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(Circulation Research. 1996;79:857-863.)
© 1996 American Heart Association, Inc.

Articles

Sustained Increase in Aortic Endothelial Nitric Oxide Synthase Expression In Vivo in a Model of Chronic High Blood Flow

Sophie Nadaud, Monique Philippe, Jean-Francois Arnal, Jean-Baptiste Michel, Florent Soubrier

INSERM U 358 (S.N., F.S.), Hopital Saint Louis, and INSERM U 367 (M.P., J.-F.A., J.-B.M.), Paris, France.

Correspondence to F. Soubrier, INSERM U 358, Hopital Saint Louis, 1 av Claude Vellefaux 75475 Paris Cedex 10, France.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Physiological adaptation of normal blood vessels to acute or chronic changes in blood flow is endothelium dependent. In vitro studies have shown that, among other genes, NO synthase (NOS) 3 mRNA and protein expression is enhanced by acute elevation of shear stress in endothelial cells. We have investigated the effect of chronic high blood flow on NOS3 mRNA and protein expression in rat aorta. NOS3 mRNA levels were measured by quantitative polymerase chain reaction (PCR) in the aortas of 12 rats with arteriovenous fistulas and 9 sham-operated control rats. The PCR assay indicated that NOS3 mRNA levels were significantly enhanced (twofold) during high blood flow. Western blots showed that immunoreactive NOS3 levels were also increased to a similar extent. Furthermore, the Ca²⁺-dependent NOS activity, measured by the L-arginine to L-citrulline conversion assay, and the cGMP content were also significantly increased in the proximal aortic wall submitted to the arteriovenous shunt. These results indicate that NOS3 mRNA and protein expression is enhanced in vivo during chronic high blood flow.

Key Words: NOS3 mRNA • shear stress • gene expression • aortocaval fistula

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial cells constitute an important physiological biomechanical sensor to flow-related hemodynamic forces. Shear stress is directly linked to blood flow and blood vessel diameter: it is elevated when blood flow is high but can be reduced by increasing the vessel diameter.¹ The intracellular transduction pathway of shear stress is not fully elucidated but likely involves a structural modification of proteins of the endothelial cell membrane, which may be transmitted to cytoskeletal elements,² or a modification of local concentrations of various factors (such as ATP) due to variations of blood flow.³ These signals induce short-term⁴ and long-term responses of the endothelial cell⁵ and participate in blood vessel remodeling.⁶

The transient response to acute increase in shear stress seems to be mediated in part by a Ca²⁺ influx in endothelial cells.⁷ Other intracellular signals such as K⁺ influx⁸ and protein phosphorylation of, for example, integrins also appear to be involved (for review see Reference 9). Stimulated endothelial cells produce the relaxing factors NO and prostacyclin,¹⁰ which, in turn, induce vascular smooth muscle cell relaxation¹¹ and therefore blood vessel enlargement to reduce shear forces.

A sustained increase in shear stress appears to cause more profound modifications of endothelial functions. The morphology and position of endothelial cells are modified to allow a realignment to blood flow¹² and a modulation of the expression of various genes, such as PDGF, endothelin-1,¹³ thrombomodulin,¹⁴ and adhesion molecules,¹⁵ induces prolonged variations in the production of these endothelial factors. These chronic changes of endothelial functions could be involved in functional and structural vessel wall remodeling in situations involving high shear stress, such as chronic exercise adaptation,¹⁶ atherosclerosis, arterial wall response to balloon injury,¹⁷ and arteriovenous fistula.^{1 18}

Using the arteriovenous fistula model, Miller and colleagues initially showed that chronic high blood flow induces enhanced endothelium-dependent vasorelaxation¹⁹ and enhanced EDRF release.²⁰ Subsequently, an increase in NOS3 mRNA levels was observed in cultured endothelial cells exposed to artificial shear stress for 24 hours.^{21 22} However, these experiments were performed in vitro on cultured cells isolated from their physiological (mechanical and hormonal) regulatory environment. Therefore, we have investigated NOS3 expression and activity in the arterial wall of animals with an arteriovenous shunt as a model of chronic high blood flow.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Design
An aortocaval fistula was prepared as described previously^{23 24 25} on 12 normotensive male Wistar rats (300 to 320 g) anesthetized with ether; these will be referred to as fistula rats. Briefly, the fistulas (1.2 to 1.5 mm long) were made through a caval venotomy approach, 10 mm distal to the renal arteries. Sham operations (9 rats [control]) were performed by exposing and temporarily clamping (10 minutes) the aorta and inferior vena cava without cutting or suturing. After 6 weeks, rats were killed by decapitation, and the thoracic aorta was rapidly excised within 2 to 3 minutes. They were then rinsed in cold phosphate-buffered saline solution, frozen in liquid nitrogen, and stored at -80°C. The procedure followed in the care and euthanasia of the study animals was in accordance with the European community standards on the care and use of laboratory animals (Ministere de l'Agriculture, authorization No. 00577).

Materials
Acrylamide, reverse transcriptase, and yeast tRNA were from GIBCO BRL. DHEBA and -³⁵S-ATP were from ICN Biochemicals. Chemicals including ß-NADPH, FAD, FMN, tetrahydrobiopterin, citrulline, calmodulin, L-NAME, and Dowex AG50W-X8 (H⁺ form) were generally purchased from Sigma Chemical Co. Protease inhibitors and Taq DNA polymerase were from Boehringer Mannheim. The dNTP kit was from Pharmacia, and the RNase inhibitor was from Promega. PCRs were performed on a Perkin Elmer Cetus apparatus. Oligonucleotides were synthesized with an Applied Biosystem synthesizer.

RNA Preparation
RNAs were prepared from frozen aortas according to the acidic phenol/chloroform procedure as described previously²⁶ using a polytron homogenizer and stored in DEPC-treated water at -20°C. RNA concentration was measured in triplicate before and after dilution with a spectrophotometer at 260 nm. In addition, mRNA concentrations were checked and corrected after dot blotting and hybridization with probes specific for GAPDH mRNA and 28S ribosomal RNA.

Plasmid Constructions and Internal Standard Preparation
Primer A is a sense primer, and primer B is an antisense primer. We used oligonucleotides derived from the human cDNA²⁷ to amplify a 616-bp fragment of the rat NOS3 cDNA. The sense primer IX-A (5'-TGCCTGCCCCACTGCTCCTC-3') is located within exon 21, and the antisense primer NO-B 3430 (5'-TGCACGGTCTGCAGGACGTTGGT-3') is located within exon 25 of the human gene.²⁸ Rat aorta RNA (5 µg) was reverse-transcribed using oligo dT 12-18 as primer and M-MLV reverse transcriptase (160 U) in the presence of RNase inhibitor (30 U). PCR amplification was performed in 25 µL containing 2 µL of the RT solution, 10 pmol of primers IX-A and NO-B 3430, 1x Taq buffer (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, and 1.5 mmol/L MgCl₂), 0.1 mmol/L dNTP, and 1 U of Taq DNA polymerase (Boehringer). Samples were subjected to 5 minutes of initial denaturation at 94°C; then 30 cycles of 30 seconds at 94°C, 30 seconds at 63°C, and 1 minute at 72°C; and then 1 minute at 63°C and 10 minutes at 72°C. The amplified fragment was purified on an agarose gel, phosphorylated, filled in with T4 DNA polymerase, and subcloned in the EcoRV site of pBluescript. The plasmid pReNOS1 obtained was sequenced to analyze the rat sequence and to design PCR primers. pReNOS1 was then used to obtain the internal standard construct: a 64-bp double-stranded oligonucleotide corresponding to a fragment of polylinker (5'-CAAATCGCGAATTTTTTGCGGCCGCTCGAGCCATGTGGTTG GTCCAAACTAGTTTGGACCAACC-3') was inserted in the Sac II site (bp 526) of the rat NOS3 cDNA plasmid pReNOS1, resulting in pReNIS5. A cRNA was synthesized in vitro as sense probe from 10 µg of the EcoRI-linearized plasmid pReNIS5 using T3 RNA polymerase and the mRNA capping kit (Stratagene) in the presence of 10 µCi of -³⁵S-UTP. After the reaction, the templates were digested with RNase-free DNase, purified by phenol/chloroform extraction, ethanol-precipitated twice to eliminate free nucleotides, and resuspended in 100 µL of DEPC-treated water. A fraction of cRNA was precipitated with trichloroacetic acid to determine the proportion of free nucleotides. The cRNA concentration was measured by spectrophotometry at 260 nm and corrected for the free nucleotide level.

Quantitative PCR
Endothelial NOS mRNA was quantified by coamplifying rat aorta RNA with defined amounts of internal standard cRNA.²⁹ The two RNAs (aorta and standard) were reverse-transcribed in the same reaction to avoid variations in the RT efficiency. Two primers, A 228 (5'-TTCCGGCTGCCACCTGATCCTAA-3') and B 568 (5'-AACATGTGTCCTTGCTCGAGGCA-3'), surrounding the 64-bp fragment insertion site were designed to allow the distinct amplification of NOS3 mRNA (340 bp) and of the internal standard cRNA (404 bp). Furthermore, these primers were chosen to avoid hybridization with the rat inducible or neuronal NOS and to encompass several introns in order to avoid amplification of contaminating genomic DNA. The specificity of amplification was checked by hybridizing the PCR product with an internal oligonucleotide, B 464 (5'-AAAGGCGGTGAGGACTTGTCCAA-3'), derived from pReNOS1 sequence. A negative control was used for each set of samples to check the RT and the PCR amplification reagents for any contamination.

Reverse Transcription
RT was performed for 1 hour at 37°C on 100 to 400 ng of rat aorta RNA in the presence of 1 µg of yeast tRNA, 10 pmol of primer B 568, and increasing amounts of internal standard cRNA (10 to 140 fg) using M-MLV reverse transcriptase.

Primer Radiolabeling (³⁵S Primer Mix)
Quantification of the PCR products was done by using radiolabeled PCR primers and by counting the radioactivity of the amplified fragments. Primers A 228 and B 568 (100 pmol) were phosphorylated with -³⁵S-ATP and T4 polynucleotide kinase, ethanol-precipitated twice, and resuspended in 70 µL of water (300 000 to 400 000 cpm/µL).

PCR
RT solution (3 µL) was mixed with the PCR mix (1x Taq buffer, 8 pmol of A 228 and B 568, and 400 000 cpm of the ³⁵S primer mix) and subjected to 5 minutes of initial denaturation at 94°C; then 28 cycles of 30 seconds at 94°C, 30 seconds at 62°C, and 1 minute at 72°C; and then 1 minute at 62°C and 10 minutes at 72°C. Taq DNA polymerase (1 U) and dNTP (0.1 mmol/L) were added at the 72°C step of the first cycle.

Quantitative Analysis
The PCR products were run for 30 minutes on an 8% acrylamide/DHEBA (29:1) gel in 1x TBE buffer (0.1 mol/L Tris, 0.09 mol/L boric acid, 1 mmol/L EDTA, pH 8.4) using a miniprotean II cell apparatus (Bio-Rad Laboratories). The gel was stained with ethidium bromide, and the bands corresponding to the amplified fragments were excised and dissolved for 2 hours at 50°C in 25 mmol/L periodic acid. After addition of 16 mL of scintillation liquid, the radioactivity was counted for 5 minutes in a ß scintillation counter.

Western Blotting of NOS3
Aortas from another set of control and fistula rats were homogenized in 5 vol of boiling homogenization buffer (50 mmol/L Tris-HCl [pH 7.4] and SDS 2%), microwaved for 15 seconds, and centrifuged at room temperature (5 minutes, 10 000g). The supernatant was used for determination of protein concentrations, and equal amounts (120 µg) of total solubilized proteins were run on a 7.5% acrylamide gel and transferred to nitrocellulose membranes. NOS3 and GAPDH protein blotting was realized using monoclonal antibodies (Transduction Laboratories for NOS3 and Chemicon International for GAPDH). The secondary anti-mouse immunoglobulin antibody (Amersham) was revealed using ECL reagents (Amersham).

NOS Assay
NOS activity was assayed by measuring the stoichiometric formation of L-[¹⁴C]citrulline from L-[¹⁴C]arginine.³⁰ Frozen aortas were weighed and then homogenized at 4°C in a 3-mL glass pestle (Kontes) with 900 µL of 50 mmol/L Tris-HCl (pH 7.4), 2 mmol/L EDTA, 10 mmol/L CHAPS containing 1 mmol/L dithiothreitol, 0.1 g/L phenylmethylsulfonyl fluoride, 10 mg/L pepstatin, 10 mg/L soybean trypsin inhibitor, and 2 mg/L aprotinin. Tissue extracts were centrifuged at 10 000g for 15 minutes (4°C), and the supernatant was stored on ice until used. The enzymatic assay was performed within 1 hour after homogenization. The supernatant (180 µL) was added to 200 µL of a solution containing 0.2 µCi L-[¹⁴C]arginine (Amersham), 1 mmol/L ß-NADPH, 4 µmol/L FAD, 4 µmol/L FMN, 10 µmol/L tetrahydrobiopterin, 2 mmol/L citrulline, 10 000 U/L calmodulin, and either 5 mmol/L CaCl₂ or 5 mmol/L EDTA. Each assay, with CaCl₂ or with EDTA, was performed in duplicate. Samples were incubated for 30 minutes at 37°C, and the reaction was stopped by adding 1 mL of the stop solution (30 mmol/L HEPES [pH 5.5] and 3 mmol/L EDTA). L-[¹⁴C]Arginine was removed from the sample on a 1-mL Dowex AG50W-X8 (Na⁺ form) column equilibrated with 1 mL of stop solution. L-[¹⁴C]Citrulline was specifically eluted with 2x 0.5 mL of water. The radioactivity of the eluate was measured by counting 1 mL with 10 mL of scintillation liquid for 5 minutes. Ca²⁺-dependent NOS activity was determined from the difference between the L-[¹⁴C]citrulline produced in the presence of Ca²⁺ and in the presence of EDTA.

Determination of Lung Water Content and Aortic cGMP and Protein Concentrations
Lung water content was the difference between wet and dry weights after dehydration in acetone diethyl ether and drying under air current for 24 hours. Lung water content was expressed as a percentage of lung wet weight.

cGMP concentrations in the aortas were determined in another set of rats. Aortas were homogenized in 10 vol of 0.1 mol/L HCl with an all-glass homogenizer at 4°C and centrifuged at 15 000g for 30 minutes at 4°C, and aliquots were stored at -20°C. cGMP was measured radioimmunologically as previously described.³¹

Soluble protein concentrations were estimated on aliquots of the different extracts of aorta by using the Bio-Rad/Coomassie brilliant blue G-250 protein assay (Bio-Rad) with bovine serum albumin as a standard.

Statistics
Regression lines were estimated by the least squares regression method. Results are expressed as mean±SEM (except for the Western blot). Comparison of data between groups was made by ANOVA when n9 (all data except Western blot). For the Western blot (n=5), levels of significance were calculated with the nonparametric Wilcoxon-Mann-Whitney rank sum test for independent groups because the number of samples was too small to approximate a normal distribution. The test was performed under the null hypothesis of no difference between the means of the two groups. The Wilcoxon-Mann-Whitney W value (rank sum value) was calculated by hand, and the statistic was evaluated using tables. A value of P.05 was considered significant (P is two-sided).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Establishment of the Quantitative PCR
A rat NOS3 cDNA fragment was amplified using primers designed from the human cDNA sequence. The sequence obtained (616 bp) was 90% homologous to the human sequence and 66% and 67% homologous to rat neuronal NOS and rat inducible NOS, respectively. Primers specific for the rat NOS3 sequence (A 228 and B 568) were designed in order to avoid amplification of the other NOS isoforms mRNAs. PCR on rat aorta mRNA produced a single band, which specifically hybridized with an internal probe B 464. The internal standard was constructed by inserting a 64-bp oligonucleotide within the rat NOS3 cDNA fragment. This results in a 404-bp PCR product compared with the 340-bp PCR product for NOS3 mRNA.

The quantitative assay was performed according to the method developed by Gilliland et al.²⁹ For each sample, a defined quantity of total RNA was reverse-transcribed and amplified with five different concentrations of internal standard (competitor) cRNA. The quantitative analysis was performed by measuring the radioactivity incorporated in the PCR products. The results for one sample were plotted as the logarithm of the ratio of the competitor to the target values versus the logarithm of the known quantity of competitor cRNA at each point. When the quantity of NOS3 mRNA in the sample is equivalent to the quantity of competitor cRNA, the PCR values are equal, and the log ratio is 0 (Fig 1).

View larger version (34K): [in this window] [in a new window]   Figure 1. NOS3 mRNA quantification by competitive PCR. A constant amount of total RNA from rat aorta was mixed with increasing amounts of internal standard RNA (competitor), reverse-transcribed, and amplified by PCR with oligonucleotides specific for NOS3. A, The PCR products, indicated as NOS3 target (340 bp) and NOS3 standard (404 bp), were run on a polyacrylamide gel with a molecular weight DNA standard, stained with ethidium bromide, and visualized by UV radiation. B, The bands corresponding to NOS3 target and standard were cut, dissolved in periodic acid, and counted after addition of scintillation liquid. Each point was plotted as the logarithm of the ratio of the standard to the target counts per minute (CPM) values versus the logarithm of the known amount of standard RNA. At the competition equivalence point (log ratio=0), the amount of target mRNA corresponds to the amount of standard RNA used. In this case, 300 ng of aorta total RNA contained 67 fg of NOS3 mRNA.

In a preliminary study (data not shown), we determined the optimal range of total RNA amounts to ensure a correct amplification between 26 and 30 cycles with the minimal amount of aorta RNA (100 to 600 ng). Control experiments showed that the PCR amplification was exponential from 26 to 30 cycles. Therefore, to allow quantitative analysis the experiments were performed at 28 cycles. A preliminary study was performed to check the linearity of the method: NOS3 mRNA was quantified in various dilutions of the same sample RNA (50 to 600 ng) using increasing amounts of internal standard cRNA. As shown in Fig 2, the results indicate that NOS3 mRNA quantification is linear between 50 and 600 ng of total RNA.

View larger version (10K): [in this window] [in a new window]   Figure 2. Linearity of the quantification by competitive PCR of NOS3 mRNA. Different amounts of total aorta RNA from the same sample were quantified for NOS3 mRNA. Each point was measured using the method described in Fig 1.

NOS3 mRNA in Control and Fistula Rats
Using this method, NOS3 mRNA levels were determined in thoracic aortas from control rats (9 animals) and from rats with aortocaval fistulas (12 animals). Total RNA concentrations were measured by spectrophotometry but were also corrected for GAPDH mRNA and 28S ribosomal RNA content after dot blotting. As shown in the Table, in all cases, the amount of NOS3 mRNA per 100 ng of total RNA is significantly increased in the fistula rats compared with the control rats: 2.2-fold increase (P<.0001) when total RNA was measured by spectrophotometry (Fig 3A), 2.7-fold increase (P<.0001) when total RNA amounts were corrected for GAPDH mRNA content, and 1.9-fold increase (P<.001) when total RNA amounts were corrected for 28S ribosomal RNA content.

View this table: [in this window] [in a new window]   Table 1. Comparison of NOS3 mRNA Content in Aortas From Control Rats and Rats With Aortocaval Fistulas

View larger version (43K): [in this window] [in a new window]   Figure 3. Comparison of NOS3 mRNA content, NOS activity, protein content, and cGMP content between proximal aorta from rats with an aortocaval fistula (dark bars) and control rats (open bars). A, NOS3 mRNA amounts (femtograms) were determined by competitive PCR in total aorta RNA (100 to 400 ng, measured by spectrophotometry) from 12 fistula rats and 9 control rats. B, Ca2+-dependent NOS activity was measured by using the L-[14C]arginine to L-[14C]citrulline conversion assay. Aortas from 9 control rats and 7 fistula rats were used. C, Protein content was determined in the samples used for the NOS activity assay with the Bio-Rad/Coomassie brilliant blue assay. D, cGMP concentrations were measured by radioimmunoassay after succinylation in 17 fistula rats and 19 control rats. Data are mean±SEM.

NOS3 Protein Levels
Aortic NOS3 protein levels were semiquantitatively measured by immunoblotting using a monoclonal antibody. NOS3 immunoreactive protein abundance was compared after scanning densitometric analysis of the autoradiogram and normalization for GAPDH levels. GAPDH content was not significantly different between the two groups. As shown in Fig 4, we found that NOS3 immunoreactive protein levels are increased to the same extent (3.5-fold) as NOS3 mRNA in aortas from fistula rats compared with control rats (W=20, P.05).

View larger version (33K): [in this window] [in a new window]   Figure 4. Comparison of NOS3 protein content in the aortas of control and fistula rats. A, Equal amounts of total aortic proteins from control and fistula rats were immunoblotted using a monoclonal anti–NOS3 antibody. Immunoreactive proteins were revealed using a peroxidase-linked secondary antibody and ECL chemiluminescent reagents (Amersham). B, Immunoreactive NOS3 protein abundance normalized for GAPDH content is shown. Quantification was performed by scanning densitometry of autoradiograms.

Thoracic Aorta NOS Activity and cGMP Contents Are Increased in Fistula Rats
Ca²⁺-dependent NOS activity was measured in the upper parts of aortas of 7 animals among those used for the quantitative PCR analysis (Fig 3B). The specificity of the NOS dosage was observed in one control experiment by inhibiting the conversion of L-arginine to L-citrulline with L-NAME. Ca²⁺-dependent NOS activity was calculated as the difference between the total activity and the activity in the presence of EDTA (2.5 mmol/L). The activity of control rats (11±1 fmol of L-citrulline/min per milligram of tissue) was significantly lower (P<.001) than that of fistula rats (18±2 fmol of L-citrulline/min per milligram of tissue).

Soluble protein content in thoracic aorta (Fig 3C) was determined in both groups of rats and indicated that it is significantly (P=.02) increased in fistula rats (42±9 µg/mg of tissue) compared with control rats (16±1 µg/mg of tissue).

cGMP content was measured in another set of experiments in thoracic aortas (Fig 3D). The results show that the cGMP content of the arterial wall is increased (P<.001) in fistula rats (3928±429 fmol/mg of protein) compared with control rats (2500±157 fmol/mg of protein).

Body weight was not influenced by the aortocaval fistula (341±8 g for control rats and 336±10 g for fistula rats). There was a significant increase in heart weight in fistula rats: 1181±49 mg compared with 891±22 mg for control rats (P<.001). Lung water content was not different between the two groups of rats (78±1.7% in control versus 75±1.8% in fistula rats, P=NS), indicating no sign of heart failure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have used the aortocaval shunt as an in vivo model of increased blood flow in the thoracic aorta. In the present study, we demonstrate that a sustained high flow–induced shear stress results in a physiological adaptation of the aortic endothelium detectable at four levels: increases in (1) NOS3 mRNA, (2) Ca²⁺-dependent NOS activity, (3) NOS3 immunoreactivity, and (4) aortic wall cGMP contents. The aortocaval fistula is a classic model of blood flow and volume overload that is used to chronically increase blood flow in a defined arterial segment.^{1 20 31} It induces a biventricular hypertrophy. Nevertheless, 6 weeks after induction of the model, there are no signs of congestive heart failure.

In order to measure NOS3 mRNA in small amounts of aortic tissues, we developed a quantitative assay based on competitive RT-PCR.²⁹ This method allows measurements of RNA from minute amounts of tissue, such as vessels, and the use of an internal standard makes the method suitable for quantitative studies. The design of primers took advantage of sequence differences between NOS genes, in order to specifically amplify the endothelial isoform. Our data show a chronic increase of the NOS3 mRNA expression in response to increased blood flow, thus illustrating that NOS3 is not only regulated at the level of its enzymatic activity but also at the level of the protein expression.

Increase of Ca²⁺-dependent NOS total activity was also observed (approximately twofold) in aortic walls from aortocaval fistula animals and was comparable to the level of mRNA increase. As the constitutive neuronal NOS isoform is also Ca²⁺ dependent, we cannot exclude that an increased expression of this isoform participates in the observed increase of NOS activity. However, according to results obtained with isolated endothelial cells,^{21 22} the enhancement of NOS3 expression most likely accounts for the major part of this increase. Moreover, this induction is probably underestimated, because the increase of arterial wall cross-sectional area and mass associated with high-flow remodeling involves quantitatively more hypertrophy of smooth muscle cells than of endothelial cells.¹

The increase in cGMP content of the arterial wall attests to the potential functional consequences of the enhanced NOS3 expression, detected at the mRNA and protein level. This cGMP increase is specifically inhibited by L-NAME in this model, showing that this is due to the NOS pathway.³²

Hemodynamic parameters measured at the initiation of the arteriovenous fistula indicate dramatic variations compared with the steady state 2 months later: in particular, the mean shear stress is acutely increased by 10-fold in the aorta above the fistula.¹⁸ In this environment, NOS3 expression is probably highly enhanced during the first few days, in order to counterbalance the blood flow increase by a sustained vasodilation. Before structural adaptation of the vessel wall, the endothelial cell number is enhanced, and their morphology is modified: the cells are radially thickened¹² and adopt a fusiform aligned structure to minimize mechanical stress.³³ In the arteriovenous shunt conditions, the structure of the blood vessel wall is modified to stably enlarge the vessel diameter with a medial expansive remodeling.¹⁸ Conversely, chronic decrease in blood flow is associated with an opposite arterial wall remodeling.⁶ During sustained high blood flow, enhanced NOS3 expression could participate in the mechanisms involved in the chronic expansive remodeling of the arterial wall. Nevertheless, it is not clear how NO could modulate vascular remodeling.

This structural adaptation tends to reverse the high shear stress level on the arterial wall but probably does not normalize it in the model of aortocaval fistula in rats. Indeed, despite this arterial lumenal enlargement, experiments conducted on arteriovenous fistula animals suggest a chronic enhanced NO/EDRF pathway. Miller and Vanhoutte²⁰ have shown that a continuous increased blood flow produces long-term modifications of EDRF secretion. Arterial cGMP content, which seems to depend mainly on NOS activity (since cGMP level decreases with L-NAME infusion), is also chronically augmented upstream from the shunt.³² Our results show that these variations are due in part to a chronic enhanced NOS3 expression, resulting in an increased basal and stimulated enzyme activity upstream from the fistula.

Three studies have reported that, in vitro, shear stress increases NOS3 mRNA and protein expression in cultured bovine^{21 34} and human²² endothelial cells. These experiments indicate the ability of endothelial cells to respond to low shear stress, per se, in a short period of time (24 hours). However, in vivo, endothelial cells are submitted to a higher shear stress at basal state (15 dyne/cm²)³⁵ and to neuronal, autocrine, or paracrine counterregulatory mechanisms, which may modify the long-term response. We show here that, in vivo, NOS3 mRNA and protein expression is modulated by high blood flow–induced high shear stress despite structural adaptation. Indeed, the chronic enlargement of the arterial lumen observed in this model was not able to normalize shear stress and therefore to reestablish NOS3 expression at its basal levels. These results could be seen in connection with the study of Sessa et al,¹⁶ showing an increase in NOS3 mRNA expression in the coronary artery of dogs submitted to chronic exercise.

NOS3 increase is part of the physiological response to a sustained high flow–induced high shear stress. Another important feature is the modulation of the secretion of other endothelium-derived factors to allow the vasodilation of vascular smooth muscle cells in order to adapt the vessel lumen and wall to high flow. Thus, in addition to NOS3 expression enhancement, in vitro studies indicate that several other genes are regulated by shear stress in endothelial cells: vasoactive molecules, growth factors, modulators of thrombosis and fibrinolysis, and adhesion molecules (for review, see References 9 and 33). Shear stress upregulates mRNA levels of various factors, such as PDGF-B, PDGF-A,³⁶ and c-fos,³⁷ and downregulates others, such as thrombomodulin¹⁴ and endothelin-1.¹³ These patterns of expression have been tested under acute shear stress, but their regulation might be different when chronically exposed to shear stress. Thus, thrombomodulin mRNA is transiently increased before downregulation by shear stress in endothelial cells in culture.¹⁴ Different pathways, such as cAMP, Ca²⁺, protein kinase C, and tyrosine phosphorylation, could be implicated.^{9 33}

Further experiments will allow the distinction between transcriptional and posttranscriptional mechanisms of regulation of NOS3 by shear stress. Transcriptional induction may be mediated by the shear stress responsive element present in the NOS3 gene promoter, which has been functionally demonstrated for PDGF-B.³⁸ Other transcriptional factors, such as nuclear factor-B³⁹ or AP-1, could be involved, since protein kinase C activity and c-fos expression³⁷ are induced in shear stress–stimulated cells. In addition to these pathways, shear stress stimulation might stabilize mRNA, as demonstrated for the NOS3 gene in hypoxic endothelial cells.⁴⁰

In conclusion, the high sensitivity of quantitative PCR allowed us to measure NOS3 mRNA directly from rat aortas. This method constitutes a useful tool to detect variations of NOS3 mRNA levels in various physiological models. We have shown in the present study that a chronic high flow–induced high shear stress increases in vivo NOS3 mRNA and protein expression. Therefore, chronic increase in NOS3 expression and activity could be mainly involved in the expansive remodeling of the arterial wall observed in association with high blood flow. Future studies will have to address the molecular mechanisms involved in this regulation.

	Selected Abbreviations and Acronyms

 

DEPC = diethyl pyrocarbonate DHEBA = N-N'-(1,2-dihydroxyethylene)-bis-acrylamide EDRF = endothelium-derived relaxing factor FAD = flavin adenine dinucleotide FMN = flavin mononucleotide L-NAME = N-nitro-L-arginine methyl ester M-MLV = Moloney murine leukemia virus NOS = NO synthase PCR = polymerase chain reaction PDGF = platelet-derived growth factor RT = reverse transcription

	Acknowledgments

 
This study was supported by INSERM and by Bristol Myers Squibb. We are grateful to Dr Jean-Michel Le Moullec, Dr Marie-Therese Chauvet, Dr Francois-Xavier Galen, and Annie Michaud for very helpful discussions. We also thank the laboratory of photography of the Saint-Louis Hayem Institute (LPH-Saint-Louis) for artwork.

Received September 18, 1995; accepted July 15, 1996.

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