-induced surface Fas expression. Consistent with this, Western blot analysis showed that DETA/NO did not increase cellular Fas in VSMCs over the same time course (not shown). Thus, NO donors upregulated VSMC surface Fas expression from a preformed intracellular (Golgi) pool.
NO Donor DETA/NO Sensitizes Plaque and Coronary Medial VSMCs to Fas Activation
We tested whether the concentration of DETA/NO used to study the upregulation of VSMC surface Fas would sensitize HCMED1-E6 and nonimmortalized plaque-derived VSMCs to apoptosis induced by an agonistic anti-Fas antibody (please see online Figure IV, available at http://atvb.ahajournals.org).
Data are expressed as mean±SEM apoptotic indices: basal 1.9±0.4%, DETA/NO-treated 1.9±0.3%, anti-Fas 2.6±0.5%, and anti-Fas+DETA/NO 50±6.6%. Only the combination of agonistic anti-Fas antibody and DETA/NO induced significant HCMED1-E6 VSMC apoptosis (P<0.05 by ANOVA), indicating that DETA/NO sensitizes HCMED1-E6 VSMCs to Fas-induced apoptosis.
Data are expressed as mean±SEM apoptotic indices (n=8 per data point, duplicate values for plaque VSMCs from 4 donors): basal 6.6±1.8%, DETA/NO (100 μmol/L) 6.3±1.5%, agonistic anti-Fas antibody 26±1.8%, and anti-Fas+DETA/NO 44±6.9%. DETA/NO had no effect on its own (P>0.05 by ANOVA). Thus, DETA/NO, at a concentration insufficient to induce apoptosis directly, sensitizes plaque VSMCs to Fas-induced apoptosis.
Macrophage-Derived Autocrine NO Is Required for Expression of Surface Fas-L
To examine whether NO also promoted macrophage-induced VSMC apoptosis through effects on the macrophages themselves, we examined NO-dependent regulation of surface Fas-L on macrophages in monoculture by using iNOS inhibitors and NO donors. We have previously shown that Fas-L appears on the surface of cultured macrophages between culture day 4 and culture day 6. Therefore, whether NO induces surface Fas-L was tested with macrophages at culture day 4, and whether NO inhibition reduces Fas-L was tested with macrophages at culture day 6. DETA/NO induced surface Fas-L expression in day-4 macrophages (please see online Figure V, available at http://atvb.ahajournals.org; n=3 experiments on macrophages from 3 donors). In contrast, L-NAME (100 μmol/L) inhibited surface expression of Fas-L on day-6 macrophages (please see online Figure V; n=3 experiments with different macrophage donors). Thus, macrophage surface Fas-L expression with differentiation in culture is dependent on NO.
Expression of iNOS in Plaques
Our data suggested that macrophage iNOS generates NO, inducing Fas-L on macrophages and Fas on VSMCs. The consequent NO/Fas-induced VSMC apoptosis may then promote plaque rupture. To study whether iNOS expression colocalized with macrophages at sites of plaque rupture, an area associated with VSMC apoptosis, we examined the expression of iNOS in ruptured atherosclerotic plaques by immunohistochemistry (please see online Figure VI, available at http://atvb.ahajournals.org). At sites of rupture, human coronary atherosclerotic plaques contained macrophages immunoreactive for CD68, iNOS, and Fas-L. We also stained ruptured plaques by using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) reaction. However, rupture-prone plaques contain few VSMCs. There were <10 VSMCs in the vicinity of the fibrous cap macrophage infiltrates, none of which showed specific TUNEL labeling, consistent with estimates of a TUNEL index <10%. Thus, these data are consistent with the hypothesis that macrophages in ruptured plaques coordinately expressed Fas-L and iNOS.
The present study demonstrates a pivotal role for iNOS in macrophage-induced VSMC apoptosis. Namely, NOS inhibition reduces macrophage-induced apoptosis of VSMCs in a dose-dependent manner; NO donors upregulated VSMC surface Fas from a Golgi pool and promoted Fas-induced apoptosis. Macrophage surface Fas-L expression is NO dependent, and Fas-L and iNOS are colocalized in plaque macrophages.
Potential roles of NO in atherogenesis have been studied previously.22 However, the emphasis has previously been on protective roles of NO derived from the low-output isoenzyme eNOS.22 This and other recent reports depart from this concept.23 The implication of the data in the present study is that NO may (sometimes) be deleterious in atherogenesis, akin to its destructive effects in other inflammatory conditions.11,24⇓ NOS II (iNOS) has been most closely linked to cytotoxicity.11 This is probably related to NO concentration: NO is well understood to be a bifunctional mediator of apoptosis and at low concentrations may even inhibit apoptosis.25 In the present study, we show that cultured macrophages express iNOS at high levels but do no not express eNOS or nNOS and that macrophage-induced apoptosis is sensitive to the iNOS-selective inhibitor L-NIL at an EC50 appropriate for the iNOS isoenzyme.26,27⇓ This implicates iNOS in macrophage-induced VSMC apoptosis, consistent with a view of iNOS as a damaging pathway.
To confirm that inhibition of macrophage-induced apoptosis is iNOS-related, it was necessary to show that iNOS is expressed and generates NO. In agreement with the literature,11 iNOS protein was expressed, and NO was secreted by cultured macrophages but not cultured VSMCs. Although the levels of nitrite were extremely low, they were entirely in accord with the levels previously obtained with human macrophages, which are very much lower than the levels reported in rodents.12 Expression of iNOS was concomitant with surface expression of HLA-DR and CD16, which are recognized macrophage activation markers in culture.28 Thus, in culture, human blood–derived macrophages become activated, express iNOS, and synthesize NO, which promotes cell-cell proximity-dependent VSMC apoptosis in addition to Fas-L/Fas interactions. Because VSMCs did not express detectable nitrite or iNOS, macrophage-derived NO acted on VSMCs themselves or on both to promote apoptosis.
L-NAME effectively blocked macrophage-induced HCMED1-E6 VSMC apoptosis, indicating an important role for NO in macrophage-induced VSMC apoptosis. With the plaque VSMCs, L-NAME (100 μmol/L) blocked macrophage NO secretion but only inhibited macrophage-induced plaque VSMC apoptosis by 60%, suggesting an NO-independent mechanism accounting for ≈40% of apoptosis. Similarly, inhibition of Fas/Fas-L inhibits macrophage-induced plaque VSMC apoptosis by up to 60%.7 These data, along with the strong synergy seen between NO and Fas in plaque VSMC apoptosis and also with NO-induced surface Fas expression, suggest an NO/Fas-L pathway in macrophage-induced VSMC apoptosis.
We have previously found that macrophage-induced apoptosis depends in part on Fas/Fas-L. This suggested that there may be cross talk between apoptosis stimulated through Fas and NO. Although human plaque VSMCs, which are highly sensitive to apoptosis, express high levels of surface Fas, human coronary medial VSMCs mainly express Fas intracellularly in the Golgi complex, where it is effectively sequestered from Fas-L binding.7,15⇓ In the present study, we confirmed, with double immunofluorescence, that human coronary medial VSMCs contain Fas in a Golgi location. Translocation from the Golgi apparatus may be inhibited by the drug brefeldin A, which disrupts Golgi trafficking.15,29,30⇓⇓ With coronary medial VSMCs, surface expression was induced by NO donors DETA/NO and SNP and appeared to be via translocation from the preformed Golgi pool, inasmuch as it was blocked by brefeldin A but not protein synthesis inhibitors.15 NO donors upregulated Fas on nonimmortalized human aortic VSMCs, which express the majority of Fas intracellularly, widening the relevance of this finding. Furthermore, previous studies have shown that HCMED1-E6 VSMCs show identical levels of macrophage-induced apoptosis to untransfected plaque-derived and aortic-derived VSMCs and identical requirements of macrophage-induced apoptosis for Fas-L and caspases.7 Thus, NO donors upregulate VSMC surface Fas from an intracellular Golgi pool. There is a precedent for this, inasmuch as previous studies have shown that inflammatory cytokines prime VSMCs for Fas-induced apoptosis.31
NO exerts proapoptotic and antiapoptotic effects.25 Several mechanisms have been suggested for NO-induced cytotoxicity, including disruption of mitochondrial function,32 activation of p53,14 upregulation of Fas,33 activation of c-Jun N-terminal kinase,34 and induction of ceramide synthesis.34 Because we have shown that p53 may induce trafficking of Fas from the Golgi zone to the cell surface,15 our data showing that NO mediates macrophage-induced apoptosis by trafficking Fas to the cell surface and sensitizing cells to Fas-L confirm and extend previous work.
Macrophage expression of the surface death ligand, Fas-L, was NO dependent. The activation of monocytes has previously been shown to induce secretion of soluble Fas-L from an intracellular Fas-L store, and activation of neutrophil leukocytes with cGMP has been shown to stimulate the exocytosis of intracellular granules.17 Taken together, this raises the possibility that NO is also capable of stimulating monocyte/macrophage translocation of intracellular Fas-L to the cell surface, where it would be available for Fas ligation.
Our observations have potential implications for VSMC apoptosis in plaque rupture. In atherogenesis, peripheral blood monocytes migrate into the plaque and are exposed to oxidized lipoproteins, which activate them through scavenger receptors, thus forming tissue macrophages and foam cells.1 Activation to macrophages in the vessel wall may increase the expression of iNOS and NO secretion. Macrophage NO may upregulate macrophage surface Fas-L; VSMCs adjacent to these tissue macrophages may respond by increasing surface Fas expression; and the joint effect of proapoptotic signals through Fas/Fas-L and NO may induce VSMC apoptosis. This paradigm is consistent with our coimmunoreactivity of macrophages in ruptured plaques for iNOS and Fas-L, which is reasonably consistent with studies on unruptured plaques.35,36⇓ Although VSMCs in plaques sit within matrix cages37 that might be expected to impede cell-cell contact, activated macrophages within plaques are armed with proteases, such as metalloproteinases, that are important in plaque rupture6 and could lyse the matrix around VSMCs, permitting cell-cell proximity.
NO donors such as glyceryl trinitrate and isosorbide mononitrate are standard and effective therapies for stable and unstable angina.38 These are very highly characterized and work by a venodilator action, reducing cardiac preload and oxygen demand.38 This does not, per se, indicate that they have an isolated beneficial effect on plaque stability in acute coronary syndromes. However, the amount of NO released by these agents is low and (in essence) designed to simulate NO from the low-output pathway, eNOS. We anticipate from our in vitro data that the NO concentration from the iNOS pathway in the vicinity of an activated macrophage would be much higher and would be associated with synergistic effects of death ligands such as Fas-L. Therefore, these results do not suggest that therapeutic nitrates are deleterious for plaque rupture.
In conclusion, we have demonstrated that human macrophage–induced VSMC apoptosis is mediated by a combination of NO and Fas-L. NO or Fas-L is necessary but not sufficient for full macrophage cytotoxicity. Further studies are required to determine whether this mechanism contributes in vivo to the VSMC apoptosis associated with the rupture of advanced atherosclerotic plaques.
J.J.B. was supported by Medical Research Council Clinical Training Fellowship grant G84/4663 and by The Sackler Trust. This work was also supported by British Heart Foundation grants FS/97024 to M.R.B. and CH/9400 to P.L.W. We thank Dr D.E. Bowyer for helpful discussions.
Received April 22, 2002; revision accepted May 14, 2002.
- ↵van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994; 89: 36–44.
- ↵Bennett MR. Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture. Cardiovasc Res. 1999; 41: 361–368.
- ↵Bauriedel G, Hutter R, Welsch U, Bach R, Sievert H, Luderitz B. Role of smooth muscle cell death in advanced coronary primary lesions: implications for plaque instability. Cardiovasc Res. 1999; 41: 480–488.
- ↵Newby AC, Libby P, van der Wal AC. Plaque instability: the real challenge for atherosclerosis research in the next decade? Cardiovasc Res. 1999; 41: 321–322.
- ↵Boyle JJ, Bowyer DE, Weissberg PL, Bennett MR. Human blood-derived macrophages induce apoptosis in human plaque- derived vascular smooth muscle cells by Fas-ligand/Fas interactions. Arterioscler Thromb Vasc Biol. 2001; 21: 1402–1407.
- ↵Martin JH, Edwards SW. Changes in mechanisms of monocyte/macrophage-mediated cytotoxicity during culture: reactive oxygen intermediates are involved in monocyte-mediated cytotoxicity, whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing. J Immunol. 1993; 150: 3478–3486.
- ↵Albina JE. On the expression of nitric oxide synthase by human macrophages: why no NO? J Leukoc Biol. 1995; 58: 643–649.
- ↵Forrester K, Ambs S, Lupold SE, Kapust RB, Spillare EA, Weinberg WC, Felley BE, Wang XW, Geller DA, Tzeng E, Billiar TR, Harris CC. Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. Proc Natl Acad Sci U S A. 1996; 93: 2442–2447.
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- ↵Smith RJ, Ignarro LJ. Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of guanosine 3′:5′-monophosphate and calcium in stimulus-secretion coupling. Proc Natl Acad Sci U S A. 1975; 72: 108–112.
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- ↵Geng YJ, Henderson LE, Levesque EB, Muszynski M, Libby P. Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997; 17: 2200–2208.
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