This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adams, M. R. Articles by Celermajer, D. S. Search for Related Content PubMed PubMed Citation Articles by Adams, M. R. Articles by Celermajer, D. S.

(Circulation. 1997;95:662-668.)
© 1997 American Heart Association, Inc.

Articles

L-Arginine Reduces Human Monocyte Adhesion to Vascular Endothelium and Endothelial Expression of Cell Adhesion Molecules

Mark R. Adams, MBBS, FRACP; Wendy Jessup, PhD; Deborah Hailstones, PhD; David S. Celermajer, MBBS, PhD, FRACP

the Department of Cardiology, Royal Prince Alfred Hospital (M.R.A., D.S.C.) and the Heart Research Institute (W.J., D.H., D.S.C.), Sydney, Australia.

Correspondence to Dr Mark R. Adams, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown 2050, Sydney, Australia.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Monocyte adhesion to endothelial cells is a key early event in atherogenesis. Because L-arginine has been shown to reduce atheroma and to decrease monocyte–endothelial cell adhesion in an animal model of atherosclerosis, we studied the effects of L-arginine on human monocyte adhesion to human endothelial cells and endothelial expression of cell adhesion molecules.

Methods and Results Human umbilical vein endothelial cells (HUVECs) were grown to confluence, then incubated for 24 hours with arginine-deficient media to which was added saline (control), 100 or 1000 µmol/L L-arginine, 100 µmol/L D-arginine, 100 µmol/L N^G-monomethyl-L-arginine (L-NMMA), or 100 µmol/L L-arginine with 100 µmol/L L-NMMA. Human monocytes obtained by elutriation were incubated for 1 hour with HUVECs, and adhesion was measured by light microscopy. Compared with control, monocyte adhesion was reduced by L-arginine (59±10%, P=.01) and increased by L-NMMA (123±20%, P=.01). Surface expression of cell adhesion molecules by HUVECs was assessed by an ELISA under the above conditions with and without stimulation with interleukin-1ß. Expression of ICAM-1 was reduced with both concentrations of L-arginine compared with control in both the basal (43±12%, P<.01), and stimulated (46±15%, P<.01) states, which correlated with decreased levels of mRNA. Expression of VCAM-1 was reduced only in the stimulated state and only in the presence of 1000 µmol/L L-arginine (72±24%, P=.02).

Conclusions L-Arginine reduces human monocyte adhesion to endothelial cells and decreases expression of certain endothelial cell adhesion molecules.

Key Words: atherosclerosis • endothelium-derived factors • leukocytes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The adherence of monocytes to the vascular endothelium is an important early event in atherogenesis.¹ This may be, in part, a consequence of the functional loss of nitric oxide production by endothelial cells.^{2 3} Enhanced monocyte adherence to endothelial cells is evident within 1 week of commencement of a high-cholesterol diet, possibly mediated by the increased expression of endothelial cell adhesion molecules.^{4 5} Expression of cellular adhesion molecules is an important step in leukocyte adhesion to vascular endothelium^{6 7 8} and can be affected by a variety of stimuli.^{9 10 11} It has recently been shown that this process can be modulated in vitro by factors such as antioxidants and nitric oxide donors.^{12 13 14}

Nitric oxide is an important vascular mediator, released continuously by endothelial cells in the basal state,^{15 16} and is formed by nitric oxide synthase from the physiological substrate L-arginine.¹⁷ In the hypercholesterolemic rabbit model, dietary supplementation with L-arginine reduces atheroma formation, improves endothelium-dependent dilation,^{18 19} decreases platelet aggregation,²⁰ and decreases monocyte adherence to aortic endothelium.²¹ In hypercholesterolemic humans, L-arginine supplementation may also improve endothelium-dependent dilation,^{22 23} and we have recently shown that L-arginine supplementation inhibits platelet aggregation in healthy young adults via the nitric oxide pathway.²⁴

The effects of L-arginine on monocyte/endothelium interactions have not been studied in human cells. We aimed, therefore, to study the effects of L-arginine on the interaction between human monocytes and endothelium and to assess the potential role of vascular endothelial cell adhesion molecules in this interaction.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Variable amino acid RPMI cell culture medium was obtained from Life Technology. L-Arginine HCl was obtained from Sigma Chemical Co. D-Arginine and L-NMMA were obtained from Calbiochem-Novabiochem Corp. SIN-1 was a gift from Casella AG. IL-1ß was obtained from Genzyme Corp. Mouse anti-human monoclonal antibodies against VCAM-1, ICAM-1, and E-selectin were obtained from Becton Dickinson, and isotype mouse IgG1 and IgG2 not directed against endothelial cell antigens were obtained from ICN Immunobiologicals. Sheep anti-mouse antibody/horseradish peroxidase conjugate was obtained from Amersham International. A 1-kb fragment of ICAM-1 cDNA from pGEM7 was kindly provided by Dr Andrew Boyd (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia).

Endothelial Cell Culture
HUVECs were harvested enzymatically with a type II collagenase under sterile conditions as described by Minter et al²⁵ and established as primary cell cultures in M199 (Trace Biosciences) containing 20% heat-inactivated human serum, L-glutamine 2 mmol/L (ICN Biomedicals), 0.5% endothelial cell growth promoter (Starrate Products), penicillin 100 U/mL, and streptomycin 0.1 mg/mL. All media were prepared with endotoxin-free water (Baxter) and filtered with Zetapore filters (Cuno Life Sciences Division). Endotoxin-free plasticware and glassware were used in all experiments.

For experimental studies, confluent HUVEC monolayers (passages 1 to 4) were trypsinized and replated onto gelatin-coated 24-mm-diameter tissue culture plates for monocyte adhesion studies and gelatin-coated 96-well plates (Falcon, Becton Dickinson) for studies of cell adhesion molecule expression. Plates were gelatin-coated by addition of 1 mL/5 cm² Haemaccel (Behringwerke AG) diluted 1:250 in PBS, incubation for 1 hour at 37°C, then decanting of the solution before use. HUVECs were grown to confluence and used within 72 hours. Before use, each monolayer was inspected microscopically to ensure that only endothelial cells were present (identified by their typical cobblestone pattern); the purity of the cultures was periodically confirmed by cell staining with a monoclonal antibody specific for von Willebrand factor.

Isolation of Human Monocytes
White cell concentrate (Red Cross Blood Bank) was obtained from peripheral blood of human volunteers who were normocholesterolemic (total cholesterol, 140±31 mg/dL) and had no clinical evidence of cardiovascular disease. Within 24 hours of collection, monocytes were isolated by density gradient separation of white cell concentrate anticoagulated with 0.07% EDTA (Merck Pty Ltd) on Lymphoprep (Nycomed Pharma) at 20°C followed by counterflow centrifugation elutriation as described by Garner et al.²⁶ A Beckman J2-21M/E centrifuge equipped with a JE-6B elutriation rotor and a standard 4.2-mL elutriation chamber (Beckman Instruments, Inc) was used. The elutriation buffer was HBSS without calcium or magnesium (Trace Biosciences) with EDTA (0.1 g/L) and 1% heat-inactivated human serum added. After rinsing of the system with 250 mL 70% ethanol, 250 mL endotoxin-free water, 250 mL 6% hydrogen peroxide, another 250 mL endotoxin-free water, and 250 mL of elutriation buffer, the mononuclear cell fraction taken from the Lymphoprep density gradient at the Lymphoprep/plasma interface was loaded at 9 mL/min into the elutriation rotor (2020 rpm at 20°C). Flow rate was increased by 1 mL/min every 10 minutes, and fractions of eluted cells were collected. Monocytes were eluted between 16 and 17 mL/min. Collected cell fractions were examined with a Cytospin system (Shandon) and Wright's stain (Diff-Quik, Lab-Aids). Monocyte suspensions were used only if purity was >90% on light microscopy, with <1% contamination by neutrophils, and viability was >95% by Trypan blue exclusion. Monocytes were then resuspended in RPMI containing 10% heat-inactivated human serum, penicillin (100 U/mL), and streptomycin (0.1 mg/mL) and stored in Teflon containers at a concentration between 1.5x10⁶ and 2.0x10⁶/mL at 37°C under 5% CO₂ in air and were used within 3 hours.

Monocyte–Endothelial Cell Adhesion Studies
Confluent endothelial cell monolayers or monocytes were incubated with various concentrations of arginine for 24 hours before adhesion assays. Arginine-deficient RPMI prepared with RPMI without arginine but with 10% heat-inactivated human serum (which had been filtered and dialyzed to remove low-molecular-weight compounds) was used for these experiments. This medium had an arginine concentration of <2 µmol/L measured by deproteinizing media with 2% sulfosalicylic acid and analyzing for free L-arginine with high-performance liquid chromatography. To each 10 mL of arginine-deficient media we added 100 µL of the following agents to give the final concentrations shown in parentheses: (1) PBS; (2) L-arginine (100 µmol/L), similar to the normal human plasma arginine level; (3) L-arginine (1000 µmol/L); (4) D-arginine (100 µmol/L); (5) L-arginine (100 µmol/L)+L-NMMA (100 µmol/L); (6) L-NMMA (100 µmol/L); (7) D-arginine (1000 µmol/L); and (8) the nitric oxide donor SIN-1 (100 µmol/L). At the end of the 24-hour incubation, cell viability was >95% (by Trypan blue exclusion) for each condition. Separate experiments were performed after 24 hours of incubation of endothelial cells or monocytes under each of these conditions or with each of these conditions present only during the adhesion assay. Experiments were repeated on at least two occasions, with four separate wells used for each condition.

The adhesion assay was performed by addition of 1 mL of monocyte suspension at a concentration of 1.0x10⁶ to 1.5x10⁶/mL to each endothelial cell monolayer and incubation for 1 hour at 37°C under 5% CO₂ in air. After 1 hour, nonadherent cells were removed by standardized gentle washing with a 1000-µL automatic pipette (Gilson), and the suspension was stored on ice until the cell concentration was counted with a Neubauer hemocytometer (Weber Scientific). The initial suspensions and the suspensions from each well were counted four times by an observer blinded to the incubation conditions. The percentage of adherent monocytes was then calculated by comparison with the initial monocyte concentration. The experiment was repeated with endothelial cell monolayers that had been preincubated with IL-1ß (25 U/mL) as well as the above conditions for 24 hours.

Before this study, preliminary experiments were conducted to assess the effect of various times after elutriation and various incubation periods on monocyte adhesion to HUVECs. These studies were carried out three times during three separate weeks with 12 wells for each condition. Intraobserver error was also assessed in these initial studies, with repeated measurements taken on separate wells under the same conditions and repeated measurements taken on the same wells separated by time. Basal monocyte adhesion to HUVECs was not significantly different at 1 hour (38%), 24 hours (35%), or 48 hours (35%) after elutriation. Monocyte adhesion to HUVECs was maximal after 1 hour of incubation, increasing from 30 minutes (22%) to 1 hour (38%) and remaining unchanged at 2 hours (38%). This technique had a low intraobserver error, with a coefficient of variation of <5%.

Cell Adhesion Molecule Detection on Endothelial Monolayers
Endothelial cell surface expression of adhesion molecules was assessed with an ELISA method. Confluent endothelial monolayers were preincubated for 24 hours in 96-well plates at various arginine concentrations or with SIN-1 (100 µmol/L) as described above for the adhesion assays and with or without IL-1ß (25 U/mL). After the monolayers had been washed with HBSS, monoclonal antibodies to ICAM-1, VCAM-1, E-selectin, and isotype mouse IgG (0.1 µg in 100 µL HBSS with 10% heat-inactivated human serum) were added and allowed to stand for 30 minutes; the monolayers were then washed, and sheep anti-mouse antibody/horseradish peroxidase conjugate (1:500 in 100 µL HBSS with 10% heat-inactivated human serum and 0.05% Tween 20) was added and left for 30 minutes. After further washing, 150 µL ABTS substrate (Kirkegaard and Perry Laboratories) was added to each well and allowed to develop for 15 minutes. Optical density was measured at 414 nm with an ELISA reader (Titertek Multiscan, Flow Laboratories). After these experiments, this technique was used to assess the effect of various arginine conditions on ICAM-1 expression in human monocytes and fibroblasts. Monocytes from elutriation were allowed to adhere to 96-well plates and were incubated for 24 hours under the conditions described above before ELISA. Human skin fibroblasts isolated from a skin explant and used at the third passage were grown in 96-well plates before 24 hours of incubation under the above conditions and ELISA for ICAM-1.

Northern Analysis
RNA was isolated from confluent HUVECs exposed to various arginine conditions for 24 hours by the guanidinium isothiocyanate procedure of Chomczynski and Sacchi.²⁷ Samples of total RNA were electrophoresed on a 1% agarose gel and transferred to a nylon membrane (Hybond N+, Amersham Corp) under vacuum with 50 mmol/L NaOH. Hybridization was performed as described elsewhere²⁸ with a random-primed 800-bp fragment of ICAM coding sequence (specific activity of 10⁹ dpm/µg). After hybridization, the filter was washed sequentially with 2xSSC (0.015 mol/L NaCl/0.015 mol/L trisodium citrate)/0.1% SDS for 30 minutes at 42°C, 0.5xSSC/0.1% SDS for 30 minutes at 42°C, and 0.2xSSC/0.1% SDS for 30 minutes at room temperature and exposed to Kodak Biomax MS film. To demonstrate the quantity and quality of the RNA transferred, the filter was subsequently hybridized at 55°C to an end-labeled oligonucleotide complementary to the 18S ribosomal RNA subunit (in an excess of unlabeled oligonucleotide) and washed twice at room temperature and once at 55°C in 4xSSC/0.1% SDS.

Statistical Analysis
Data were analyzed with SPSS for Windows 6.0. All descriptive data are expressed as mean±SD. Because results for each experiment use cells from different subjects with varied basal adhesion, each condition is expressed as a percentage of the control condition for the experiment. Groups were compared by a one-way ANOVA with Scheffe's test for multiple comparisons. Assessment of intraobserver error used the mean and SD to calculate the coefficient of variation as , as described elsewhere.²⁹ Statistical significance was inferred at a two-sided value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Monocyte–Endothelial Cell Adhesion
Monocyte adhesion to endothelial cell monolayers was reduced when endothelial cells were incubated for 24 hours in the presence of 100 µmol/L L-arginine compared with incubation in arginine-deficient media (68.7±9.4% compared with control, P=.01) (Fig 1). Monocyte adhesion to endothelium was reduced further in the presence of 1000 µmol/L L-arginine (58.9±10.0%, P=.04 compared with 100 µmol/L L-arginine) (Fig 2). D-Arginine did not affect adhesion at either 100 µmol/L (106.6±15.8% compared with control) or 1000 µmol/L (104.9±8.7% compared with control); however, addition of the arginine analogue L-NMMA or L-NMMA and L-arginine for 24 hours before adhesion resulted in increased monocyte adhesion compared with control (122.8±19.5% and 112.7±16.0% compared with control, P=.01). Similarly, the addition of 100 µmol/L SIN-1 resulted in a reduction in monocyte adhesion to HUVECs (57±18% compared with control, P=.01). These results are consistent with an L-arginine–related decrease in monocyte–endothelial cell adhesion dependent on the nitric oxide pathway.

View larger version (67K): [in this window] [in a new window]   Figure 1. Photomicrographs showing monocyte adhesion to HUVEC monolayers taken randomly from the center of 25-mm wells. Monolayer on left (A) has been incubated for 24 hours in arginine-deficient media before adhesion study. Monolayer on right (B) has been incubated for 24 hours in the same medium, to which 1000 µmol/L L-arginine has been added, and shows a reduction in monocyte adhesion.

View larger version (44K): [in this window] [in a new window]   Figure 2. Histogram showing the effect of L-arginine (L-ARG) 100 and 1000 µmol/L, D-arginine (D-ARG) 100 and 1000 µmol/L, L-arginine 100 µmol/L with L-NMMA 100 µmol/L (L-ARG+L-NMMA), and L-NMMA 100 µmol/L (L-NMMA) on human monocyte adhesion to HUVEC monolayers. Results are derived from five separate experiments, each using four wells for each condition. All results are expressed as a percentage of the control condition (see "Methods"). *P=.01.

Monocyte adhesion to endothelial cells was not altered significantly if monocytes were preincubated for 24 hours in the presence of 100 µmol/L or 1000 µmol/L L-arginine (85±5.8% and 98±1.7% compared with control) or D-arginine (83.5±5.0% compared with control) and was unaffected by inhibition of nitric oxide synthase for 24 hours before adhesion assays with L-NMMA or L-NMMA and L-arginine (102±6.0% and 106±4.5%, respectively, compared with control) (P=.08 by ANOVA).

Varied L-arginine concentrations had no effect on monocyte adhesion to endothelial cells when the condition was present only at the time of the adhesion assay (99±5.0% and 95±7.0% compared with control). Similarly, no effect was seen with the addition of D-arginine (95±7.0% compared with control), L-NMMA (93±4.5% compared with control), or L-NMMA and L-arginine (96±3.6% compared with control) (P=.57 by ANOVA).

Monocyte adhesion to endothelial cell monolayers was increased when endothelial cells were preincubated for 24 hours with IL-1ß (from 26±3.3% at baseline to 52±1.6% with stimulation, P<.01). Under this stimulated condition, L-arginine was still associated with reduced monocyte adhesion compared with arginine-deficient media at both 100 µmol/L (79±3.0% compared with control, P<.01) and 1000 µmol/L (69±9.0% compared with control, P<.01). The presence of 100 µmol/L D-arginine (102±1.6% compared with control, P=NS), 1000 µmol/L D-arginine (99±2.2% compared with control, P=NS), or L-NMMA (102±2.1% compared with control, P=NS) did not alter monocyte adhesion to endothelium.

Cell Adhesion Molecule Expression
In the basal state, L-arginine was associated with decreased expression of ICAM-1 compared with arginine-deficient medium (Fig 3); however, no effect was seen on the expression of VCAM-1 (Fig 4). The reduced expression of ICAM-1 with L-arginine was seen at both 100 µmol/L (43±12% compared with control, P<.01) and 1000 µmol/L (57±15% compared with control, P<.01) but not with D-arginine at 100 µmol/L (100±30% compared with control, P=NS) or 1000 µmol/L (101±22% compared with control, P=NS) and was completely blocked by the presence of L-NMMA (107±30% compared with control, P=NS). ICAM-1 expression was also decreased with 100 µmol/L SIN-1 (60±14% compared with control, P<.01), as was the expression of VCAM-1 (70±16% compared with control, P<.01).

View larger version (81K): [in this window] [in a new window]   Figure 3. Histogram showing effect of L-arginine (L-ARG) 100 and 1000 µmol/L, D-arginine (D-ARG) 100 and 1000 µmol/L, L-arginine 100 µmol/L with L-NMMA 100 µmol/L (L-ARG+L-NMMA), and L-NMMA 100 µmol/L (L-NMMA) on the surface expression of ICAM-1 by HUVECs. Top, Basal differences and bottom, differences after 24 hours of stimulation with IL-1ß. All results are expressed as a percentage of the control condition. *P<.01.

View larger version (90K): [in this window] [in a new window]   Figure 4. Histogram showing effect of L-arginine (L-ARG) 100 and 1000 µmol/L, D-arginine (D-ARG) 100 and 1000 µmol/L, L-arginine 100 µmol/L with L-NMMA 100 µmol/L (L-ARG+L-NMMA), and L-NMMA 100 µmol/L (L-NMMA) on the surface expression of VCAM-1 by HUVECs. Top, Basal differences and bottom, differences after 24 hours of stimulation with IL-1ß. All results are expressed as a percentage of the control condition. *P=.02.

Treatment of endothelial cell monolayers for 24 hours with IL-1ß resulted in increased surface expression of ICAM-1 (optical density, 0.26±0.07 versus 0.34±0.08; P=.03), VCAM-1 (optical density, 0.22±0.02 versus 0.29±0.02; P=.01), and E-selectin (optical density, 0.14±0.03 versus 0.21±0.03; P<.01).

In the stimulated state, L-arginine was associated with decreased expression of ICAM-1 when L-arginine was present at both 100 µmol/L (46±15% compared with control, P<.01) and 1000 µmol/L (49±22% compared with control, P<.01) (Fig 2). This effect was not seen with D-arginine 100 µmol/L (99±38% compared with control, P=NS) or 1000 µmol/L (102±29% compared with control, P=NS) and was blocked by L-NMMA (120±39% compared with control, P=NS). L-Arginine preincubation was also associated with reduced expression of VCAM-1 (Fig 3) in the stimulated state but only when L-arginine was present at 1000 µmol/L (72±24% compared with control, P=.02); this was not seen with 1000 µmol/L D-arginine (104±20% compared with control, P=NS). SIN-1 was also associated with reduced expression of ICAM-1 (58±13% compared with control, P<.01) and VCAM-1 (65±19% compared with control, P=.02) in the stimulated state.

L-Arginine had no effect on E-selectin expression either in the basal state or after stimulation by IL-1ß (data not shown). There was no significant binding of isotype IgG to endothelial cell monolayers in either the basal or stimulated state. L-Arginine had no effect on the expression of ICAM-1 in human monocytes or fibroblasts either in the basal state or after stimulation with IL-1ß.

Northern Analysis
Northern blot analysis of RNA extracted from HUVECs and probed with ICAM-1 coding sequence detected two predominant mRNAs in the control cells at about 3.3 and 2 kb (Fig 5). In contrast, in the presence of either 100 or 1000 µmol/L L-arginine these mRNAs were not detectable. This finding is not due to degradation of total RNA in these lanes, as shown by their strong 18S rRNA signal (Fig 5, bottom). The presence of D-arginine or the addition of L-NMMA to L-arginine did not affect ICAM-1 mRNA expression compared with control (note that the lanes containing L-NMMA are slightly underloaded compared with control, as shown in Fig 5, bottom). These findings are consistent with the decreased surface expression of ICAM-1 seen with the presence of L-arginine and suggest that these effects occur at the transcriptional level.

View larger version (36K): [in this window] [in a new window]   Figure 5. Northern blot analysis showing effect of L-arginine on mRNA levels of ICAM. Total RNA (10 µg RNA/lane) from HUVECs incubated for 24 hours with either control medium (low L-arginine concentration), L-arginine (L-ARG) 100 and 1000 µmol/L, D-arginine (D-ARG) 100 µmol/L, L-arginine 100 µmol/L with L-NMMA 100 µmol/L (L-ARG+L-NMMA), or L-NMMA 100 µmol/L (L-NMMA) was electrophoresed, transferred to nylon, and hybridized to a coding sequence probe from ICAM cDNA (top). Major mRNA species and location of 28S and 18S rRNA bands are indicated. Bottom, Same filter after hybridization to an 18S rRNA oligonucleotide to correct for errors in loading and transfer (see "Methods").

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
L-Arginine, the physiological precursor of nitric oxide, has been shown to reduce atheroma formation, endothelial dysfunction, platelet aggregation, and monocyte adhesion to endothelial cells in a hypercholesterolemic rabbit model of atherosclerosis.^{18 19 20 21} In humans, it is recognized that there is decreased nitric oxide availability in the early stages of atherosclerosis and in the presence of vascular risk factors such as hypercholesterolemia.^{22 30 31} Both acute parenteral and chronic oral therapy with L-arginine have recently been shown to improve endothelium-dependent dilation in hypercholesterolemic subjects,^{22 23} and oral L-arginine has been shown to inhibit platelet aggregation in healthy young adults.²⁴ The effect of L-arginine on monocyte adhesion using human cells, however, has not been studied, nor has the mechanism whereby L-arginine might attenuate monocyte adhesion to endothelial cells.

In the present study, we have investigated the effect of L-arginine in vitro using highly purified populations of human monocytes and endothelial cells to assess monocyte adhesion and demonstrated that L-arginine reduces monocyte adhesion to HUVECs via an L-arginine–dependent pathway. These effects were seen only when HUVECs were incubated for 24 hours with L-arginine and not when L-arginine was present only for a short time or when monocytes were incubated for 24 hours, suggesting that the mechanism responsible for decreased monocyte adhesion involves modulation of some process within the endothelial cells that does not change in the short term.

L-Arginine was also associated with decreased surface expression of cell adhesion molecules and decreased levels of ICAM-1 mRNA, suggesting that at least some of the antiadhesion effects of L-arginine may be mediated by reduced cell adhesion molecule expression. It has been shown in some models that endothelial cells express ICAM-1 constitutively.^{10 12} In our study, incubation with the arginine analogue and nitric oxide synthase inhibitor L-NMMA was associated with increased expression of ICAM-1 (but not VCAM-1 or E-selectin), suggesting that tonic production of nitric oxide by endothelial cells may modulate ICAM-1 expression in the basal state. Stimulation of endothelial cells with IL-1ß is known to increase monocyte adhesion to endothelial cells³² and to induce endothelial cell surface expression of ICAM-1, VCAM-1, and E-selectin,^{6 33} and synthesis of these molecules has also been demonstrated by arterial cells in atherosclerosis.³⁴ In this study, IL-1ß also resulted in increased expression of all cell adhesion molecules studied. After stimulation, L-arginine was still associated with decreased expression of ICAM-1 at both 100 and 1000 µmol/L but was associated with decreased expression of VCAM-1 only at 1000 µmol/L, suggesting that the reduction in monocyte adhesion seen in this model with higher concentrations of L-arginine may be mediated, at least in part, by reduced expression of VCAM-1.

Nitric oxide has been proposed by some investigators as an antiatherogenic molecule,³⁵ in part because of an inhibitory effect on adhesion of monocytes to endothelial cells. In the hypercholesterolemic rabbit model, Tsao et al^{21 36} demonstrated that both endogenous and exogenous nitric oxide reduce monocyte binding to endothelial cells. Bath et al³⁷ also showed that nitric oxide donors reduce human monocyte adhesion to cultured porcine aortic endothelial cells. The mechanism of this effect may relate to nitric oxide-induced changes in cell adhesion molecules. Certain nitric oxide donors decrease both the basal and stimulated expression of mRNA for monocyte chemotactic protein-1,³⁸ the expression of ICAM-1 and P-selectin by arteriolar endothelial cells in hypercholesterolemic rats,¹³ and the expression of VCAM-1, ICAM-1, and E-selectin in human endothelial cells stimulated by a number of cytokines.¹² This may be due, at least in part, to decreased VCAM-1 gene transcription by inhibition of NF-B.^{12 39}

In contrast to these studies using pharmacological nitric oxide donors, L-arginine is the physiological substrate of nitric oxide, is a naturally occurring compound, can be taken orally in doses that significantly increase plasma levels without lowering blood pressure, and appears to have potentially antiatherogenic properties in animals and humans.^{18 22 23 24} The beneficial effect of L-arginine on monocyte binding to endothelial cells may be due to attenuation of the increased expression of the cell adhesion molecules that are present in animal models of atherosclerosis and in established human disease,⁴⁰ and this may be mediated by increasing the availability of nitric oxide within the vessel wall. It is most likely that the effect seen in our study is mediated via the nitric oxide pathway, because L-NMMA blocks the attenuation of ICAM-1 and VCAM-1 induced by L-arginine, and D-arginine has no effect on cell adhesion or adhesion molecule expression. It is also possible that L-arginine, directly or via the production of nitric oxide, alters the redox state of vascular cells. Expression of cell adhesion molecules is redox-sensitive and can be stimulated by H₂O₂ and attenuated by the presence of antioxidants through inhibition of NF-B.⁴¹ Also, L-arginine supplementation has been shown in the rabbit model both to increase the production of nitric oxide and to decrease the production of radicals such as PMA-stimulated O₂^- by the vessel wall.¹⁹ Because such radicals may play a role in inactivating nitric oxide, both of these effects of L-arginine may be important in increasing the bioavailability of nitric oxide. Some investigators have also proposed a direct antioxidant action of L-arginine and have shown that L-arginine in vitro is able to inhibit copper-mediated oxidation of LDL (estimated indirectly by diene conjugation).⁴² This is less likely to be the mechanism in our study, since the changes seen with L-arginine were completely eliminated by L-NMMA and were not seen with D-arginine.

Our experiment uses an in vitro model of HUVECs and monocytes obtained from peripheral blood by elutriation, which may differ from the in vivo situation. Endothelial cells do not usually express significant levels of ICAM-1, VCAM-1, or E-selectin in vivo,^{40 43} whereas the HUVECs used in our experiment expressed both ICAM-1 and VCAM-1, presumably stimulated by tissue culture conditions. Although this may differ from arterial endothelial cells from normal subjects in vivo, it may be similar to the situation seen in atherosclerosis, when cell adhesion molecules are expressed by endothelial and smooth muscle cells.⁴⁰ Similarly, stimulation with IL-1ß may not precisely represent the pathophysiological changes seen in atherosclerosis; however, it is a powerful stimulator of cell adhesion molecule expression⁵ and is present in human atherosclerotic lesions.³⁴ Furthermore, since these studies did not use sera or cells from hypercholesterolemic subjects, the results may not necessarily be similar in the presence of oxidized LDL or other lipoproteins.

L-Arginine has been used acutely in several studies of human arterial physiology and has been shown to restore endothelium-dependent dilation in subjects with hypercholesterolemia.^{22 30} Oral supplementation with L-arginine may also improve endothelium-dependent dilation in hypercholesterolemic humans when given for 4 weeks.²³ There is some evidence, however, to suggest that L-arginine may not improve endothelial function once atherosclerosis is very advanced; Otsuji and colleagues⁴⁴ observed restoration of endothelium-dependent dilation with L-arginine infusion in subjects with smooth coronary arteries but not when obstructive atherosclerotic plaque was present. Much interest, however, has focused on the potential therapeutic use of L-arginine and newer, exogenous nitric oxide donors earlier in the disease process.⁴⁵ As a therapeutic agent, L-arginine has the advantage that it may have little or no effect on hemodynamics,⁴⁶ whereas it possesses many of the advantageous qualities of other nitric oxide donors.

Conclusions
In a model of human cells, L-arginine reduces monocyte adhesion to endothelial cells. This effect is associated with decreased endothelial expression of the cell adhesion molecules ICAM-1 and VCAM-1.

	Selected Abbreviations and Acronyms

 

HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule-1 IL-1ß = interleukin-1ß L-NMMA = NG-monomethyl-L-arginine NF = nuclear factor SIN-1 = 3-morpholino sydnonimine VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This work was supported in part by grants from the National Health and Medical Research Council of Australia (NHMRC) and the National Heart Foundation of Australia (NHF). Dr Adams is supported by the NHF, Dr Hailstones by the Heart Research Institute, and Drs Jessup and Celermajer by the NHMRC. The authors are indebted to Mandy Edwards and Emily Oates for their excellent technical assistance.

Received May 8, 1996; revision received August 28, 1996; accepted September 4, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]
   
2. Lefer AM, Ma XI. Decreased basal nitric oxide release in hypercholesterolemia increases neutrophil adherence to rabbit coronary artery endothelium. Arterioscler Thromb. 1993;13:771-776.[Abstract/Free Full Text]
   
3. Cohen RA, Zitnay KM, Haudenschild CC, Cunningham LD. Loss of selective endothelial cell vasoactive functions caused by hypercholesterolemia in pig coronary arteries. Circ Res. 1988;63:903-910.[Abstract/Free Full Text]
   
4. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwarz C, Fogelman AM. Minimally oxidized low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990;87:5134-5138.[Abstract/Free Full Text]
   
5. Jang Y, Lincoff AM, Plow EF, Topol EJ. Cell adhesion molecules in coronary artery disease. J Am Coll Cardiol. 1994;24:1591-1601.[Abstract]
   
6. Graber N, Gopal TV, Wilson D, Beall LD, Polte T, Newman W. T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1. J Immunol. 1990;145:819-830.[Abstract]
   
7. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol. 1986;137:245-254.[Abstract]
   
8. Carlos TM, Schwartz BR, Kovach NL, Yee E, Rosa M, Osborn L, Chi-Rosso G, Newman B, Lobb R, Rosso M. Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured endothelial cells. Blood. 1990;76:965-970.[Abstract/Free Full Text]
   
9. Kim JA, Wayner TE, Parhami CF, Smith CW, Haberland ME, Fogelman AM, Berliner JA. Partial characterization of leukocyte binding molecules on endothelial cells induced by minimally oxidized LDL. Arterioscler Thromb. 1994;14:427-433.[Abstract/Free Full Text]
   
10. Takahashi M, Ikeda U, Masuyama J-I, Kitagawa S-I, Kasahara T, Saito M, Kano S, Shimada K. Involvement of adhesion molecules in human monocyte adhesion to and transmigration through endothelial cells in vitro. Atherosclerosis. 1994;108:73-81.[Medline] [Order article via Infotrieve]
    
11. Luscinskas FW, Kansas GS, Ding H, Pizcueta P, Scheiffenbaum BE, Tedder TF, Gimbrone MA. Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, ß₁-integrins and ß₂-integrins. J Cell Biol. 1994;125:1417-1427.[Abstract/Free Full Text]
    
12. De Caterina R, Libby P, Peng H-B, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation. J Clin Invest. 1995;96:60-68.
    
13. Gauthier TW, Scalia R, Murohara T, Guo J-P, Lefer AM. Nitric oxide protects against leukocyte-endothelium interactions in the early stages of hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1995;15:1652-1659.[Abstract/Free Full Text]
    
14. Faruqi R, de la Motte C, DiCorleto PE. -Tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest. 1994;94:592-600.
    
15. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990;323:27-36.[Medline] [Order article via Infotrieve]
    
16. Stamler JS, Loh E, Roddy MA, Currie KE, Creager MA. Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation. 1994;89:2035-2040.[Abstract/Free Full Text]
    
17. Palmer RMJ, Ferridge AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524-526.[Medline] [Order article via Infotrieve]
    
18. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.
    
19. Boger RH, Bode-Boger SM, Mugge A, Kienke S, Brandes R, Dwenger A, Frolich JC. Supplementation of hypercholesterolemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis. 1995;117:273-284.[Medline] [Order article via Infotrieve]
    
20. Tsao PS, Theilmeier G, Singer AH, Leung LK, Cooke JP. L-Arginine attenuates platelet reactivity in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:1529-1533.[Abstract/Free Full Text]
    
21. Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation. 1994;89:2176-2182.[Abstract/Free Full Text]
    
22. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ, Cooke JP. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248-1253.
    
23. Clarkson P, Adams MR, Powe AJ, Donald AE, McCredie R, Robinson J, Betteridge DJ, Keech A, Celermajer DS, Deanfield JE. Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults. Circulation. 1995;92(suppl I):I-366. Abstract.
    
24. Adams MR, Forsyth CJ, Jessup W, Robinson J, Celermajer DS. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilation in healthy young adults. J Am Coll Cardiol. 1995;26:1054-1061.[Abstract]
    
25. Minter AJ, Dawes J, Chesterman CN. Effects of heparin and endothelial growth supplement on hemostatic functions of vascular endothelium. Thromb Haemost. 1992;67:718-723.[Medline] [Order article via Infotrieve]
    
26. Garner B, Dean RT, Jessup W. Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production. Biochem J. 1994;301:421-428.
    
27. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1986;162:156-159.
    
28. Hailstones D, Gunning P. Regulation of nonmuscle myosin light chain 3 gene expression in response to exogenous MLC3nm mRNA. Cell Mol Biol Res. 1994;40:53-62.[Medline] [Order article via Infotrieve]
    
29. Wendelhag I, Wiklund O, Wikstrand J. Arterial wall thickness in familial hypercholesterolemia. Arterioscler Thromb. 1992;12:70-77.[Abstract/Free Full Text]
    
30. Drexler H, Zeiher AM, Meisner K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolemic patients by L-arginine. Lancet. 1991;338:1546-1550.[Medline] [Order article via Infotrieve]
    
31. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340:1111-1115.[Medline] [Order article via Infotrieve]
    
32. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA. Interleukin-1 activation of vascular endothelium: effects on procoagulant activity and leukocyte adhesion. Am J Pathol. 1985;121:393-403.
    
33. Pober JS, Gimbrone MJ, Lapierre LA, Mendrick DL, Fiers W, Rothlein R, Springer TA. Overlapping patterns of activation of human endothelial cells by interleukin-1, tumour necrosis factor, and immune interferon. J Immunol. 1986;137:1893-1896.[Abstract]
    
34. Moyer CF, Sajuthi D, Tulli H, Williams JK. Synthesis of IL-1 and ß by arterial cells in atherosclerosis. Am J Pathol. 1991;138:951-960.[Abstract]
    
35. Cooke JP, Tsao PS. Is NO an endogenous antiatherogenic molecule? Arterioscler Thromb. 1994;14:653-655.[Free Full Text]
    
36. Tsao PS, Wang B-Y, Chan JR, Cooke JP. Endogenous and exogenous nitric oxide inhibits lipid- and cytokine-induced monocyte-endothelial cell interactions. Circulation. 1995;92(suppl I):I-422. Abstract.
    
37. Bath PMW, Hassall DG, Gladwin A-M, Palmer RMJ, Martin JF. Nitric oxide and prostacyclin: divergence of inhibitory effects on monocyte chemotaxis and adhesion to endothelium in vitro. Arterioscler Thromb. 1991;11:254-260.[Abstract/Free Full Text]
    
38. Zeiher AH, Fisslhalter B, Schray-Utz B, Busse R. Nitric oxide modulates the expression of monocyte chemotactic protein-1 in cultured human endothelial cells. Circ Res. 1995;76:980-986.[Abstract/Free Full Text]
    
39. Kahn BV, Harrison DG, Olbrych MT, Alexander RW, Medford RM. Nitric oxide regulates VCAM-1 gene expression and redox-sensitive transcriptional events in vascular endothelial cells. Circulation. 1995;92(suppl I):I-506. Abstract.
    
40. O'Brien KD, Allen MD, McDonald TO, Chait A, Harlan JM, Fishbein D, McCarty J, Ferguson M, Hudkins K, Benjamin CD, Lobb R, Alpers CE. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques: implications for the mode of progression of advanced coronary atherosclerosis. J Clin Invest. 1993;92:945-951.
    
41. Weber C, Erl W, Pietsch A, Strobel M, Zeigler-Heitbrock HWL, Weber PC. Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. Arterioscler Thromb. 1994;14:1665-1673.[Abstract/Free Full Text]
    
42. Philis-Tsimikas A, Witztum JL. L-Arginine may inhibit atherosclerosis through inhibition of LDL oxidation. Circulation. 1995;92(suppl I):I-422. Abstract.
    
43. Wood KM, Cadogan MD, Ramshaw AL, Parums DV. The distribution of adhesion molecules in human atherosclerosis. Histopathology. 1993;22:437-444.[Medline] [Order article via Infotrieve]
    
44. Otsuji S, Nakajima O, Waku S, Kojima S, Hosokawa H, Kinoshita I, Okubu T, Tamoto S, Takada K, Ishihara T, Osawa N. Attenuation of acetylcholine-induced vasoconstriction by L-arginine is related to the progression of atherosclerosis. Am Heart J. 1995;129:1094-1100.[Medline] [Order article via Infotrieve]
    
45. Mehta JL. Endothelium, coronary vasodilation, and organic nitrates. Am Heart J. 1995;129:382-391.[Medline] [Order article via Infotrieve]
    
46. Hind JM, Doodson AC. Oral L-arginine supplementation has no effect on cardiovascular responses to lower body negative pressure in man. Clin Autonom Res. 1994;4:293-297.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				J. G. Tidball Inflammatory processes in muscle injury and repair Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R345 - R353. [Abstract] [Full Text] [PDF]

				C.-F. Lin, S.-C. Chiu, Y.-L. Hsiao, S.-W. Wan, H.-Y. Lei, A.-L. Shiau, H.-S. Liu, T.-M. Yeh, S.-H. Chen, C.-C. Liu, et al. Expression of Cytokine, Chemokine, and Adhesion Molecules during Endothelial Cell Activation Induced by Antibodies against Dengue Virus Nonstructural Protein 1 J. Immunol., January 1, 2005; 174(1): 395 - 403. [Abstract] [Full Text] [PDF]

				S. H. Wilson, R. D. Simari, P. J. M. Best, T. E. Peterson, L. O. Lerman, M. Aviram, K. A. Nath, D. R. Holmes Jr, and A. Lerman Simvastatin Preserves Coronary Endothelial Function in Hypercholesterolemia in the Absence of Lipid Lowering Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 122 - 128. [Abstract] [Full Text] [PDF]

				R. H. Boger, S. M. Bode-Boger, P. S. Tsao, P. S. Lin, J. R. Chan, and J. P. Cooke An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes J. Am. Coll. Cardiol., December 1, 2000; 36(7): 2287 - 2295. [Abstract] [Full Text] [PDF]

				D. Tomasian, J. F. Keaney Jr., and J. A. Vita Antioxidants and the bioactivity of endothelium-derived nitric oxide Cardiovasc Res, August 18, 2000; 47(3): 426 - 435. [Full Text] [PDF]

				Y. Ding, N. D. Vaziri, R. Coulson, V. S. Kamanna, and D. D. Roh Effects of simulated hyperglycemia, insulin, and glucagon on endothelial nitric oxide synthase expression Am J Physiol Endocrinol Metab, July 1, 2000; 279(1): E11 - E17. [Abstract] [Full Text] [PDF]

				A. Blum, L. Hathaway, R. Mincemoyer, W. H. Schenke, M. Kirby, G. Csako, M. A. Waclawiw, J. A. Panza, and R. O. Cannon III Effects of oral L-arginine on endothelium-dependent vasodilation and markers of inflammation in healthy postmenopausal women J. Am. Coll. Cardiol., February 1, 2000; 35(2): 271 - 276. [Abstract] [Full Text] [PDF]

				J. A. McCrohon, W. Jessup, D. J. Handelsman, and D. S. Celermajer Androgen Exposure Increases Human Monocyte Adhesion to Vascular Endothelium and Endothelial Cell Expression of Vascular Cell Adhesion Molecule-1 Circulation, May 4, 1999; 99(17): 2317 - 2322. [Abstract] [Full Text] [PDF]

				D. Corseaux, T. Le Tourneau, I. Six, M. D. Ezekowitz, E. P. Mc Fadden, T. Meurice, P. Asseman, C. Bauters, and B. Jude Enhanced Monocyte Tissue Factor Response After Experimental Balloon Angioplasty in Hypercholesterolemic Rabbit: Inhibition With Dietary L-Arginine Circulation, October 27, 1998; 98(17): 1776 - 1782. [Abstract] [Full Text] [PDF]

				A. M. Lefer Nitric Oxide: Nature's Naturally Occurring Leukocyte Inhibitor Circulation, February 4, 1997; 95(3): 553 - 554. [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Adams, M. R. Articles by Celermajer, D. S. Search for Related Content PubMed PubMed Citation Articles by Adams, M. R. Articles by Celermajer, D. S.