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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1922-1926.)
© 1998 American Heart Association, Inc.

Original Contributions

Upregulation of Prostacyclin Synthesis–Related Gene Expression by Shear Stress in Vascular Endothelial Cells

Kazuhiro Okahara; Bing Sun; Jun-ichi Kambayashi

From the Maryland Research Laboratories, Otsuka America Pharmaceutical, Inc, Rockville, Md.

Correspondence to Jun-ichi Kambayashi, MD, PhD, Maryland Research Laboratories, Otsuka America Pharmaceutical, Inc, 9900 Medical Center Dr, Rockville, MD 20850. E-mail junichik{at}mrl.oapi.com

	Abstract

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Abstract—Prostacyclin (prostaglandin I₂, PGI₂) has a variety of functions, including inhibition of smooth muscle cell proliferation, vasodilation, and antiplatelet aggregation. PGI₂ production in endothelial cells has been reported to increase biphasically after shear loading, but the underlying mechanism is not well understood. To clarify the mechanism for the second phase of PGI₂ upregulation, we examined the gene expression of the enzymes involved in PGI₂ production in human umbilical vein endothelial cells (HUVECs) after shear-stress (24 dyne/cm²) loading. The production of 6-keto-PGF₁, a stable metabolite of PGI₂, increased time-dependently under shear stress. The arachidonic acid liberation from membrane phospholipids in HUVECs after 12 hours of shear loading was increased significantly compared with the static condition. No change was observed for cytosolic phospholipase A₂ expression, as detected by reverse transcription–polymerase chain reaction and Western blotting. Cyclooxygenase (COX)-1 mRNA increased after 1 hour of shear loading, and the increase lasted for 12 hours, the longest time tested, whereas COX-2 mRNA increased after 1 hour of shear loading and peaked at 6 hours. An increase of COX-1 expression was detected at 12 hours of shear loading by Western blotting. No expression of COX-2 was detected in the static control, but induced expression was observed at 6 hours after shear loading. PGI₂ synthase was also found to be upregulated. These results suggest that the elevated PGI₂ production by shear stress is mediated by increased arachidonic acid release and a combination of increased expression of COXs and PGI₂ synthase.

Key Words: shear stress • prostacyclin • gene regulation • endothelial cells

	Introduction

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Vascular endothelial cells were once considered to be a passive, antithrombogenic barrier; however, they are now known to have a variety of functions, such as regulation of vascular tone, atherogenic progression, and smooth muscle cell proliferation. Endothelial functions are regulated not only by humoral factors, such as growth factors and cytokines, but also by mechanical forces, such as shear stress, pressure, and stretch. Shear stress in particular plays an important role in endothelial functions. Shear stress can mediate a change in morphology and the release of various vasoactive substances from endothelial cells.^{1 2} Decreased shear stress has been suggested to be involved in the pathogenesis of atherosclerosis. It has been reported that areas of low shear stress are more likely to become sites of atherosclerosis³ and that low shear stress promotes the formation of intimal hyperplasia.⁴ Previous studies have shown that the release of NO, C-type natriuretic peptide, and prostaglandin I₂ (PGI₂) is augmented by shear stress through elevated intracellular Ca²⁺ and increased transcription of endothelial NO synthase mRNA and C-type natriuretic peptide mRNA.^{5 6 7}

Frangos et al⁷ reported that shear stress increases PGI₂ production in a biphasic manner: the first peak was seen within several minutes of shear loading at 10 dyne/cm², and the second peak started at 2 hours and was sustained for several hours. Recently, the enzymes related to PGI₂ biosynthesis have been reported to be regulated by cytokines such as transforming growth factor (TGF-ß) and interleukin-1 at the transcriptional level.⁸ Cyclooxygenase (COX)-2 mRNA expression has also been found to be induced by shear stress.⁹ In the present study, we investigated the mechanism of the second phase of shear stress–induced PGI₂ production by examining the gene expression of PGI₂ synthesis–related enzymes by use of reverse transcription–polymerase chain reaction (RT-PCR) and Western blot analysis.

	Methods

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Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from the umbilical veins by use of collagenase, as described previously.⁶ Cells were cultured at 37°C in medium 199 containing 10% FCS, 90 µg/mL heparin, 150 µg/mL bovine brain extract, 100 U/mL penicillin, and 100 µg/mL streptomycin under humidified 5% CO₂/95% air. The cells were subcultured with 0.02% trypsin and used within 5 passages for all experiments.

Shear Loading
The system we used was modified from the cone-plate viscometer device first described and characterized by Bussolari et al,¹⁰ which allowed us to expose cultured HUVECs to a well-defined laminar fluid flow. The shear-stress device consists of a cone that rotates above a stationary base plate containing the cultured HUVECs. The base plate is made from a 35-mm-diameter gelatin-coated polystyrene dish (Corning). The cone makes an angle of 0.5° with the culture plate and is coupled to a variable-speed motor. The distance between the cone and the culture dish was adjusted to 50 µm. The shear-stress device was operated in a CO₂ incubator.

Confluent HUVECs in a 35-mm dish coated with 0.2% gelatin (Sigma Chemical Co) were washed once with PBS, and 1 mL of culture medium was added. The cells were then exposed to shear stress (24 dyne/cm²) for 1, 3, 6, and 12 hours after 30 minutes of preincubation in the incubator. Control samples were obtained by the same procedure but without exposure to shear stress.

6-Keto-prostaglandin F₁ Measurement by Enzyme Immunoassay
After shear loading, the cells were washed 3 times with PBS, and 1 mL of medium 199 medium without phenol red was added. The cells were incubated in a CO₂ incubator for 30 minutes, and then the medium was collected. After centrifugation at 1500g for 5 minutes, supernatant was collected and the 6-keto-prostaglandin F₁ (6-keto-PGF₁) concentration was measured with a 6-keto-PGF₁ enzyme immunoassay kit (Amersham) according to the manufacturer's protocol.

Arachidonic Acid Liberation Assay
HUVECs were preincubated with 0.5 mCi of ³H-labeled arachidonic acid (AA; 0.1 mCi/mL, DuPont NEN) in 1 mL of culture medium for 24 hours and then washed 3 times with PBS. After the addition of 1 mL of culture medium, the control cells were placed in the CO₂ incubator, and the cells for shear loading were exposed to shear stress (24 dyne/cm²) for 12 hours. Then the cells were washed with PBS and fed with culture medium. After incubation for 30 minutes at 37°C, the medium was collected and the cells were lysed in 2% SDS; the medium and cell lysate were counted in a liquid scintillation counter (Wallac). The liberation rate was calculated by dividing the count from the medium by the total count from both medium and lysate.

RT-PCR
Total RNA from HUVECs was extracted with RNA Stat-60 (Tel-test).¹¹ The purity and degradation of RNA were checked by electrophoresis in 0.8% agarose gel containing ethidium bromide and by spectrophotometric measurements at 260 and 280 nm. Total RNA (2 µg) from HUVECs was transcribed into cDNA with a Supertranscriptase II kit (Gibco BRL) and random hexamers. Aliquots of 1/50 RT reaction were used for each PCR amplification with cytosolic phospholipase A₂ (cPLA₂), COX-1, COX-2, PGI₂ synthase (PGIS), and GAPDH specific primers and a thermal cycler (model 480, Perkin-Elmer). The sequences of the sense and antisense primers used were cPLA₂ sense, 5'-CAGTATTCCCACAAGTTTACG-3'; cPLA₂ antisense, 5'-ACCATTTCAGTGACTTGGTTG-3'; COX-1 sense, 5'-AGAAGCAGTTGCCAGATGC-3'; COX-1 antisense, 5'-AGCCGCAGTTGATACTGAC-3'; COX-2 sense, 5'-CAGTGCA- CTACATACTTACC-3'; COX-2 antisense, 5'-TCTAGCCAGAG- TTTCACCG-3'; PGIS sense, 5'-CAGCTTCCTCACGAGGAT-GA-3'; PGIS antisense, 5'-CAGCCACTGCCTGCTTCTG-3'; GAPDH sense, 5'-GTGCCAAAAGGGTCATCATCTC-3'; and GAPDH antisense, 5'-GATGGCATGGACTGTGGT CATG-3'. The expected sizes of these products were cPLA₂, 360 bp; COX-1, 235 bp; COX-2, 461 bp; PGIS, 324 bp; and GAPDH, 200 bp. PCR was performed for 25 cycles for GAPDH and 30 cycles for all others. The amplification products were quantified by separation on the 1.5% agarose gel and transferred to nylon membranes (Hybond N⁺, Amersham). Specific oligonucleotide probes were labeled with [-³²P]ATP by T4 polynucleotide kinase reaction and hybridized to the membrane at 37°C overnight. The membranes were washed 3 times with 2x SSC for 10 minutes and autoradiographed. Autoradiographs were scanned, and the bands were quantified by use of SigmaScan software. All values were normalized to GAPDH.

Western Blotting
The cells were homogenized in lysis buffer (HEPES 50 mmol/L, NaCl 150 mmol/L, EGTA 2.5 mmol/L, EDTA 1 mmol/L, Triton X-100 1%, PMSF 200 mmol/L, aprotinin 2 mg/mL, and leupeptin 1 mg/mL), and the protein concentration was determined with a BCA protein assay kit (Pierce). Loading buffer was added to achieve a final concentration of Tris-HCl 62.5 mmol/L, SDS 2%, glycerol 10%, and 2-mercaptoethanol 5% before being heated to 95°C for 5 minutes. Twenty micrograms of each sample was used and separated by 10% SDS–polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to membranes (PVDF, Bio-Rad). After incubation for 1 hour at room temperature or overnight at 4°C in TBS/Tween-20 (Tris 25 mmol/L, NaCl 0.5 mol/L containing Tween-20 0.2%, pH 7.4) with 5% skim milk and 1% BSA to reduce nonspecific binding, the membranes were incubated with anti-cPLA₂ polyclonal antibody (Santa Cruz, 1:200), anti–COX-1 monoclonal antibody (Cayman Chemical, 0.5 µg/mL), anti–COX-2 monoclonal antibody (Cayman Chemical, 0.5 µg/mL), and anti-PGIS monoclonal antibody (Cayman Chemical, 0.4 µg/mL) for 2 hours at room temperature. The second antibodies used were anti-mouse peroxidase-conjugated IgG (1:2000) for monoclonal antibody or anti-rabbit peroxidase-conjugated IgG (1:5000) for polyclonal antibody. Finally, the membranes were washed in TBS/Tween-20 and visualized with enhanced chemiluminescence detection reagent (ECL, Amersham).

Statistical Analysis
All values are expressed as mean±SEM. Statistical analysis between 2 groups was performed with unpaired Student's t test. Values of P<0.05 were considered statistically significant.

	Results

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6-Keto-PGF₁ Production Under Shear Stress
PGI₂ production increased under shear stress, as measured by its stable metabolite 6-keto-PGF₁ (Figure 1). The basal release of 6-keto-PGF₁ during the 30-minute incubation at 37°C was 7.9 pg/well. After 1 minute of shear loading at 24 dyne/cm², 6-keto-PGF₁ generation increased to 1.7 times the basal level. After 1 hour of shear loading, 6-keto-PGF₁ production dropped slightly (1.4 times basal). The sustained phase of the 6-keto-PGF₁ increase began after 3 hours of shear loading with 6.6 times the control level and increased further to 15.3 times at 12 hours. This biphasic pattern of PGI₂ production in shear-loaded HUVECs is similar to previous findings.⁷

View larger version (25K): [in this window] [in a new window]   Figure 1. Amount of 6-keto-PGF1 released from HUVECs under shear stress over time. Bar graph shows 6-keto-PGF1 release into the medium for 30 minutes before (basal control) and after shear loading (24 dyne/cm2), measured by enzyme immunoassay. Cells were washed after shear stress exposure and incubated with serum-free medium 199 for 30 minutes. Values are mean±SEM (n=4 each). *P<0.05.

Arachidonic Acid Liberation
The release of AA from HUVECs was examined. It was found that the AA liberation rate (see Methods) was 0.60±0.05% in the static control (n=6). The rate increased significantly (1.5-fold) after 12 hours of shear loading at 24 dyne/cm² (0.91±0.09%, n=5, P<0.05).

Expression of cPLA₂, COX-1, COX-2, and PGIS
cPLA₂, COX-1, COX-2, and PGIS are the enzymes involved in PGI₂ synthesis. The present study showed that cPLA₂ mRNA was expressed at a low level in static conditions, as shown by RT-PCR (Figure 2). No increase was observed after 1, 3, 6, and 12 hours of shear loading at 24 dyne/cm². COX-1 mRNA was also detected at a low level in the static control. The expression increased significantly (1.4-fold) at 1 hour and remained at that level up to 12 hours of shear loading. COX-2 mRNA was detected as a faint band in the static control; its expression became apparent at 1 hour of shear loading and peaked at 6 hours (2.0-fold). However, the expression returned to basal levels at 12 hours. The expression of PGIS was also detected in the static control; its expression increased at 1 hour with a peak at 6 hours (2.2-fold, P<0.05), and then the mRNA levels decreased at 12 hours of shear loading.

View larger version (37K): [in this window] [in a new window]   Figure 2. mRNA expression of cPLA2, COX-1, COX-2, PGIS, and GAPDH (for normalization) in HUVECs determined by RT-PCR. A, Representative set from 4 individual RT-PCR/Southern blotting experiments after shear stress loading at 24 dyne/cm2. Lane 1, static control; lane 2, 1 hour; lane 3, 3 hours; lane 4, 6 hours; lane 5, 12 hours. B, Normalized mRNA level determined by RT-PCR/Southern analysis. Values of static control are taken as 1. Data are mean±SEM (n=4 each). *P<0.05.

Western blot analysis revealed similar levels of cPLA₂, COX-1, COX-2, and PGIS protein expression in HUVECs under shear stress. It was found that cPLA₂ expression was not changed by shear stress. One typical set of results (n=3) is presented in Figure 3. The expression of COX-1 at the protein level was increased only at 12 hours of shear loading, which is different from mRNA expression. COX-2 was not detectable in the static control. After 6 and 12 hours, COX-2 expression was detected by Western blotting, with higher expression at 6 hours, which is consistent with the RT-PCR results. Western blotting also showed PGIS expression to be increased after shear stress. The increase was observed after 6 hours of shear loading. The expression returned to static levels at 12 hours. The pattern of increased PGIS expression at the protein level is correlated with the increased mRNA levels.

View larger version (65K): [in this window] [in a new window]   Figure 3. Western blotting of cPLA2, COX-1, COX-2, and PGIS. Expression of cPLA2, COX-1, COX-2, and PGIS in HUVECs before and at 6 and 12 hours after shear loading (24 dyne/cm2). Twenty micrograms of total protein per lane was loaded for polyacrylamide gel electrophoresis. Lane 1, static control; lane 2, 6 hours after shear loading; and lane 3, 12 hours after shear loading.

	Discussion

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The existence of a second phase of increased PGI₂ production in ECs under shear stress was confirmed in the present study by measurement of 6-keto-PGF₁. Several types of phospholipase have been reported to be involved in AA liberation. Among these, cPLA₂ is considered to play a major role.¹² Activation of cPLA₂ has been suggested to play a role in the early phase of shear stress–induced PGI₂ production.¹³ cPLA₂ is a calcium-dependent phospholipase and is activated by micromolar concentrations of intracellular calcium. Because shear stress elevates the intracellular calcium of vascular ECs during the first several minutes of shear loading,¹⁴ cPLA₂ is believed to be involved in the initial burst of shear-induced AA liberation and PGI₂ production. However, the mechanism of the prolonged second phase of PGI₂ production under shear stress remains unclear.

It has been reported that shear stress activates diacylglycerol lipase.¹⁵ It was found that diacylglycerol lipase inhibitor inhibits shear stress–induced PGI₂ production during the prolonged second phase. The present results are in agreement with previous observations. Although AA liberation increased 1.5-fold after 12 hours of shear loading, no increase in either mRNA or protein levels for cPLA₂ expression was observed, which also suggests that shear stress augments AA liberation through activation of other lipase(s) or increased activity of cPLA₂,¹³ rather than through increased cPLA₂ expression. Nevertheless, the augmented AA liberation, as observed after 12 hours of shear loading in the present study, may contribute to the enhanced second phase of PGI₂ production.

COX has both cyclooxygenase and peroxidase catalytic activity. COX utilizes AA as its substrate and catalyzes the conversion of AA to PGG₂ and further to PGH₂.¹⁶ Numerous reports have indicated that COX plays a major regulatory role in prostanoid biosynthesis.^{17 18 19} Two isoforms (COX-1 and COX-2) have been molecularly cloned and characterized biochemically.^{20 21} COX-1 is considered a constitutive enzyme, which is expressed in most cell types. Recently, it was found that the expression of COX-1 is regulated by several growth factors and cytokines, such as TGF-ß, interleukin-1, and phorbol-12-myristate 13-acetate in ECs.^{8 22 23} By DNA sequence analysis, we found that the sequence of shear stress–responsive elements (5'-GAGACC/GGTCTC-3') is present in the promoter region of the COX-1 gene (–261 to –256 and –492 to –487).²⁴ Our results also clearly demonstrated that the expression of COX-1 at the levels of both transcription and translation in HUVECs was increased by shear stress. The second cyclooxygenase, COX-2, is undetectable in most mammalian cells.^{25 26} COX-2 expression was also reported to be induced by cytokines and growth factors.²⁷ Using a differential display technique, Topper et al⁹ recently reported that shear stress induced COX-2 mRNA expression. In the present study, COX-2 was not detected in HUVECs by Western blotting in the static control. After shear loading for 1 hour at 24 dyne/cm², the level of COX-2 mRNA was found to be elevated. Although it is unclear whether the increase in COX-1 and COX-2 mRNA levels is due to increased transcription or decreased degradation, the elevated mRNAs induced by shear stress resulted in increased COX expression at the protein level. The total increase of COX expression may also contribute to the prolonged second phase of PGI₂ production under shear stress.

The final enzyme involved in PGI₂ biosynthesis is PGIS, which catalyzes the conversion of PGH₂ to PGI₂.²⁸ PGIS is also reported to have the shear stress–responsive element sequence in the promoter region.²⁹ Our results showed that PGIS was significantly increased by shear stress at both the mRNA and protein levels. PGH₂ is also a substrate of thromboxane (TX) synthase, which produces TXA₂. Measurement of TXB₂, a metabolite of TXA₂, in the same samples revealed that a shear stress–induced increase in PGI₂ production was not accompanied by a concomitant increase in TXA₂ (data not shown). Therefore, shear stress appears to selectively augment PGI₂ production in HUVECs.

In summary, our results suggest that increases in the liberation of AA and the expression of COX and PGIS may contribute to the prolonged second phase of PGI₂ production in HUVECs under shear stress. The level of shear stress used in this study is within the physiological range of the arterial system (10 to 55 dyne/cm²).³⁰ Therefore, we postulate that physiological shear stress stimulates PGI₂ production without affecting TXA₂ formation. Lowered shear stress, which has been observed at sites of atherosclerosis, may result in decreased PGI₂ production and may be implicated in acceleration of atherosclerosis formation in vivo.

	Acknowledgments

 
We thank S. Lockyer and P. Randolph for reviewing the manuscript.

Received May 19, 1998; accepted May 29, 1998.

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