Gene transfer of nitric oxide synthase

Gene transfer of nitric oxide synthase

Journal of the American College of Cardiology © 1999 by the American College of Cardiology Published by Elsevier Science Inc. Vol. 34, No. 4, 1999 IS...

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Journal of the American College of Cardiology © 1999 by the American College of Cardiology Published by Elsevier Science Inc.

Vol. 34, No. 4, 1999 ISSN 0735-1097/99/$20.00 PII S0735-1097(99)00304-6

Endothelial Function

Gene Transfer of Nitric Oxide Synthase Effects on Endothelial Biology Josef Niebauer, MD,*† Jo´zef Dulak, PHD,* Jason R. Chan, BS,* Philip S. Tsao, PHD,* John P. Cooke, MD, PHD, FACC* Stanford, California, and Leipzig, Germany OBJECTIVES

The purpose of the study was to investigate the role of nitric oxide (NO) in monocyteendothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.

BACKGROUND Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC. METHODS

Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription– polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.

RESULTS

The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by ⫹60% (n ⫽ 6), which increased further when EC were stimulated by shear stress (24 h) by ⫹137% (n ⫽ 5) as compared with untransfected, unstimulated EC (both p ⬍ 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r ⫽ ⫺0.5669, p ⬍ 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.

CONCLUSIONS Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation. (J Am Coll Cardiol 1999;34:1201–7) © 1999 by the American College of Cardiology

Accumulating evidence indicates that endothelium-derived nitric oxide (NO) is a potent inhibitor of endothelial interaction with monocytes (1– 8). The effect of NO to inhibit endothelial adhesiveness for monocytes appears to have an acute and a delayed mechanism. The NO donors acutely inhibit monocyte adherence to the endothelium, an effect mimicked by cyclic guanosine monophosphate (cGMP) analogues (1). The acute effect of exogenous or From the *Section of Vascular Medicine, Division of Cardiovascular Medicine, Stanford University, Stanford, California, and †Herzzentrum der Universita¨t Leipzig, Kardiologie, Leipzig, Germany. This work was supported in part by a grant from the National Heart, Lung, and Blood Institute (1RO1-HL-58638), and was done during the tenure of a Grant-in-Aid Award from the American Heart Association, with additional funding from Sanofi Winthrop, and Roche Bioscience. Dr. Niebauer is a recipient of a stipend award from the Deutsche Forschungsgemeinschaft, Bonn, Germany (Ni 456/1-1). Dr. Dulak is a recipient of a research grant from the Polish Committee of Scientific Research (No 4 P05A 131 14). Dr. Tsao is the recipient of a National Service Research Award (1F32-HL-08779). Dr. Cooke is an Established Investigator of the American Heart Association and is the founder of Cooke Pharmaceuticals. Manuscript received September 24, 1998; revised manuscript received May 17, 1999, accepted June 10, 1999.

endogenous NO to inhibit monocyte adhesion occurs in the absence of any change in endothelial adhesion molecules for monocytes (5). It is possible that the acute effect of NO on monocyte-endothelial interaction is mediated by a cGMPdependent inhibition of adhesion signaling. However, a sustained exposure to NO donors, or to increased NO biosynthesis, appears to inhibit monocyte binding by altering the expression of endothelial adhesion molecules and chemokines. Endothelial cells exposed to cytokines or oxidized lipoprotein elaborate superoxide anion (3,4). This endothelial oxidative stress is associated with activation of nuclear factor ␬B (NF␬B) and its translocation to the nucleus, where it activates the expression of vascular cell adhesion molecule (VCAM), monocyte chemotactic protein (MCP) and other gene encoding proteins involved in endothelial-monocyte interaction (4,6 – 8). Nitric oxide turns off this oxidant-sensitive transcriptional pathway by reducing endothelial generation of superoxide anion, and by increasing the expression of I␬B␣, the cytoplasmic inhibitor of NF␬B (4,6 –9). In this study we set out to provide

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Abbreviations and Acronyms ANOVA ⫽ analysis of variance EC ⫽ endothelial cells eNOS ⫽ endothelial cell nitric oxide synthase MCP-1 ⫽ monocyte chemotactic protein-1 NF␬B ⫽ nuclear factor ␬B NO ⫽ nitric oxide NOS ⫽ nitric oxide synthase RT-PCR ⫽ reverse transcription–polymerase chain reaction TNF-␣ ⫽ tumor necrosis factor-alpha VCAM ⫽ vascular cell adhesion molecule

JACC Vol. 34, No. 4, 1999 October 1999:1201–7 PRIMERS. The eNOS-specific primers (see Table 1), localized in exon 12 (sense) and exon 16 (antisense), were used to generate 422 base pairs (bp) product. Because of the similarity of the cDNA sequence, the primers were able to amplify both the human and bovine endothelial NOS. The primers for human MCP-1 (Table 1) were localized in exons 1 and 3, and generated 256 bp product. Primers for the human glyceraldehyde phosphate dehydrogenase (housekeeping, reference gene; Table 1) were used to produce 587 bp product as a control for RNA isolation and amplification. REVERSE TRANSCRIPTION–POLYMERASE CHAIN REACTION

A qualitative analysis of mRNA expression was performed by RT-PCR assay, using 500 ng of total RNA obtained from cells collected 48 h after transfection (including the 24-h shear stress treatment). A 30-min reverse transcription step was performed at 70°C (1 U Tth DNA polymerase, 1 mmol/liter MnCl2, 1 ␮mol/liter downstream [3⬘] primer). Afterwards, 20 ␮l of chelating buffer (750 ␮mol/liter EGTA, 0.5 U Taq polymerase, 2.5 mmol/ liter MgCl2, 250 nmol/liter upstream [5⬘] primer) was added and 30 cycles of PCR were performed (94°C/40 s, 62°C/40 s, 72°C/40 s). Final elongation step was 10 min at 72°C. The amplification was performed in a thermocycler (MJ Research Inc., Waltham, Massachusetts). The PCR products were analyzed electrophoretically on ethidium bromide-stained 2% agarose gel. (RT-PCR).

additional evidence for the important role of NO in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells with endothelial cell (EC) nitric oxide synthase (eNOS) DNA.

METHODS Transfection. Transformed human umbilical vein endothelial cells (ECV 304) were transfected at 70% to 80% confluency with eNOS DNA (eNOS DNA: T. Michel, Harvard Medical School; pUC-CAGGS expression plasmid: J. Miyazaki, Tokyo; engineered vector: H. von der Leyen) or pSV40-beta-gal reporter plasmid (beta-gal; negative control) mediated by cationic liposomes (Lipofectamine, Gibco BRL). Once cells reached confluency (approx. 96 h after transfection) the medium was changed to serum-free M199. After 1 h of incubation, cells were either exposed to static or flow conditions and in some experiments stimulated with cytokines (tumor necrosis factoralpha [TNF-alpha] for 4 h; 250 U/ml; Sigma). Expression of eNOS, MCP-1 and GAPDH mRNA. ISOLATION OF RNA. Total cellular RNA was isolated from VSMC (10) using the Total RNA Extraction Kit (Promega, Madison, Wisconsin). The RNA concentration and purity were controlled spectrophotometrically by the optical density measured at 260/280 nm. RNA was diluted in RNAsefree water and kept at ⫺70°C. The samples of RNA used for reverse transcription–polymerase chain reaction (RTPCR) were treated with DNASe (1 U/␮g of RNA) for 15 min at 37°C, followed by phenol-chloroform extraction of RNA from digestion mixture.

Total RNA Extraction kit, Tth polymerase and Taq polymerase were purchased from Promega. RNAsefree DNAse and agarose were from Gibco BRL. The primers were synthesized by Keystone Laboratories (Menlo Park, California). REAGENTS.

FLUID FLOW. Flow was induced by placing culture dishes on a mixing table (Thermylene) rotating at 120 rpm for 24 h. Compared with the well-defined cone-plate viscometer, this technique induces qualitatively similar changes in cell alignment, NO production and NOS mRNA transcription (4,5,10). NITRIC OXIDE MEASUREMENT. Following exposure to either static conditions or stimulation with flow or cytokines, medium was collected from each well for measurements of the stable breakdown products NO2 and NO3 (2). In brief, nitrogen oxides are measured with a commercially available

Table 1. eNOS and MCP-1 Specific Primers Primer

Sequence

eNOS sense eNOS antisense MCP-1 sense MCP-1 antisense GAPDH sense GAPDH antisense

5⬘CTG ATG GCC AAG CGA GTG AA 5⬘CCC AGT CCG AAC ACA CAG AA 5⬘TGC CTG CTG CTC ATA GCA GCC 5⬘GTT TGC TGT TCC AGG TGG TCC 5⬘CGT ATT GGG CGC CTG GTC ACC 5⬘GGG ATG ATG TTC TGG AGA GCC C

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Figure 1. Expression of eNOS and GAPDH mRNA in ECV cells. Note the augmented eNOS expression in eNOS-transfected cells.

chemiluminescence apparatus (model 2108, Dasibi, Glendale, California), after reduction of the samples in boiling acidic vanadium (III) chloride at 98°C. Boiling acidic vanadium quantitatively reduces the oxidative metabolites of NO (NO2 and NO3) to NO, which is quantified by the chemiluminescence detector after reaction with ozone. Signals from the detector were analyzed by a computerized integrator and recorded as areas under the curve. Standard curves for NaNO2/NaNO3 were linear over the range of 50 pmol/liter to 10 nmol/liter. FUNCTIONAL BINDING ASSAY. Endothelial adhesiveness for monocytes was assessed with a functional binding assay as we have previously described (5). Briefly, confluent monolayers of venous endothelial cells were washed with HBSS (Irvine Scientific, Santa Ana, California) containing (in mmol/liter) CaCl2 2, MgCl2 2 and HEPES 20. Culture dishes were then placed on a rocking platform, and human monocytoid cells (THP-1) were incubated with ECVs for 30 min, with dishes rotated 120° clockwise every 10 min to ensure even distribution of cells. Medium was aspirated and replaced with fresh HBSS to remove nonadherent cells. After a second washing, dishes were returned to the rocker platform for an additional 5 min. Medium was again aspirated and replaced with HBSS containing 2% glutaraldehyde. After overnight fixation, adherent mononuclear cells were quantitated using a computer analysis program (Image Analyst, Billerica, Massachusetts).

Data reported are expressed as a percentage of values obtained in the control group (i.e., untransfected, unstimulated cells), which represent 100%. For all statistical tests, differences were considered statistically significant if the two-sided probability of the observed result under the null hypothesis was ⱕ0.05. Results are expressed as mean value ⫾ SD. All calculations were performed using SPSS (SPSS, Chicago, Illinois). Analysis of variance (ANOVA) was performed to identify a significant difference among the mean values of a variable mea-

STATISTICAL ANALYSES.

sured in more than two groups. When the ANOVA was significant, comparisons of the mean values were made by a paired Student t test with the Fisher exact test correction. Correlation coefficients were calculated by Pearson productmoment correlation.

RESULTS Efficiency of transfection. To assess the efficiency of transfection, ECs transfected with the beta-gal construct were stained by X-gal 96 h after the transfection (at the time point at which the data described below were collected). After exposure to X-gal, the EC monolayer was assessed by light microscopy. Approximately 20% to 30% of EC were stained by X-gal, which is consistent with the range of transfection efficiencies typically achieved using cationic liposomal technique in vitro. All EC expressed eNOS mRNA, as demonstrated by RT-PCR (Fig. 1). However, the expression of eNOS was higher in cells transfected with eNOS plasmid than in cells transfected with beta-galactosidase gene or treated with liposomes (Fig. 1). To rule out that eNOS product in eNOS-transfected cells were not derived from amplification of plasmid DNA, samples were treated with RNAse-free DNAse before RT-PCR. The RT-PCR performed on RNA isolated after such a digestion revealed the presence of eNOS mRNA in transfected cells (Fig. 2). The transfection procedure itself did not affect the parameters of endothelial biology assessed in this study. Specifically, we observed no difference in NO synthesis or endothelial adhesiveness for monocytes in nontransfected cells, cells exposed only to the cationic liposome solution or cells exposed to the mixture of cationic liposomes and the beta-gal plasmid construct (data not shown). NO release and monocyte binding. Endothelial cells transfected with eNOS exhibited an increase (158 ⫾ 19%; n ⫽ 6) in NO production in comparison to nontransfected

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Figure 2. RT-PCR for eNOS mRNA was performed on the same RNA isolation before and after its digestion with RNAse-free DNAse. The PCR product was obtained in both cases, which indicates the presence of eNOS mRNA in the transfected cells.

cells (n ⫽ 7) or -gal-transfected cells (99 ⫾ 2%; n ⫽ 2) (all p ⬍ 0.05) (Fig. 3). When nontransfected EC were exposed to shear stress, an increase in NO biosynthesis was observed (134 ⫾ 27%, n ⫽ 6) in comparison to quiescent conditions. When EC transfected with eNOS were also exposed to shear stress there was a greater increase in NO levels observed (237 ⫾ 78%; n ⫽ 5; p ⬍ 0.05) compared with quiescent nontransfected cells. These results were mirrored by those obtained from monocyte-endothelial binding assays. Transfection with eNOS tended to reduce endothelial adhesiveness for monocytes (91 ⫾ 6%; n ⫽ 2), compared with the untransfected, unstimulated cells (100%; n ⫽ 2) or the beta-gal-transfected group (102 ⫾ 2; n ⫽ 2; p ⫽ NS) (Fig. 4). When nontransfected EC were exposed to a 24-h period of shear stress (before the binding assay), their adhesiveness declined significantly compared with quiescent conditions (78 ⫾

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Figure 4. Histogram showing effect of eNOS transfection on endothelial monocyte binding. Transfection with eNOS reduced monocyte-endothelial binding in comparison to untransfected control cells or beta-gal transfected cells. Monocyte-endothelial binding was further inhibited by prior exposure of endothelial cells to shear stress (all p ⬍ 0.05). *p ⬍ 0.05 vs. control and beta-gal.

16%; n ⫽ 2). The effect of shear stress was even more pronounced in the eNOS-transfected cells (70 ⫾ 3% of values achieved in nontransfected cells exposed to quiescent conditions, p ⬍ 0.05). Furthermore, ECV cells treated with liposomes or pSVbeta-gal plasmid expressed higher amounts of MCP-1 mRNA than did eNOS-transfected cells (Fig. 5), suggesting that augmented NO generation by eNOS-transfected cells induces downregulation of MCP-1 expression and thus decreased adhesion of monocytes to eNOS-transfected EC. To ensure that these observations with NOS gene transfection were not unique to this cell line, these studies were repeated using transformed murine brain EC (bEnd3 cells) and murine monocytoid cells (WEHI 274.1). In the murine EC we also observed a significant increase in NO release in eNOS-transfected cells (146% of the response in nontransfected cells; n ⫽ 2). The eNOS-transfected murine EC also manifested a greater increase in NO release when exposed to shear stress (198%; n ⫽ 4 p ⬍ 0.05). This was associated

Figure 3. Histogram showing effect of eNOS transfection on NO production. The eNOS-transfected cells manifest a significant increase in basal NO production, which is further augmented by exposure to shear stress. Control ⫽ nontransfected cells; beta-gal ⫽ cells transfected with a plasmid construct encoding the gene for NO synthase; SS ⫽ control cells exposed to shear stress; eNOS ⫹ SS ⫽ eNOS cells exposed to shear stress. *p ⬍ 0.05 vs. control and ␤-gal.

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Figure 5. Expression of MCP-1 mRNA in ECV cells. Note the diminished MCP-1 expression in eNOS-transfected cells.

with reduced monocyte binding to eNOS-transfected cells (⫺23%; p ⬍ 0.05). NO release and monocyte binding after cytokine stimulation. In the following series of experiments, EC were incubated with tumor necrosis factor-alpha (TNF-alpha) 96 h after transfection. Exposure to TNF-alpha reduced levels of NO synthesis in cells that were transfected with beta-gal to a level of NO production (82 ⫾ 12%) of control cells (Fig. 6). The effect of TNF-alpha was reversed by prior transfection with eNOS (108 ⫾ 8% of the NO elaborated by nontransfected cells not exposed to TNF-alpha). There was a significant difference in NO synthesis between eNOS and beta-gal-transfected cells (p ⬍ 0.02). These results were again mirrored by data obtained from monocyte binding studies. The TNF-alpha stimulation led to an increase in monocyte binding in beta-gal-transfected cells (134 ⫾ 19%; n ⫽ 6) but not in the eNOS-transfected cells (94 ⫾ 9%; n ⫽ 2; p ⬍ 0.02) (Fig. 7). Bivariate correlation. Data obtained from monocyte binding assays and NO measurements were entered into a bivariate correlation analysis; a significant inverse correlation was detected between NO release and monocyte binding (r ⫽ ⫺0.5669; p ⬍ 0.002).

DISCUSSION The salient findings of this investigation are:

1. The eNOS transfection increased NO elaboration by transformed human umbilical venous endothelial cells in quiescent and shear-stress conditions; 2. The enhancement of NO synthesis was associated with reduced endothelial adhesiveness for monocytes; and 3. The eNOS transfection increased the resistance of EC to cytokine-induced adhesion. In the current study, we have demonstrated that human umbilical venous EC or murine brain EC generate more NO when transfected with a plasmid construct encoding NOS. The increase in NO generation was associated with a diminished MCP-1 expression and a reduction of endothelial adhesiveness for monocytes. In the NOS-transfected cells, shear stress produced a greater rise in NO elaboration, consistent with previous observations that the tractive force of fluid flow activates endothelial NOS (5,11,12). The combination of shear stress and eNOS overexpression had additive effects to suppress monocyte adhesion. This latter observation may be due to the fact that, in addition to enhancing NO release, shear stress may have NOindependent effects to reduce endothelial adhesiveness (13,14). Cells transfected with NOS were resistant to the effects of TNF-alpha to induce endothelial adhesiveness for monocytes. These findings are consistent with previous observations that NO inhibits endothelial-monocyte interaction (1,3– 8). Our findings are in keeping with previous observations indicating that modulation of endogenous NO synthesis

Figure 6. TNF-alpha stimulation reduced NO production in those cells transfected with beta-gal but not in those transfected with eNOS (p ⬍ 0.02). *p ⬍ 0.02 vs. ␤-gal ⫹ TNF-␣.

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Figure 7. TNF-alpha stimulation increased in monocyte binding in beta-gal transfected cells. This increase in endothelial adhesiveness was blocked in eNOS-transfected cells (p ⬍ 0.02). *p ⬍ 0.05 vs. control. #p ⬍ 0.02 vs. ␤-gal ⫹ TNF-␣.

affects endothelial monocyte interaction and atherogenesis. It has recently been shown that a circulating antagonist of NOS is elevated in the presence of atherosclerosis or risk factors for atherosclerosis (15–19). This competitive inhibitor of NOS is asymmetric dimethylarginine, the effects of which can be reversed by supplemental L-arginine (15,19 – 21). The diminution of NO activity not only affects vascular tone but also affects endothelial-monocyte interaction. Exogenous administration of NO synthase antagonists to New Zealand White rabbits increases endothelial adhesiveness for monocytes, and accelerates atherogenesis (2,9,11,12,15– 24). Endothelial cells exposed to NOS antagonists generate less NO, and more superoxide anion (25). This may be because NO normally suppresses oxidative enzyme activity (9). The imbalance between NO and superoxide anion leads to activation of an oxidant-sensitive transcriptional pathway that induces the expression of endothelial adhesion molecules and chemokines mediating monocyte adherence (3,4,6 – 8). This atherogenic pathway can be abrogated by enhancing endogenous NO production (23,24). New Zealand White rabbits fed a 1% cholesterol diet for 10 weeks develop intimal lesions that involve 40% of the thoracic aortae; administration of supplemental L-arginine enhances endothelial synthesis of NO, reduces MCP-1 elaboration and inhibits lesion formation by 75% (3,26,27). The vessel wall will be a likely target for gene therapy in the future. Because of its strategic location, the endothelium may be genetically engineered to have local effects on the vessel wall; to condition circulating blood elements; or to secrete substances into the lumen so as to have systemic effects. To date, most experimental applications have been targeted to affect vessel structure, particularly to inhibit restenosis after balloon angioplasty. These approaches have included the use of antisense oligonucleotides to inhibit genes involved in vascular smooth muscle proliferation (28); oligonucleotide “decoys” to neutralize transcriptional factors involved in cell cycle (29); or plasmid constructs encoding “vasoprotective” genes that enhance endothelial regenera-

tion or vascular generation of NO (30). Gene therapy directed at enhancement of endothelial regeneration and angiogenesis may also prove fruitful in the treatment of atherosclerosis and restenosis (31,32). Indeed, in vivo adenovirus-mediated endovascular delivery of neuronal NO synthase has been most recently shown to enhance vascular NOS activity and to reverse hyperlipidemia-induced vascular dysfunction (33). Conclusions. Transfection of cultured EC with a plasmid construct encoding the endothelial isoform of NOS increases the elaboration of NO by quiescent or shear-stressed cells. The increase in NO elaboration reduced endothelial adhesiveness for monocytes, and increased the resistance of the endothelium to cytokine-stimulated monocyte adhesion. Reprint requests and correspondence: Dr. John P. Cooke, Director, Vascular Medicine, Falk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5246. E-mail: [email protected]

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