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(Circulation Research. 1996;79:512-523.)
© 1996 American Heart Association, Inc.

Articles

Monocyte- and Cytokine-Induced Downregulation of Angiotensin-Converting Enzyme in Cultured Human and Porcine Endothelial Cells

Andreas Papapetropoulos, Alexander Antonov, Renu Virmani, Frank D. Kolodgie, David H. Munn, Nandor Marczin, James W. Ryan, Ross G. Gerrity, John D. Catravas

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

Correspondence to Dr John D. Catravas, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We investigated the effects of monocytes on endothelial cell (EC) ectoenzyme activity. Coculture of human aortic ECs with human monocytes (2x10⁵ monocytes per 2-cm² well) led to a decrease in EC angiotensin-converting enzyme (ACE) activity (64.5±3.5% of control) but not aminopeptidase N, aminopeptidase P, and 5'-nucleotidase activities. Similar results were obtained using human umbilical vein EC–human monocyte and porcine aortic EC–porcine monocyte cocultures. The decrease in ACE activity was monocyte concentration and coculture time dependent, reaching a maximum of 65% decrease in activity at 120 hours. Monocyte-mediated reduction in ACE activity did not require cell to cell contact, since exposure of ECs to conditioned medium from cocultures (CCCM) or from monocyte cultures (MCM) produced a decrease in ACE activity similar to that observed in EC-monocyte cocultures. Exogenously added tumor necrosis factor (TNF)- and interleukin (IL)-1, two known secretory products of monocytes, simulated the effects of monocytes on ACE activity. Western blot analysis revealed a decrease in the amount of ACE protein in TNF-–treated and CCCM-treated ECs compared with control ECs. Both TNF- and IL-1 were present in CCCM and MCM but not EC-conditioned medium. Incubation of the cocultures with a mixture of neutralizing antibodies against TNF- and IL-1 totally abolished the monocyte-induced decrease in ACE activity. In conclusion, monocytes decrease ACE activity in cultured ECs through the release of cytokines such as TNF- and IL-1.

Key Words: angiotensin-converting enzyme • 5'-nucleotidase • surface peptidase • tumor necrosis factor- • interleukin-1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased adherence of monocytes to the endothelium and subendothelial migration are early events of atherogenesis, resulting from and/or contributing to endothelial dysfunction and smooth muscle migration and proliferation.¹ Monocytes and monocyte-derived cytokines have been shown to modulate endothelial functions with respect to vascular tone, thrombosis, and inflammation.^{1 2} TNF- and IL-1 are monocyte/macrophage-derived cytokines with pleiotropic actions on the morphology and biochemistry of various cell types.^{2 3 4} Endothelium exposed to TNF- or IL-1 displays increased expression of adhesion molecules, including P-selectin and intercellular adhesion molecule-1.^{3 4} Moreover, TNF- and IL-1 stimulate the secretion of other cytokines, such as IL-6, IL-8, and monocyte chemoattractant protein, as well as growth factors, including MCSF, granulocyte-MCSF, and platelet-derived growth factor.^{2 5 6} Moreover, TNF- and IL-1 enhance the production of plasminogen activator inhibitor-1, von Willebrand factor, and prostacyclin.⁶

ACE, AmN, and NCT are ectoenzymes located on the cell surface, each with its catalytic domain facing the extracellular space. It has been postulated that surface peptidases may participate in the regulation of cell growth and differentiation by changing local concentrations of peptide hormones and growth factors.⁸ ACE, a target enzyme for a novel class of antihypertensive agents, is found in large amounts on the surface of vascular endothelium, where it catalyzes the conversion of the biologically inactive decapeptide angiotensin I to angiotensin II and converts bradykinin to inactive fragments⁹ ; AmN is believed to participate in the degradation of angiotensin III and Lys-bradykinin.¹⁰ NCT dephosphorylates 5'-AMP to adenosine, a known vasorelaxant and antithrombogenic agent.^{11 12} Angiotensin II is a potent vasoconstrictor that induces smooth muscle migration and proliferation,^{13 14} whereas the vasodilator bradykinin increases vascular permeability and is believed to be involved in inflammatory processes.^{15 16}

Recent reports have indicated that EC ACE activity is reduced by TNF-¹⁷ and lipopolysaccharide¹⁸ and is increased by IL-1¹⁹ and platelet-activating factor.²⁰ The aim of the present study was to investigate whether interaction of monocytes with ECs alters endothelial ectoenzyme activities. IL-1 and TNF- have been shown to affect EC receptor expression and coupling to second messenger systems.^{2 3} An alternative means for monocyte and monocyte-derived cytokine modulation of vasoactive peptide and growth factor action on vascular cells would be alteration of the activity of enzymes involved in peptide formation and degradation. Effects on endothelial ectoenzyme activity could provide a previously unrecognized mechanism for monocyte modulation of cardiovascular homeostasis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Medium 199, Earle's balanced salt solution, 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 Laboratories. EC growth factor and dispase (neutral protease from Bacillus polymyxa) were purchased from Boehringer Mannheim GmbH. Culture plastics were from Costar. Universal Immunostaining kit was from Signet Laboratories, Inc. HAM-56 antibody to monocytes was from Enzo Diagnostics. Transwells were from Millipore Products Division. [³H]Benzoyl-phenylalanine-alanine-proline (22 Ci/mmol) and Ventrex cocktail No. 1 were from Ventrex; [¹⁴C]5'-AMP (53.8 mCi/mmol) was from Amersham International; toluene was from Baxter Healthcare Corp; and Omnifluor was from NEN Research Products. Ecoscint A was from National Diagnostics. Human recombinant MCSF (1.9x10⁶ U/mL), IL-1 (2x10⁴ U/mL), and IL-6 (5x10⁶ U/mL) were gifts from Genetics Institute, Cambridge, Mass; TNF- (3.2x10⁷ U/mL) was a gift from Genentech, San Francisco, Calif. Interferon- (6x10⁵ U/mL) was purchased from Hoffman–La Roche. The TNF-– and IL-1–neutralizing antibodies and the IL-1 immunoassay kit were from R&D Systems; the TNF- immunoassay kit was purchased from Biosource International. PVDF membrane, dry milk, and Tween 20 were from Bio-Rad Laboratories. The monoclonal antibody 9B9 against ACE²¹ was from Biotrack, Inc. Matrigel was from Collaborative Research Inc. The ECL system was from Amersham Life Sciences, and the x-ray film was from Eastman Kodak Co. Nonidet P-40, NaCl, aprotinin, phenylmethylsulfonyl fluoride, superoxide dismutase, catalase, Tiron, bovine serum albumin, and dipyridamole were purchased from Sigma Chemical Co. Arg-Phe-[³H]anilide (22 Ci/mmol) and Arg-Pro-Pro-[³H]benzylamide (22 Ci/mmol) were prepared as described previously.^{22 23}

EC and Monocyte Cultures and Cocultures
Primary HAEC cultures were established from thoracic aortas of male victims of sudden traumatic death and were obtained at autopsy within 12 hours of death. HAECs were isolated and cultured as previously described.^{24 25} Briefly, vessels were separated from connective tissue, cut longitudinally, 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 resulting cell suspensions were pelleted at 600g for 10 minutes and resuspended in growth medium (medium 199 supplemented with 15% 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 endothelial cell growth factor, and 30 µg/mL heparin). ECs were seeded at a density of 5 to 8x10⁴ cells/cm² in plastic flasks previously coated with 0.2% gelatin and were subcultured (1:3) using trypsin. Experiments were performed using confluent ECs (4 to 7 days in culture, passages 2 to 5) from nine 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, and uptake of acetylated low-density lipoprotein.^{24 26 27} Growth characteristics of ECs under these conditions have been reported previously.²⁶ HUVEC cultures were prepared according to Gimbrone et al²⁸ with minor modifications, using dispase instead of collagenase,²⁶ and were cultured identically to HAECs. 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 endothelial cell growth factor. HUVECs and PAECs were used at passages 2 to 5 and 2 to 12, respectively. Smooth muscle cells from porcine aorta were isolated by incubation of vessels with 0.2% collagenase in DMEM for 3 hours and cultured in growth medium (DMEM) with 10% FCS.

Human and porcine monocytes were isolated by counterflow centrifugal elutriation²⁹ from mononuclear cell layers prepared by centrifugation of heparinized blood (500 g, 40 minutes at 18°C) on Ficoll-Paque (d 1.077). Cells were diluted (5 to 20x10⁶ cells per tube) in FCS containing 10% dimethyl sulfoxide, frozen, and stored in liquid nitrogen. The purity of monocyte fractions determined by differential counting of cytospin preparations stained with Villanueva stain (porcine) or monocyte antibody (human) was >95%.

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 were washed three times with 2 mL of medium 199 (37°C); then growth medium supplemented with 200 U/mL MCSF was added. Monocytes (2 to 50x10⁴ in a final volume of 0.5 mL per well) were added directly to the EC monolayers or to gelatin-coated wells without endothelium. Agents to be tested were added at time 0. After 2 hours of incubation, nonadherent cells (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 48 to 120 hours. The numbers of adherent monocytes were determined by flow cytometry of trypsinized monocyte/EC suspensions stained with HAM 56 or were determined directly in the culture dish using computerized image analysis of HAM 56–stained cultures. Monocyte adherence differed with the monocyte and EC donor used, ranging from 15 to 30 monocytes per 100 endothelial cells. To determine whether alteration in endothelial ectoenzyme activity was dependent upon direct contact between monocytes and ECs or the secretion of soluble products by monocytes or endothelium, experiments with both CM and transwell plates were carried out. CM was collected from confluent ECs, monocytes (5x10⁵ per well), or monocyte/EC cocultures. CM from all three sources was collected after 24 hours (day 0) and on days 4, 9, and 12. In the case of cocultures and monocytes alone, nonadherent cells were removed after 24 hours. Fresh growth medium was added after each collection. The CM were centrifuged for 10 minutes at 400g, filtered through 0.22-µm filters, and stored at -20°C if not immediately used. To analyze CM for their abilities to decrease ACE activity with parallel assay for the presence of TNF- and IL-1, CM were pooled from four to six 2-cm² wells. Cytokine concentrations were determined in triplicate. CM effects on ACE activity were measured in quadruplicate. The transwells (diameter, 6.5 mm; distance from the bottom, 1.5 mm) were coated with matrigel, rinsed three times with medium 199, and then inserted into 24-well plates containing confluent EC cultures. Monocytes (2x10⁵ cells per well) were either added into transwells or directly onto ECs, and ACE activity was estimated 48 hours later. Migration of monocytes across transwell membranes coated with matrigel was not observed. To visualize EC borders, cultures were fixed for 10 minutes with 3.7% paraformaldehyde, stained with 0.2% silver nitrate,³⁰ and counterstained with pararosaniline.

EC Ectoenzyme Activity Assays
EC ACE, AmN, AmP, and NCT assays were performed in separate 2-cm² wells under first-order reaction conditions. Confluent control- or cytokine-treated ECs or cocultures of ECs with monocytes were incubated with the appropriate substrate for 30 minutes (ACE, AmN, and NCT) for HUVECs and HAECs and for 60 minutes for PAECs. Incubation time was 4 hours for AmP activity measurements for both porcine and human cells. After two gentle washes with Earle's salt solution to remove traces of serum, cytokines, and other chemical treatments, enzymatic reactions were carried out at 37°C in a final volume of 0.6 mL Earle's salt solution. The substrates used were [³H]benzoyl-Phe-Ala-Pro (0.2 µCi/mL) for ACE,³¹ Arg-Phe-[³H]anilide (0.2 µCi/mL) for AmN,²³ Arg-Pro-Pro-[³H]benzylamide (0.2 µCi/mL) for AmP,²² and [¹⁴C]5'-AMP (50 nCi/mL, 53.8 mCi/mmol) for NCT. Dipyridamole (0.1 mmol/L) was included in the last reaction mixture to prevent the cellular uptake of [¹⁴C]adenosine, the ¹⁴C-labeled product formed by NCT. Reactions were stopped by either placing the plate into an ice bath or by removing samples into acid or base.

For the peptidase activity assays, radiolabeled products ([³H]benzoyl-Phe, Phe-[³H]anilide, and Pro-Pro-[³H]benzylamide for ACE, AmN, and AmP, respectively) were separated from parent compounds by extraction into toluene. An aliquot from each supernatant was transferred to a 7-mL polyethylene scintillation vial containing 5 mL of scintillation cocktail (Ecoscint A). Total ³H radioactivity was measured in a Beckman LS7500 liquid scintillation spectrometer (Beckman Instruments). [³H]Benzoyl-Phe was separated from unreacted substrate by transferring 0.1-mL aliquots, in duplicate, to 7-mL scintillation vials containing 2.9 mL of 0.12N HCl, to which 3 mL of toluene supplemented with 4 g/L omnifluor was added. For the extractions of Phe-[³H]anilide and Pro-Pro-[³H]benzylamide, 0.2-mL aliquots were added to 0.2 mL of 0.2N NaOH. To that, 3 mL of Ventrex cocktail No. 1 (a toluene-based cocktail) was added. Products were preferentially extracted into the organic phase, and radioactivity was determined by liquid scintillation spectrometry after storing the samples overnight (AmN and AmP) or for 48 hours (ACE) in the dark. Since a small amount (5% to 15%) of the parent compounds was also extracted into toluene, correction for this extraction was undertaken as previously described.³² [¹⁴C]Adenosine was separated from [¹⁴C]AMP on a disposable anion-exchange column. Three 0.3-mL aliquots were transferred into 7-mL polyethylene scintillation vials, each containing 6 mL of scintillation cocktail (Ecoscint A), and total ¹⁴C activity was measured. Duplicate 0.3-mL aliquots were transferred to disposable chromatography columns containing 2.1 mL of Dowex 1x8 400-mesh (Cl^-) anion-exchange resin retained on a glass wool plug. The columns bind [¹⁴C]5'-AMP, whereas [¹⁴C]adenosine is eluted with 3 mL of 1 mmol/L NaCl in 20% ethanol. Radioactivity was measured in 6 mL Ecoscint A. In this way, 70% of the dephosphorylated compound (ie, [¹⁴C]adenosine) was eluted, whereas <5% of the parent ([¹⁴C]5'-AMP) appeared in the eluent.

Enzyme activity was calculated as follows:

where [S_o] and [S] are the initial and final substrate concentrations, respectively, and t is incubation time.³³ Enzyme activity is presented in units: one unit is the V_max/K_m value equivalent to 1% substrate metabolism in 1 minute under first-order reaction conditions.

Protein Measurements
Protein content of the supernatant of the centrifuged (2000 rpm for 5 minutes at room temperature) NaOH-solubilized samples was measured by the Bradford method.³⁴ Bovine albumin in NaOH was used as the standard. To determine the protein concentration of samples that were used for Western blotting, the standard curve was constructed in appropriate buffer.

Immunoblotting
Cells were cultured in 100-mm-diameter dishes, incubated with vehicle, TNF- (1000 U/mL, 4x10⁵ monocytes/mL), or day 0 CCCM for 48 hours, and then lysed in lysis buffer (20 mmol/L HEPES/NaOH buffer, pH 7.0, containing 1% Nonidet P-40, 150 mmol/L NaCl, 1 mmol/L EDTA, 10 µg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride). Cell lysates were centrifuged at 20 000 rpm, the supernatant fraction was collected, and protein concentration was measured. Samples (50 µg of protein per lane) were electrophoresed in a 4% to 20% gradient polyacrylamide gel and transferred to a PVDF membrane at 60 V for 3 hours at 4°C in a buffer containing 25 mmol/L Tris and 700 mmol/L glycine. Membranes were then incubated overnight at 4°C with 5% dry milk in buffer containing 0.1% (vol/vol) Tween 20 in Tris-buffered solution (TTBS) to block nonspecific binding. The following day, membranes were incubated with a monoclonal antibody for ACE in 1% milk in TTBS, washed three times with TTBS for 20 minutes each time, blocked for an additional hour with 5% milk in TTBS, and finally incubated for 1 hour with a horseradish peroxidase–conjugated anti-mouse IgG. Immunoreactive protein bands were visualized using the ECL system after 10 minutes of 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. Enzyme activity data are expressed as percentage of the control EC 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 Materials and Methods Results Discussion References

 
Morphology of ECs in Coculture
After removal of nonadherent monocytes, after 2 hours of EC-monocyte incubation, cocultures routinely showed 15 to 30 adherent monocytes per 100 ECs. Even these relatively low numbers of adherent monocytes markedly altered EC morphology. Many ECs lost their initial polygonal appearance (Fig 1, left panels) and became elongated and spindlelike (Fig 1, right panels). These changes in EC morphology were observed at 24 and 48 hours after the addition of monocytes and were maintained for at least 4 to 5 days in coculture. The interaction between ECs and monocytes did not induce EC sloughing, and morphological alterations in EC monolayers occurred without loss of cell-cell contact, as demonstrated by silver nitrate staining (Fig 1, right panels). HAECs, HUVECs, and PAECs all demonstrated similar morphology under these conditions.

View larger version (602K): [in this window] [in a new window]   Figure 1. Changes in appearance of EC monolayers produced by monocytes in coculture. Top left, Confluent monolayer of control PAECs. Top right, Monolayer of ECs after 48-hour coculture with monocytes. ECs are elongated compared with control. Bottom left, Confluent monolayer of control HUVECs. Bottom right, Monolayer of elongated HUVECs after 48-hour coculture with monocytes. Top panels are phase contrast; bottom panels show silver nitrate staining and pararosaniline counterstain.

Ectoenzyme Activities in EC-Monocyte Cocultures
Cultured HAECs, HUVECs, and PAECs expressed ACE (0.88±0.03, 6.17±0.23, and 0.32±0.01 U per monolayer), AmN (0.49±0.03, 0.63±0.01, and 0.17±0.02 U per monolayer), and NCT (3.81±0.18, 5.83±0.26, and 0.41±0.01 U per monolayer) activities, respectively. AmP activity was 2.02±0.1x10^-2 and 0.2±0.01x10^-2 U per monolayer for HAECs and PAECs, respectively. Human monocytes when cultured on plastic displayed smaller activities for ACE, AmN, and NCT than did ECs. No AmP activity was detected in human monocytes. Porcine monocytes on the other hand exhibited eightfold higher NCT activity, similar AmN activity, and 60% of the ACE activity compared with PAECs. Coculture of HAECs and HUVECs with freshly isolated human monocytes for 2 to 4 days led to a decrease in ACE activity (to 64.5±3.5% and 59.4±1.1% of control values in HAEC and HUVEC monocyte cocultures) without affecting NCT and AmN activities (Fig 2A and 2B). AmP activity was not different in HAEC and HAEC-monocyte cocultures (2.02±0.1x10^-2 and 2.05±0.1x10^-2 U per monolayer, respectively). Similar results were obtained when activities were expressed per cell number or per milligram protein. Protein content per monolayer remained unaltered upon cocultivation of ECs with monocytes (75.9±2.1 and 72.7±2.9 µg per monolayer) for HAEC and HAEC-monocyte cocultures, respectively. Similarly, EC cell number per monolayer was not different in EC and EC-monocyte cocultures (150 000 per monolayer). ACE activity was also diminished in PAEC and porcine-monocyte cocultures (to 56.7±5.3% of control values, Fig 2C), whereas AmP was found to be unaltered, and NCT and AmN activities were increased in cocultures compared with ECs, probably because of the high NCT and AmN activities of monocytes.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of monocytes on endothelial ectoenzyme activities. NCT, ACE, and AmN activities of HAECs (A), HUVECs (B), and PAECs (C) cultured alone or in coculture with monocytes (200x103) for 2 to 4 days are shown. Enzyme activities of human (A and B) and porcine (C) monocytes were determined using monocytes cultured on plastic. Values are mean±SEM (n=4 wells). *P<.05 vs EC ACE activity; #P<.05 vs EC NCT activity; and **P<.05 vs EC AmN activity.

The remainder of the studies focused on the mechanism of monocyte-mediated reduction in endothelial ACE activity. Since porcine and human ECs showed similar diminishment in ACE activity upon coculture with monocytes, the three different EC types were used interchangeably. ACE activity in PAEC-monocyte cocultures (2x10⁵ monocytes and 2x10⁵ ECs per coculture) decreased in a time-dependent manner, reaching significance at 48 hours. After 120 hours, monocyte-induced decline in ACE activity reached a maximum, with only 30.4±4.0% and 34.8±3.0% of control ACE activity remaining in the presence or absence of MCSF. Addition of MCSF (200 U/mL) did not affect EC ACE activity, nor did it alter the time course of decline in ACE activity in the presence of monocytes (Fig 3A). The decrease in ACE activity was also monocyte-number dependent (Fig 3B). A plateau in the monocyte-mediated decrease in ACE activity was observed at 2x10⁵ monocytes per well. Addition of MCSF potentiated the decrease in ACE activity at low monocyte concentrations (2x10⁴). Experiments were performed using both freshly isolated and freeze-thawed monocytes and yielded similar results. Subsequent experiments were thus performed with freeze-thawed monocytes.

View larger version (40K): [in this window] [in a new window]   Figure 3. A, Time course of monocyte-induced reduction in ACE activity in PAEC and porcine-monocyte cocultures. Frozen porcine monocytes (200x103) were added to confluent endothelial monolayers in the presence and absence of MCSF (200 U/mL); 2 hours after the addition of monocytes, nonadherent cells were removed by washing. Similar results were obtained using freshly isolated monocytes. B, Monocytes were incubated with PAECs for 48 hours in the presence and absence of MCSF. Values are mean±SEM (n=4 wells). *P<.05 vs control PAEC ACE activity.

We investigated whether the decrease in cell-associated PAEC ACE activity was due to monocyte protease action, releasing ACE into the culture medium. Cells were incubated with media containing 2% heat-inactivated FCS for 48 hours. The CM was collected and then centrifuged at 1000 rpm for 10 minutes to remove floating cells. Medium ACE activities from control EC and EC-monocyte cocultures were not significantly different (Table 1). Moreover, monocyte-induced free radical production was not found to be involved in the decrease in ACE activity. Addition of extracellular (300 U/mL superoxide dismutase or 2000 U/mL catalase) or intracellular (10 mmol/L Tiron or 5 mmol/L dimethylthiourea) free radical scavengers in the cocultures for the entire 48 hours did not protect against the decrease in ACE activity (Fig 4). Cell-cell contact between monocytes and ECs was not essential for the monocyte-mediated decrease in ACE activity, since CCCM and physical separation of monocytes in the cocultures by cultivation of monocytes in transwells yielded similar decreases in ACE activity (Tables 1 and 2). Freezing and thawing the CCCM did not result in loss of the medium's ability to decrease ACE activity (Table 2). To investigate whether monocytes also induced a decrease in smooth muscle ACE activity, ACE activity was determined in porcine aortic smooth muscle–monocyte cocultures. In both the presence and absence of MCSF, no alteration in ACE activity was observed (111.4±4.9% versus 108.6±7.8% of control smooth muscle cell ACE activity values for the +MCSF and -MCSF groups, respectively).

View this table: [in this window] [in a new window]   Table 1. Effects of Monocytes and EC-Monocyte CCCM on ACE Activity

View larger version (15K): [in this window] [in a new window]   Figure 4. Free radical scavenging agents do not protect against monocyte-induced decrease in ACE activity. PAECs were cocultured with porcine monocytes (200x103) in the presence of superoxide dismutase (SOD, 300 U/mL), catalase (CAT, 2000 U/mL), 1,3-dimethyl-2-thiourea (DMTU, 5 mmol/L), or Tiron (10 mmol/L) for 48 hours. Agents were added at time 0 and then resupplied after 24 hours. Values are mean±SEM (n=4 wells). *P<.05 vs PAECs.

View this table: [in this window] [in a new window]   Table 2. Effect of Cell-Cell Contact on Monocyte-Mediated Decrease in ACE Activity

Effects of Exogenously Added Cytokines on Ectoenzyme Activities
To further characterize the soluble factor(s) responsible for the decrease in ACE activity in the cocultures, we tested for effects of several monocyte-derived cytokines on ACE and NCT activities (Fig 5). Incubation of HAECs with 1000 U/mL TNF- for 48 hours decreased ACE activity to 63.3±4.4% of control without affecting NCT activity. Added TNF- induced morphological changes of ECs similar to those produced by monocytes. Similar results, though less pronounced, were obtained by adding 50 U/mL IL-1. IL-6 (1000 U/mL) had no effect on either ACE or NCT activities. IFN- (1000 U/mL) did not affect ACE but increased NCT activity. TNF- also induced a decrease in HUVEC ACE activity. This decrease was time and concentration dependent (Fig 6). Moreover, a combination of 100 U/mL TNF- and 50 U/mL IL-1 had a greater effect on ACE activity than either cytokine alone (Fig 7).

View larger version (40K): [in this window] [in a new window]   Figure 5. Effect of cytokines on HAEC ACE and NCT activities. HAECs were incubated with TNF- (1000 U/mL), IFN- (1000 U/mL), IL-1 (50 U/mL), and IL-6 (1000 U/mL). Cytokines were added to HAEC monolayers at time 0, and enzyme activities were determined 48 hours later, after removal of the cytokines. Values are mean±SEM (n=4 wells). *P<.05 vs EC ACE activity; #P<.05 vs EC NCT activity.

View larger version (24K): [in this window] [in a new window]   Figure 6. A, Time and concentration dependence of TNF-–induced ACE decrease in HUVECs is shown. HUVECs were incubated with 1000 U/mL TNF- for 12, 24, 36, and 48 hours, and ACE activity was determined as described in "Materials and Methods." B, TNF- reduces HUVEC ACE activity in a dose-dependent manner. HUVECs were incubated with the indicated concentration of TNF- for 48 hours. Values are mean±SEM (n=4 wells). *P<.05 vs HUVEC ACE activity.

View larger version (13K): [in this window] [in a new window]   Figure 7. HUVECs were incubated with monocytes (EC+MONO, 200x103), IL-1 (50 U/mL), TNF- (100 U/mL), or a combination of TNF- (100 U/mL) and IL-1 (50 U/mL), and ACE activity was determined after 48 hours. Values are mean±SEM (n=4 wells). *P<.05 vs ECs; #P<.05 vs IL-1+TNF-.

TNF- and IL-1 in CM
No TNF- or IL-1 was present in measurable amounts in EC CM (Table 3). TNF- was present only in day-0 MCM and CCCM. IL-1 secretion was more sustained than TNF- secretion, remaining detectable even in day-9 CCCM and MCM. MCM exhibited higher concentrations of TNF- and IL-1 and greater inhibition of EC ACE activity compared with CCCM. TNF- was measured in concentrations sufficient by itself to account for the decrease in EC ACE activity. IL-1 was present in lower concentrations (20% of that added exogenously) and may have contributed to decreases in ACE activity by acting additively with TNF- (Table 3, Fig 7).

View this table: [in this window] [in a new window]   Table 3. Comparison of TNF- and IL-1 Concentration With ACE Activity in ECs Exposed to CM From HUVEC-Monocyte Cocultures and Monocyte Cultures

Effects of Anti–TNF-– and Anti–IL-1–Neutralizing Antibodies on Monocyte-Induced Downregulation of ACE Activity
An anti–TNF-–neutralizing antibody was used in HUVEC-monocyte cocultures to investigate whether neutralization of TNF- released in the coculture medium could prevent the monocyte-induced decrease in ACE activity (Fig 8). The ability of exogenously added TNF- to depress EC ACE activity was fully antagonized by the anti–TNF-–neutralizing antibody (2 µg/mL). The anti–TNF- antibody did not itself affect EC ACE activity. In cocultures, the anti–TNF-–neutralizing antibody inhibited the monocyte-induced decrease in ACE activity by 25%, suggesting that TNF- is responsible, at least in part, for the monocyte-induced decrease in ACE activity (Fig 8). No additional protective effect of the antibody was observed when its concentration was doubled (4 µg/mL), as ACE activity remained at 67% of control. In HAEC-monocyte cocultures (Fig 9), a neutralizing antibody against IL-1 was ineffective in preventing the reduction in ACE activity. Neither of the antibodies, when added alone, had any effect on EC ACE activity. Combination of the neutralizing antibodies against TNF- and IL-1 fully prevented the reduction in ACE activity.

View larger version (37K): [in this window] [in a new window]   Figure 8. A neutralizing antibody (nAb) against TNF- partially protects against the monocyte-induced reduction in ACE activity. HUVECs were incubated with human monocytes (EC+MONO, 200x103) or TNF- (100 U/mL) in the presence and absence of an anti–TNF- nAb (nAbTNF-, 2 µg/mL). nAbTNF- was added to HUVECs and allowed to equilibrate; after 10 minutes, monocytes or TNF- was added to the monolayer; 24 hours later, nAbTNF- was resupplied, and ACE activity was determined at 48 hours. Values are mean±SEM (n=4 wells). *P<.05 vs ECs; #P<.05 vs EC+MONO.

View larger version (30K): [in this window] [in a new window]   Figure 9. A mixture of neutralizing antibodies (nAbs) against TNF- and IL-1 fully protects against the monocyte-induced reduction in ACE activity. HAECs were incubated with human monocytes (HAEC+MONO, 200x103) in the presence and absence of an anti–TNF- nAb (nAbTNF-, 1 µg/mL), IL-1 (nAbIL-1, 2 µg/mL), or IL-1ß (nAbIL-1ß, 2 µg/mL). nAbs were added to HAECs and allowed to equilibrate; after 10 minutes, monocytes were added to the monolayer. ACE activity was determined at 48 hours. Values are mean±SEM (n=4 wells). *P<.05 vs HAECs; #P<.05 vs HAEC+MONO.

Immunoblotting
Western blot analysis of total cell lysates revealed a 170-kD band for ACE (Fig 10). ACE protein levels were found to be decreased in HAEC-monocyte cocultures and in HAEC cultures treated with CCCM or TNF-.

View larger version (38K): [in this window] [in a new window]   Figure 10. Western blot analysis. ACE protein is reduced in HAECs exposed to monocytes. A, Total cell lysate from HAECs treated for 48 hours with vehicle (lane 1, control [CTL]), TNF- (100 U/mL, lane 2), endothelium-monocyte cocultures (lane 3), or HAECs incubated with day-0 CCCM (lane 4). B, The same membranes incubated with an anti-tubulin monoclonal antibody to check for equality in loading and transfer. Autoradiographic signals were analyzed by computer-assisted densitometry. Relative optical density values (average of two separate experiments) yielded an ACE-to-tubulin ratio of 1, 0.34, 0.46, and 0.52 for CTL, TNF-, coculture, and CCCM, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocytes are known to generate free radicals and cytokines, both of which decrease ACE activity.^{17 35 36} To investigate the effects of monocytes on ectoenzyme activities, an EC-monocyte coculture system was used. The major findings of the present study are as follows: (1) Cocultivation of ECs with peripheral blood monocytes induced distinct morphological changes to the endothelium. (2) EC monolayers displayed greater ACE activity than cocultures of EC with monocytes, whereas AmN, AmP, and NCT activities remained largely unaltered. (3) Free radical scavengers did not prevent the monocyte-mediated decrease in ACE activity. (4) Direct contact between monocytes and ECs was not required, since monocytes cultured in transwells, as well as monocyte and EC-monocyte CCCM, decreased ACE activity to a degree similar to that observed in cocultures. (5) Either exogenously added TNF- or IL-1 conferred effects similar to those of monocytes on ACE activity. (6) Culture supernatants from monocytes and EC-monocyte cocultures, but not ECs, contained TNF- and IL-1 in concentrations sufficient to decrease EC ACE activity. (7) Incubation of the EC-monocyte cocultures with a mixture of neutralizing antibodies against TNF- and IL-1 fully prevented the monocyte-induced decrease in ACE activity. (8) Reduction in EC ACE activity by monocytes and TNF- resulted from decreased levels of ACE protein.

Porcine monocytes displayed severalfold higher NCT and AmN activities comparable to PAECs (Fig 2C). As enzyme activity determined in the cocultures is the sum of the activities present on the endothelium and the monocytes, the observation that NCT and AmN activities are greater in porcine cocultures than in EC monolayers is not surprising. If one compares the combined AmN activity of ECs and monocytes to that of EC-monocyte cocultures, the latter exhibit a depressed enzyme activity (Fig 2A and 2B). Such comparisons, however, may be inappropriate since (1) the exact number of monocytes in the cocultures or when cultured alone was not determined in the present study and (2) there is a difference in the phenotype between monocytes cultured on plastic and those cultured on ECs.³⁷

On the basis of data obtained from the time course and monocyte concentration experiments, a 48-hour coculture period was used with 4x10⁵ monocytes/mL, a concentration well within the range of monocytes in blood (4.6x10⁵ monocytes/mL³⁸ ). MCSF is a cytokine affecting survival, proliferation, and differentiation of hemopoietic progenitor cells into monocytes; it also regulates some mature cell functions.^{39 40} Addition of MCSF to the cocultures did not change the time course of the monocyte effects, but it increased the action of monocytes at low monocyte concentrations. Augmentation of the monocyte effects by MCSF may be explained by an increased survival rate and/or stimulation of cytokine production by monocytes. Even though MCSF is constitutively expressed by both endothelial⁴¹ and smooth muscle cells,⁴² we routinely included 200 U/mL MCSF in experiments involving monocytes to ensure constant MCSF levels.

Smooth muscle cells possess ACE activity.^{43 44} Effects of monocytes on ACE activity were found to be endothelium selective, since smooth muscle ACE activity was not significantly reduced in the presence of monocytes. Monocytes express and release proteinases.^{45 46} It is nonetheless unlikely that the decreased EC-associated ACE activity was the result of proteolytic cleavage of the membrane-bound ACE, since EC and EC-monocyte CCCM exhibited similar ACE activities.

Monocytes produce free radicals under certain conditions.^{36 47 48 49} Enzymatically generated superoxide anion (O₂^-) by the xanthine–xanthine oxidase reaction or addition of exogenous hydrogen peroxide decreases ACE activity in cultured bovine pulmonary artery ECs.³⁵ Free radical scavengers protect against the decline in EC ACE activity caused by phorbol myristate acetate–activated neutrophils.³⁵ However, free radicals appear not to be involved in the monocyte-induced decrease in ACE activity described here. Both superoxide dismutase, an extracellular O₂^- scavenger, and Tiron, an agent that scavenges O₂^- both extracellularly and intracellularly, failed to protect the monocyte-mediated decrease in ACE activity. Moreover, catalase and dimethylthiourea, scavengers of hydrogen peroxide and hydroxyl radicals, respectively, were not effective in preventing the decrease in ACE activity induced by monocytes.

The increased ACE activity observed in control PAECs (Table 1) between days 2 and 5 is in agreement with previous reports indicating that ACE activity increases after confluence.^{50 51} Although certain actions of monocytes on ECs require cell-cell contact, other actions are mediated by soluble factors.^{7 52} CM from either monocytes or EC-monocyte cocultures added to EC monolayers for 2 or 5 days decreased EC ACE activity to an extent comparable to that observed in EC-monocyte cocultures. Similar results were obtained with monocytes cultured in transwells, thus preventing direct cell-cell contact between the two cell types. A possible soluble factor(s) mediating the decrease in ACE activity includes the monocyte secretory products IL-1 and TNF-. In the present study, TNF- reduced endothelial ACE activity in a time- and concentration-dependent manner. This observation is in agreement with Hennig et al,¹⁷ who reported that exposure of porcine EC to TNF- lowered ACE activity. Matucci-Cerinic et al,¹⁹ however, have reported that HUVEC ACE activity increases after exposure to IL-1; IL-1 has been reported by others to mimic practically every action of TNF- on cultured ECs.² In the present study, IL-1 suppressed ACE activity (Fig 7). It is interesting to note that systemic administration of TNF- or IL-1 results in hypotension, which is at least partly due to increased NO formation through induction of NO synthase in the vascular wall.^{3 4 53 54} A reduction in ACE activity with a consequent decrease in circulating angiotensin II levels could contribute to the hypotensive effect of these cytokines.

Exposure of ECs to TNF- can reduce intracellular zinc concentration as well as the activities of zinc-dependent enzymes, such as ACE.¹⁷ Reduced catalytic activity does not appear to be the mechanism of TNF- and monocyte-mediated decrease in ACE activity, since in Western blot analysis the 170-kD band for ACE was decreased in HAEC-monocyte cocultures as well as in HAECs treated with TNF- or CCCM. Further studies are required to determine whether the decreased levels of ACE protein are due to decreased synthesis or increased degradation of protein and/or mRNA.

Cultured monocytes can release cytokines into the growth medium even in the absence of pharmacological stimulation.^{55 56} On the other hand, ECs do not constitutively express IL-1 and TNF- but have been shown to do so upon activation with various cytokines.^{3 4} In the present study, EC CM did not contain detectable amounts of immunoreactive cytokines. However, day-0 CCCM and MCM contained TNF-, with the latter containing fivefold more TNF- and inhibiting ACE activity to a greater extent than CCCM. TNF- concentrations in both MCM and CCCM fell to undetectable levels after day 0. IL-1 was detectable in both MCM and CCCM even after prolonged cocultivation of EC with monocytes. To investigate the role of TNF- in the effects of monocytes on ACE activity, a TNF-–neutralizing antibody was used. The TNF-–neutralizing antibody completely inhibited the action of exogenously added TNF-. The same antibody partially prevented the effects of monocytes on EC ACE activity, suggesting that TNF- is one of the cytokines involved in the downregulation of ACE activity. In addition, incubation of the cocultures with a neutralizing antibody against IL-1ß partially prevented the effects of monocytes. A mixture of neutralizing antibodies against TNF- and IL-1 restored ACE activity, suggesting that the monocyte-induced reduction in ACE activity is mediated by the release of these cytokines.

Cocultivation of monocytes with ECs has also been shown to augment the production of prostacyclin (PGI₂), von Willebrand factor, and type I plasminogen activator inhibitor by ECs.⁷ Parallel experiments performed in our laboratory showed larger amounts of EDNO released by either PAECs or HAECs than by PAEC- or HAEC-monocyte cocultures.⁵⁷ The reduction in EDNO release in the cocultures was first observed at 6 hours, whereas the decrease in ACE activity was not evident until 48 hours. It should be stressed that the effects of monocytes on EC morphology and biochemistry (EDNO, ACE) did not require the addition of a monocyte activator, such as endotoxin.⁵⁸ Hahn et al⁵⁸ recently reported that incubation of human peripheral monocytes with concentrations of angiotensin II close to those found in blood leads to increased production of TNF- as well as increased adherence of monocytes to HUVECs. All of the aforementioned effects of monocytes on EDNO, PGI₂, von Willebrand factor, and type I plasminogen activator inhibitor were manifest within 24 hours. It is possible that the early effects (<24 hours) of monocytes on EC phenotype contribute to increased recruitment of monocytes and increased adherence of monocytes and other blood elements to the EC surface. On the other hand, the later observed inhibition of ACE activity by monocytes may represent a negative-feedback mechanism, limiting monocyte activation and adherence to ECs.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme AmN, AmP = aminopeptidases N and P CCCM = conditioned medium from cocultures CM = conditioned medium (media) EC = endothelial cell EDNO = endothelium-derived NO HAEC = human aortic EC HUVEC = human umbilical vein EC IFN = interferon IL = interleukin MCM = conditioned medium from monocyte cultures MCSF = monocyte colony stimulating factor NCT = 5'-nucleotidase PAEC = porcine aortic EC Tiron = 4,5-dihydroxy-1,3-benzene-disulfonic acid TNF = tumor necrosis factor TTBS = Tris-buffered solution containing Tween 20

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-31422 and HL-46689. We are pleased to acknowledge the expert technical assistance of Jim Parkerson, Jenifer Barrett, Livia Marczin, and Mary Snead. We thank Aretha Bogan for the preparation of the manuscript.

Received August 9, 1995; accepted June 13, 1996.

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