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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1095-1103.)
© 1996 American Heart Association, Inc.

Articles

Monocyte-Induced Downregulation of Nitric Oxide Synthase in Cultured Aortic Endothelial Cells

Nandor Marczin; Alexander Antonov; Andreas Papapetropoulos; David H. Munn; Renu Virmani; Frank D. Kolodgie; Ross Gerrity; John D. Catravas

the Vascular Biology Center and the Department of Pharmacology and Toxicology (N.M., A.P., J.D.C.), the Department of Pathology (A.A.), and the Department of Pediatrics (D.H.M.), Medical College of Georgia, Augusta; and the Armed Forces Institute of Pathology, Washington, DC (R.V., F.D.K.).

Correspondence to John D. Catravas, PhD, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2350.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell–induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by N^G-nitro-L-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200x10³ per well)–dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin- and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)- and interleukin (IL)-1. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1ß neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous L-arginine failed to improve NO release, suggesting that oxidant stress–induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.

Key Words: endothelium-derived relaxing factor • cGMP • atherosclerosis • interleukin-1 • nitric oxide synthase

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Endothelial cells are key determinants of vascular tone and intimal integrity through the generation of vasoactive and growth-regulatory substances.^{1 2} Under normal conditions, the dynamic interactions among endothelium, blood components, and vascular SM cells results in a balance of the simultaneously present vasodilators and constrictors, mitogens, and growth-inhibitory substances to maintain adequate SM tone necessary for optimal tissue perfusion and to provide a growth-inhibitory microenvironment for medial SM cells.^{1 2 3} Several pathological conditions disturb this delicate balance and cause altered vascular tone, reactivity, and changes in SM phenotype and proliferation.

Formation and release of NO is a primary mechanism whereby endothelium regulates SM tone.^{4 5 6} NO is generated under basal conditions and in response to a variety of hemodynamic and chemical stimuli from the guanidino nitrogen atom(s) of L-arginine through a reaction catalyzed by ecNOS⁵ . Several lines of evidence suggest that continuous tonic release of NO is important in maintaining vascular homeostasis. In addition to its effect on SM tone, NO is a potent inhibitor of platelet function both in vitro and in vivo.⁷ Treatment with NO synthesis inhibitors causes an immediate and long-lasting hypertension and increased adherence of platelets to vascular endothelium.^{8 9 10} Since exogenous NO–containing compounds inhibit DNA synthesis and proliferation of cultured SM cells, it is hypothesized that endogenously produced NO may also control SM growth in vivo.^{11 12 13} Indeed, L-arginine, the substrate of NO synthesis, exhibits antiatherogenic effects in cholesterol-fed rabbits, whereas inhibition of NO production potentiates intimal lesion formation in the same model.^{14 15} These studies suggest that tonic release of endothelium-derived NO might contribute not only to the apparent state of contractility but to the control of phenotype and growth properties of the SM cells in health and disease.

Atherosclerosis is associated with SM proliferation, and atherosclerotic vessels exhibit altered reactivity to endothelium-dependent vasodilators acting through NO release.^{1 16 17} Several mechanisms are hypothesized to explain the diminished release of biologically active NO, including enhanced inactivation of NO by free radical–based mechanisms and chemical reactions with low-density lipoproteins.^{18 19 20 21 22}

Interestingly, alterations in NO-mediated regulation were observed in patients even in the absence of manifest coronary artery disease, suggesting that NO dysfunction is a relatively early phenomenon preceding angiographically detectable atherosclerotic lesions.^{23 24 25} In animal models, lesion formation in areas susceptible to atherosclerosis is preceded by the preferential adherence to the endothelium of blood-borne monocytes, which subsequently migrate into the intima and become the major source of foam cells in early lesions.²⁶ During these enhanced monocyte and endothelial interactions, monocytes may modulate several endothelial functions²⁷ through either direct cell-to-cell contact or through the release of humoral factors such as TNF- and IL-1.

We hypothesized that the preferential adherence of monocytes to endothelium might alter endothelial NO formation and release and thus contribute to endothelial dysfunction in early atherosclerosis. This hypothesis is strengthened by recent observations regarding the effects of TNF on endothelium-dependent vasorelaxation and steady state levels of ecNOS mRNA.^{28 29} Accordingly, the main goal of the present study was to determine the effects of monocytes on the NO pathway in cultured ECs and to elucidate the contribution of cell contact–mediated and humoral mechanisms to the effects of monocytes.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Medium 199 (Earle's salts), DMEM with high glucose, heat-inactivated FCS, L-glutamine, sodium pyruvate, HEPES, heparin, trypsin-EDTA, Earle's balanced salt solution, penicillin, and streptomycin were from GIBCO. ECGF and dispase (neutral protease from Bacillus polymyxa) were purchased from Boehringer Mannheim GmbH. Culture plastics were from Costar. Transwells were from Millipore Products Division. The Universal Immunostaining kit was from Signet Laboratories, Inc, and HAM-56 antibody to monocytes was from Enzo Diagnostics. Human recombinant MCSF (1.9x10⁶ U/mL) and IL-1 were a gift from Genetics Institute, Cambridge, Mass; TNF- (3.5x10⁷ U/mL) was a gift from Genentech, San Francisco, Calif. TNF-, IL-1 neutralizing antibodies, and the IL-1 immunoassay kit were from R&D Systems. The TNF- immunoassay kit was purchased from N.V. Innogenetics SA. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), L-arginine, and N^G-nitro-L-arginine methyl ester were purchased from Sigma Chemical Co.

Cell Culture
ECs
Primary HAECs were established from thoracic aortas of male sudden traumatic death victims within 12 hours of death. HAECs were isolated and cultured as previously described.^{30 31} Briefly, vessels obtained from autopsies were separated from connective tissue and adventitia, cut along the dorsal side, and washed with Earle's balanced salt solution. ECs were harvested by incubation with 0.15% dispase in Medium 199 for 90 to 120 minutes at 37°C. The resultant cell suspensions were pelleted at 800g for 10 minutes and resuspended in growth medium (Medium 199 supplemented with 15% heat-inactivated FCS, 25 mmol/L HEPES, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL ECGF, and 30 µg/mL heparin). ECs were seeded at a density of 5 to 8x10⁴/cm² on plastic flasks or glass coverslips previously coated with 0.2% gelatin and subcultured (1:3) with 0.1% trypsin:2 mmol/L EDTA. Experiments were performed using confluent ECs (4 to 7 days in culture, passages 2 through 5) from at least eight different primary cell lines. Purity of cultures and EC identification were assessed by phase microscopy, visualization of Weibel-Palade bodies, presence of von Willebrand factor, angiotensin-converting enzyme activity, and uptake of acetylated low-density lipoprotein.^{30 32} PAECs were isolated by incubation with 0.15% dispase for 15 minutes at 37°C. The growth medium used was DMEM with the same additions used for human ECs but without ECGF.

RASM cells from Wistar rats (Harlan) were isolated by enzymatic dissociation using standard methods.³³ The cells were positively identified as SM by indirect immunofluorescent staining for -actin, using mouse anti–-actin antibody and anti-mouse IgG FITC conjugate. RASM cells grown in T-75 tissue-culture flasks (Corning) in 50% F-12 nutrient medium and 50% DMEM (GIBCO) supplemented with 10% fetal bovine serum, glutamine, penicillin (Sigma, 10 000 U/L) and streptomycin (Sigma, 10 000 U/L) were subcultured into 24-well tissue-culture plates (Costar) at 5000/cm² and reached confluence after 5 to 7 days.

Monocytes
Human and porcine monocytes were isolated by counterflow centrifugal elutriation³⁴ from mononuclear cell layers prepared by centrifugation (500g, 40 minutes at 18°C) on Ficoll-Paque (d=1.077). Cells were diluted (5 to 20x10⁶ per tube), frozen in FCS containing 10% DMSO, and stored in liquid nitrogen. Purity of monocyte fractions determined by differential counting of cytospin preparations stained with Villanueva stain (porcine) or monocyte antibody (human) was >95%.

EC/Monocyte Coculture
Frozen monocytes were thawed, resuspended in 50 mL of serum-free medium, centrifuged for 10 minutes at 400g, and resuspended in the growth medium used for culturing human or porcine ECs. Confluent EC monolayers cultured in 24-well plates on 12-mm-diameter glass coverslips were washed three times with 2 mL of Medium 199 (37°C), then growth medium supplemented with 200 U/mL MCSF was added. Monocytes (200x10³ per well) were added directly to the EC monolayers or to gelatin-coated wells without endothelium, and then agents to be tested were added (time 0). After 2 hours' incubation, nonadherent monocytes were removed from wells by washing three times with Medium 199. Agents were resupplied, and cocultures, control ECs, or monocytes were cultured in growth medium with 200 U/mL of MCSF for up to 72 hours. The numbers of adherent monocytes were determined by two methods: (1) flow cytometry of living monocyte/EC suspensions obtained by brief trypsinization (15 to 30 seconds) and subsequent staining with HAM-56 antibody; and (2) computerized image analysis of monocyte/endothelium cocultures fixed directly in the culture well with 2% paraformaldehyde. Monocyte/EC suspensions for flow cytometry isolated by brief trypsinization were collected in centrifuge tubes in culture medium containing 10% bovine serum to inhibit trypsin activity. The cell number was counted by hemocytometer, cells were stained in suspension with HAM-56 antibody for 30 minutes, washed three times with PBS, and the antibody was visualized with Texas red–labeled secondary antibodies to mouse IgG. For direct visualization of monocyte/EC cultures, binding of the HAM-56 primary antibody was visualized using a Universal Immunostaining kit from Signet Laboratories. In both methods, the number of HAM-56–positive cells (monocytes) per 100 ECs was determined. In additional studies, we compared antibody staining of monocytes cultured on plastic and harvested with 2 mmol/L EDTA or trypsin as described above and found no loss of antigenicity to HAM-56 antibody with the trypsinization technique. In all cases, comparable numbers of adherent monocytes (15 to 30 monocytes per 100 ECs) were found. To visualize EC borders, cultures were fixed 10 minutes with 3.7% paraformaldehyde stained with 0.2% silver nitrate and counterstained with pararosaniline as previously described.³²

Conditioned Medium
Conditioned medium from human monocytes was collected after 24 hours (day 0) and on days 4, 9, and 12. After removing nonadherent monocytes, fresh growth medium was added after each collection. The conditioned media were centrifuged for 10 minutes at 400g, filtered through 0.22-µm filters, and stored at -20°C until use. Conditioned media were analyzed for their ability to modulate ecNOS activity and protein levels and for the presence of TNF- and IL-1 using commercially available immunoassay kits according to manufacturers' instructions.

Transwell Setup
The Transwells (6.5-mm diameter, 1.5-mm distance from the bottom) were pretreated with matrigel, rinsed three times with Medium 199, then inserted into 24-well plates containing confluent EC cultures. Monocytes (200x10³ per well) were added either into transwells or directly onto ECs.

Release of Biologically Active NO From ECs
NO release was estimated by using a short-term bioassay assessing the guanylate cyclase–stimulating activity of NO, reflected as EC-induced cGMP accumulation in reporter RASM cells as described.³⁵ Briefly, ECs grown on glass coverslips were cultured alone or with monocytes or treated with experimental agents for the desired time before removal of the medium and wash in Earle's balanced salt solution. The cells were then treated with either L-arginine, the substrate of NO production, or its inhibitory analogue, N^G-nitro-L-arginine methyl ester for 30 minutes. A short-term bioassay was then established by gently transferring the coverslips with the ECs into wells containing RASM cells pretreated with 3-isobutyl-1-methylxanthine to block phosphodiesterases and to prevent breakdown of cGMP. After 15 minutes' exposure of RASM cells to ECs, the coverslips with the donor ECs were removed from the wells, and intracellular cGMP content of RASM cells was extracted into 0.1 N HCl and quantified as described.³⁵

ecNOS Western Blotting
HAECs were cultured in 100-mm dishes and incubated with either vehicle, monocytes (5x 10⁶ per dish), or TNF- (1000 U/mL) or with conditioned medium for 48 hours. They were then lysed in lysis buffer (1% NP-40, 150 mmol/L NaCl, 20 mmol/L HEPES, pH 7.0, 1 mmol/L EDTA, with protease inhibitors). Cell lysates were microcentrifuged at 20 000 rpm, the supernatant fraction was collected and protein concentration measured by the Bradford method. Fifty micrograms per lane were electrophoresed in a 4% to 20% gradient polyacrylamide gel and transferred to a polyvinylidene difluoride membrane at 60 V for 3 hours at 4°C in a buffer containing 25 mmol/L Tris and 700 mmol/L glycine. After blocking, the membranes were incubated with a monoclonal antibody raised against the human ecNOS (Transduction Laboratories). Immunoreactive protein bands were visualized using the enhanced chemiluminescence system, after 10 minutes' exposure to X-ray film. To check for equality in loading and transfer, membranes were subsequently incubated with a monoclonal antibody against tubulin and immunoreactive bands were visualized after exposure to X-ray film for 30 seconds.

Data Analysis
Data are presented as mean±SEM of the indicated number of individual cultures. cGMP levels are expressed as fold increase or percent of the control value. Statistical comparisons between groups were performed using ANOVA or Student's t test, as appropriate. Differences among means were considered significant when P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Morphology and Adherence Data
After removal of nonadherent monocytes at 2 hours, cocultures routinely showed 15 to 30 adherent monocytes per 100 ECs. Many ECs lost their initial polygonal appearance and became elongated and spindle-like (Fig 1A) compared with the typical cobblestone appearance of normal EC cultures (Fig 1B). Monocytes did not induce EC damage or sloughing, and morphological alterations in EC monolayers occurred without loss of cell-to-cell contact, as demonstrated by silver nitrate staining (Fig 1A and 1B). Both HAECs and PAECs demonstrated similar morphology under these coculture conditions.

View larger version (341K): [in this window] [in a new window]   Figure 1. Phase-contrast photomicrographs of confluent monolayers of HAECs cocultured with monocytes (arrowheads) for 48 hours (A) and HAECs before addition of monocytes (B). Both cultures stained with silver nitrate to demonstrate EC borders and maintenance of endothelial cell-to-cell contact. Note elongation of ECs cocultured with monocytes in A compared with the typical cobblestone appearance of the endothelial culture in B.

Effects of Monocytes on the Release of Biologically Active NO From ECs
RASM cells treated with coverslips devoid of ECs exhibited low levels of cGMP (9.8±0.5 pmol per milligram protein per 15 minutes). These baseline levels remained unchanged in the presence of both L-arginine (1 mmol/L) and L-NNA (0.1 mmol/L), the substrate and inhibitor of NO formation (9.8±1.0 and 9.3±0.9 pmol per milligram protein per 15 minutes, respectively). As shown in Fig 2, the presence of ECs on the coverslips produced a 183-fold increase in intracellular cGMP content of the detector SM cells. Treatment with L-arginine did not further enhance the effect of ECs (2095±130 versus 1806±183 pmol per milligram protein per 15 minutes); however, L-NNA completely abolished EC-induced cGMP accumulation (15.9±2.5 pmol per milligram protein per 15 minutes). These responses are consistent with the notion that EC-induced SM cGMP accumulation is not substrate limited during the 15-minute bioassay but is entirely due to NO production, as judged from the effect of L-arginine analogues.³⁶ ECs cocultivated with porcine monocytes (200x10³ per well) for 3 days were also capable of stimulating SM cGMP accumulation; however, these cultures exhibited markedly diminished NO release compared with ECs in the absence of monocytes (128.8±8.5 versus 1806±183 pmol per milligram protein per 15 minutes). Again, inclusion of L-arginine during the bioassay did not increase NO output from monocyte/endothelial cocultures (131.7±23.3 pmol per milligram protein per 15 minutes), whereas the action of coculture on SM cGMP levels was abolished by L-NNA (10.5±1.9 pmol per milligram protein per 15 minutes). Monocytes cultured alone in the absence of ECs had negligible effects on SM cGMP accumulation (Fig 2).

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of porcine monocytes on endothelial NO release. Shown are cGMP levels in reporter RASM cells in response to coverslips devoid of cells (control) or coverslips containing ECs cultured for 3 days either alone (PAEC) or with monocytes (PAEC+MONO) and coverslips with monocytes only (MONO). Prior to bioassay with the RASM cells, donor cells were pretreated for 30 minutes either with vehicle; with L-arginine (L-ARG), the substrate of NO synthesis; or with L-NNA, an inhibitor of NO synthesis. Bars represent mean±SEM of four cultures. *P<.05 between respective PAEC and PAEC+MONO cultures. +P<.05 from respective vehicle cultures.

NO release from endothelial/monocyte cocultures (200x10³ monocytes per well) decreased in a time-dependent manner, reaching significance as early as 2 hours in the presence of MCSF, with maximum inhibition at 24 to 48 hours (Fig 3a, control). Addition of MCSF did not affect NO release from control ECs (Fig 3a, MCSF). The decrease in NO release was monocyte-concentration dependent (Fig 3b). Incubation of PAECs with concentrations as low as 20x10³ monocytes (15:1 ratio) led to a decrease in NO release after 24 hours, with 50% inhibition at 100x10³ monocytes, both in the presence and absence of MCSF. In a separate series of experiments, freshly isolated porcine monocytes were used, and their ability to decrease NO production from PAECs was compared with that of frozen monocytes. The concentration-response results obtained with the fresh monocytes were superimposable onto those of frozen monocytes.

View larger version (48K): [in this window] [in a new window]   Figure 3. Time-dependent (a) and concentration-dependent (b) inhibitory effects of porcine monocytes on endothelial NO release. Shown are cGMP levels in reporter RASM cells in response to ECs cultured either alone or with porcine monocytes in the presence or absence of MCSF (200 v/mL). Two hours after the addition of the indicated numbers of monocytes to a confluent endothelial monolayer, nonadherent cells were removed by washing, and NO release was determined after the indicated culture time (a) or after 24 hours (b). Bars represent mean±SEM of four cultures. *P<.05 from control cGMP elicited by PAECs in the absence of monocytes.

Effects of Monocytes on Cyclic Nucleotide Accumulation in ECs
To test the selectivity of monocyte action on endothelial NO release, we investigated the effect of monocytes on atriopeptin-induced cGMP accumulation and isoproterenol-induced cAMP formation in PAECs. As summarized in Fig 4, the concentration-dependent cyclic nucleotide formation by either atriopeptin or isoproterenol was not altered by cocultivation with monocytes.

View larger version (26K): [in this window] [in a new window]   Figure 4. Lack of effect of porcine monocytes on agonist-induced and NO-independent cyclic nucleotide accumulation in ECs. Shown are atriopeptin-induced (a) and isoproterenol-induced (b) cAMP accumulation in porcine ECs cultured either alone (PAEC) or with monocytes for 24 hours (PAEC-MONO). Data represent mean±SEM of four cultures. When not seen, SEM bars are contained within the symbol.

Role of Soluble Factors and Direct Cell Contact in the Monocyte-Induced Downregulation of Endothelial NO Release
To differentiate between cell contact–mediated and soluble factor–mediated effects of monocytes, we (1) determined the effects of two monocyte-derived cytokines, TNF- and IL-1, on NO release from PAECs and HAECs, (2) quantified cytokine levels in conditioned media of human monocytes, and (3) studied the effects of monocyte-conditioned medium and the effects of monocytes physically separated from ECs on the release of biologically active NO.

Incubation of HAECs with 1000 U/mL TNF- for 24 hours decreased NO release, as reflected in HAEC-induced SM cGMP accumulation. The effect of TNF was concentration dependent in both HAECs and PAECs (Fig 5a). When HAECs were incubated for 18 hours with 50 U/mL IL-1, cGMP accumulation in the reporter SM decreased from 207±15 pmol per milligram protein per 15 minutes, for the vehicle-treated cells, to 110±10, for the IL-1–treated cells.

View larger version (89K): [in this window] [in a new window]   Figure 5. Attenuated release of biologically active NO from PAECs and HAECs by monocyte-derived soluble factors. a, cGMP levels in reporter RASM cells in response to ECs cultured for 24 hours in the presence of increasing concentrations of TNF-. b, Time course of the accumulation of immunoreactive human (h) cytokines TNF- and IL-1 in the conditioned medium of human monocytes. c, cGMP levels in RASM cells in response to ECs cultured for 24 hours in the absence (control) or presence of adherent monocytes (MONO) or monocytes on transwells (MONO TWELL). d, cGMP levels in RASM cells in response to ECs cultured for 24 hours under control conditions or in the presence of monocytes (MONO) or with monocyte-conditioned medium collected 24 hours after seeding monocytes (MONO CM). *P<.05 vs 0 (a) or control (c, d). UD indicates undetectable.

TNF- was present in conditioned medium obtained from freshly seeded monocytes but not in conditioned medium collected at later time points. IL-1 concentration was also maximal in day 0 conditioned medium, but its secretion was somewhat more sustained, remaining detectable even in day 9 conditioned medium (Fig 5b).

Physical separation of monocytes from ECs by cultivating monocytes in transwells did not prevent the attenuation in NO release from ECs (Fig 5c). Similarly, conditioned medium collected on day 0 reproduced the effects of directly adhered monocytes in both types of ECs (Fig 5d).

To investigate whether soluble TNF- mediates the effect of adhered monocytes on the NO pathway, we tested the ability of a TNF- neutralizing antibody to prevent the inhibition of NO release by exogenous TNF and monocytes. Anti–TNF- antibody (2 µg/mL) did not significantly affect NO release from control human ECs (Fig 6), whereas it completely reversed the effect of exogenous TNF- (98.9±7% and 45.7±2.7% of control in the presence and absence of neutralizing antibody, respectively). However, monocyte-induced reduction in NO release remained unaffected by the anti–TNF- neutralizing antibody (23.1±3.3% and 26.3±2.3% of control in the presence and absence of neutralizing antibody, respectively). Comparable results were obtained with porcine ECs. Similarly to the anti–TNF- antibody, a neutralizing antibody against IL-1 was also ineffective in preventing the monocyte-induced reduction in NO release. However, a neutralizing antibody against IL-1ß was able to completely prevent the decrease in the amount of biologically active NO produced in HAEC/monocyte cocultures (Fig 7).

View larger version (31K): [in this window] [in a new window]   Figure 6. Ability of anti–TNF- neutralizing antibody (TNF-NAb; 2 µg/mL) to prevent the inhibitory effect of exogenous TNF- but not of adherent monocytes on the release of biologically active NO from HAECs. Shown are cGMP levels in reporter RASM cells in response to ECs cultured for 24 hours either under control conditions or with human monocytes (MONO, 200x103 per well) or TNF- (TNF, 100 U/mL) in the absence (vehicle) or presence of TNF-NAb. The antibody was added 10 minutes prior to addition of monocytes or TNF- and was present during the 24-hour coculture. Bars represent mean±SEM of six cultures. *P<.05 from appropriate cGMP elicited by control HAECs in the absence of monocytes or TNF-.

View larger version (39K): [in this window] [in a new window]   Figure 7. Full protection against monocyte-induced reduction in NO production by neutralizing antibody against IL-1ß. HAECs were incubated with human monocytes (EC+mono; 200x103) in the presence (+) and absence (-) of neutralizing antibody against IL-1 (nAbIL-1; 2 µg/mL) and IL-1ß (nAbIL-1ß; 2 µg/mL). Neutralizing antibodies were added to HAECs and allowed to equilibrate; after 10 minutes, monocytes were added to the monolayer. cGMP was determined after 24 hours. Mean±SEM, n=4 wells.

Role of Oxidant Stress in Monocyte-Induced Downregulation of Endothelial NO Release
To investigate the possibility that the action of monocytes was mediated through reactive oxidant species generated either during the bioassay or during coculture, we tested the ability of various antioxidants to reverse or prevent the reduction in NO release. Inclusion of superoxide dismutase (300 U/mL), catalase (2000 U/mL), or 4,5-dihydroxy-1,3-benzene disulfonic acid (10 mmol/L) during the bioassay aimed at eliminating extracellular superoxide anion, hydrogen peroxide, and intracellular superoxide anion, respectively, failed to restore monocyte-induced attenuation of NO release (Fig 8a). Similarly, these antioxidants were unable to prevent the effect of monocytes even when added during the endothelial/monocyte coculture (Fig 8b). These superoxide anion scavengers were capable of preventing activated neutrophil-induced or xanthine/xanthine oxidase–induced attenuation of cGMP accumulation in response to ECs.³⁶

View larger version (61K): [in this window] [in a new window]   Figure 8. Lack of antioxidant protection against monocyte-induced attenuation of the release of biologically active NO from ECs. Shown are cGMP levels in RASM cells in response to ECs cultured for 24 hours either alone (PAEC) or with porcine monocytes (PAEC-MONO). Superoxide dismutase (SOD, 300 U/mL), catalase (2000 U/mL), or 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) were included either during the bioassay (a) or during monocyte/endothelial coculture before the bioassay (b). Bars represent mean±SEM of four cultures. *P<.05 from appropriate control cGMP elicited by PAECs in the absence of monocytes.

Effects of Monocytes, Monocyte-Conditioned Medium, and Exogenous TNF- on ecNOS Protein Levels in HAECs
Western blot analysis revealed abundance of 140 kD protein in control ECs. Coculture of ECs with monocytes or treatment with monocyte-conditioned medium or TNF- appeared to significantly reduce ecNOS protein levels (Fig 9). Reprobing the same membrane with a monoclonal antibody for ß-tubulin revealed equal amounts of tubulin among the control and experimental groups.

View larger version (51K): [in this window] [in a new window]   Figure 9. Western blot demonstrating downregulation of ecNOS by monocytes and monocyte-derived soluble factors in HAECs. Shown are ecNOS and ß-tubulin (as reference) protein levels in ECs cultured for 48 hours either under control conditions (CTR, lane 1) or with TNF- (TNF, 100 U/mL, lane 2), human monocytes (MONO, lane 3), or day 0 monocyte-conditioned medium (CM, lane 4).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The aims of the present study were to investigate how monocyte adhesion to ECs modulates the activity of ecNOS in ECs and to elucidate mechanisms mediating these effects of monocytes. The principal findings are that (1) monocyte adhesion to endothelium suppresses the release of biologically active NO in a monocyte concentration– and time-dependent manner, (2) this attenuation is not mediated by reduced substrate availability or free radical generation but is associated with reduced levels of ecNOS protein, (3) both monocyte-conditioned medium and exogenous cytokines, as well as monocytes physically separated from ECs, reproduce the effects of adherent monocytes, and (4) an IL-1ß neutralizing antibody prevents the reduction in ecNOS activity by adherent monocytes.

Interactions of ECs with monocytes play an important role in normal vascular biology and in the pathogenesis of vascular disease. Using a porcine and a human cell culture model, we observed that gravity-induced adhesion of monocytes to ECs profoundly affected release of biologically active NO from ECs. Although the onset of NO dysfunction was accompanied by shape changes in ECs, it appears to be a relatively early and selective biochemical alteration. Inhibition of NO release by monocytes occurs after a delay of several hours and at a time when other biochemical pathways, such as cyclic nucleotide synthesis through the adenylate cyclase and the particulate guanylate cyclase, remain virtually unaffected. Parallel experiments focusing on endothelial ectoenzyme functions revealed that monocytes also reduce activity of angiotensin-converting enzyme; however, this impairment occurred after longer cocultivating times (48 hours³⁷ ).

Reduction in the biological activity of NO could be explained by either increased elimination of formed NO or reduced NO formation. Superoxide anion is capable of reacting with NO and reducing its biological activity, whereas hydrogen peroxide may alter the catalytic activity of ecNOS.^{35 36 38} Activated monocytes have been shown to generate partially reduced oxygen-reactive species under certain conditions.^{39 40} In the present studies, superoxide dismutase was employed to break down extracellular superoxide anion; 4,5-dihydroxy-1,3-benzene disulfonic acid, a small-molecular-weight compound, to scavenge intracellular superoxide; and catalase to eliminate hydrogen peroxide; all these failed to restore the biological effect of NO. Both superoxide scavengers, however, have been shown to effectively prevent PMA-activated neutrophil-induced reduction in NO-mediated cGMP accumulation.³⁶ Taken together, no evidence was found for a role of reactive oxygen species in mediating the inhibitory effects of monocytes on NO release.

NO is formed enzymatically from the guanidino moiety of L-arginine, and therefore, reduction in substrate concentration could be an underlying mechanism for inhibited NO formation. Addition of L-arginine prior to the bioassay did not increase NO output from monocyte/endothelial cocultures; however, this treatment completely restored NO release in L-arginine–depleted bovine aortic ECs (unpublished observations). These results suggest that limited substrate availability is an unlikely mechanism of diminished NO output in endothelial/monocyte cultures.

The present study revealed reduced ecNOS protein levels in monocyte-treated ECs after 48 hours. Whether monocytes shorten the half-life of ecNOS protein or reduce its synthesis by altering steady state levels of ecNOS mRNA remains to be determined. In addition, it is unclear whether a similar mechanism is responsible for the early (2 hours) decrease in NO production. If the early decrease in NO release in the endothelium/monocyte cocultures is not mediated by reduced amounts of ecNOS protein, a possible mechanism that could explain the reduction in NO is cofactor (such as tetrahydrobiopterin) depletion.

While some of the actions of monocytes on ECs, such as induction of von Willebrand factor, require cell-to-cell contact, others, such as prostanoid release, are mediated by soluble factors.²⁷ In the present study, monocytes physically separated from ECs reproduced the effect of monocyte adhesion on NO release; however, monocyte-conditioned medium produced variable effects in the initial series of experiments. Analysis of cytokine content of the conditioned media showed high TNF- and IL-1 levels early after monocyte seeding, with rapid decline at later time points. Since day 0 conditioned media reproducibly attenuated NO release and reduced ecNOS protein levels, we concluded that cell-to-cell contact is not an absolute requirement for monocyte-induced ecNOS downregulation.

Since monocyte-conditioned medium contained significant amounts of TNF- and IL-1 and also downregulated ecNOS protein and activity, we investigated the ability of exogenous cytokines to influence NO release from pig and human ECs. Treatment of ECs with both TNF- and IL-1 attenuated NO release in a concentration-dependent manner. These results are in agreement with the studies of others on the effects of TNF on endothelium-dependent relaxation and on ecNOS mRNA levels.^{28 29} Interestingly, we did not find evidence of inducible NOS induction by cytokines in either HAECs or PAECs under the present experimental conditions. This finding is in agreement with published observations⁴¹ in human umbilical vein ECs and further underlines the differences in the mechanism of inducible NOS gene induction among various mammals.

To evaluate whether cytokine (TNF- and IL-1) secretion accounts for the inhibition of NO release by adherent monocytes, ECs were treated with neutralizing antibodies against these cytokines. Neutralizing antibodies against TNF- and IL-1 neutralizing antibody failed to augment the NO release in EC/monocyte cocultures, while an IL-1ß neutralizing antibody was able to completely prevent the effects of monocytes. It is possible that under the present experimental conditions, IL-1ß is first in a cascade of cytokines released by monocytes (which include among others TNF- and IL-1).

Since monocytes remain in the close proximity of ECs for prolonged periods of time during the development of atherosclerotic lesions, our observations imply that monocytes could potentially diminish NO mechanisms in vivo if control mechanisms are similar in vivo. This event would be associated with the loss of a vasodilator, antithrombotic, and antiproliferative substance and thus would predispose the vessel to develop vasospasm and thrombosis and to recruit platelets and leukocytes, with potential consequences of increased macromolecular permeability and intimal proliferation.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium EC = endothelial cell ECGF = EC growth factor ecNOS = constitutive endothelial NO synthase HAEC = human aortic EC IL = interleukin L-NNA = NG-nitro-L-arginine MCSF = monocyte colony–stimulating factor NO = nitric oxide PAEC = porcine aortic EC RASM = rat aortic SM SM = smooth muscle TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported by grants HL31422 and HL46689 from US Public Health Service, National Institutes of Health, Bethesda, Md. MCSF and IL-1 were generously provided by Genetics Institute, Cambridge, Mass; TNF- was a gift from Genentech, San Francisco, Calif. We are pleased to acknowledge the expert technical assistance of Jenifer Barrett, Livia Marczin, and Mary Snead. We thank Annie Cruz and Aretha Bogan for the preparation of the manuscript.

Received May 2, 1995; revision received March 18, 1996;

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