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Pregnancy |
Perinatal Research Laboratories, Departments of Obstetrics and Gynecology,3
Pediatrics,4 and Animal Sciences
5 University of Wisconsin-Madison, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, nitric oxide, placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Shear stress is the frictional tangential force imposed on the vessel wall when blood flows through a vessel [11]. Shear stress has a wide-range of effects on the expression of a variety of genes in endothelial cells. The proteins encoded by these genes in turn play important roles in regulating endothelial function, mediating many physiological and pathological processes, such as endothelial cell proliferation, vasodilation, vasoconstriction, and inflammatory responses. It has long been reported that, when blood flow is increased, the blood vessel dilates [12]. Removal of the endothelium, or pretreatment with L-NAME, a NOS inhibitor, greatly reduced or abolished the flow-induced dilation of isolated vessels. Moreover, NO inhibition of flow-mediated vasodilation was reversed by the NOS substrate, L-arginine [13, 14].
In vivo studies using a model of chronic high blood flow or creating fistulas to elevate arterial flow strongly suggest that prolonged increases in shear stress will stimulate endothelial expression of endothelial cell nitric oxide synthase (eNOS) at both the mRNA and protein levels [15, 16]. This physiologic response was also demonstrated in vitro, showing that steady shear stress upregulated eNOS mRNA level in bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) [17, 18].
Among the various flow types being studied in vitro, steady laminar flow has been the most investigated. Steady laminar shear stress applied to cells grown in static culture conditions induces a biphasic production of NO in cultured HUVEC [19, 20]. Although pulsatile laminar flow also was found to stimulate endothelial eNOS activity [21, 22], both eNOS mRNA and protein expression have only been shown to be increased in an endothelial cell and smooth muscle cell cocultured pulsatile system [22]. However, in endothelial cells adapted to flow conditions, the dynamic time course of NO production related to the time-dependent changes in eNOS expression during various levels of pulsatile shear stress has never been elucidated.
To investigate the effects of pulsatile shear stress on endothelial function, we grew the ovine fetoplacental artery endothelial (OFPAE) cells [23] in Cellco CELLMAX (Spectrum Laboratories, Rancho Dominguez, CA) artificial capillary modules and adapted them to an environment of pulsatile shear stress. We hypothesized that further graded elevations in shear stress would increase NO production and eNOS expression by these cells in a time-dependent manner. We observed that pulsatile shear stress induced both acute and prolonged NO production by OFPAE cells, which was shear-stress-magnitude-dependent, and was inhibited by the NOS inhibitor L-NMMA. We also reported for the first time that the acute rises in NO production were due to elevations in shear stress that occur by increasing eNOS activity, whereas the more prolonged NO changes were related to both eNOS activation and regulation of the eNOS enzyme.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pulsatile flow system
OFPAE cells (5 x 106) were inoculated into Cellco CELLMAX artificial capillary modules (Spectrum Laboratories), which were kept in a 37°C, 5% CO2 incubator. Every module contains 50 pronectin-coated permeable polyethylene capillaries, each with an internal diameter of 0.33 mm, a length of 13.0 cm, a pore size of 0.5 µm. The module and a reservoir (50 ml) were connected by silicone tubing, which functions as an O2/CO2 exchanger. The culture media (DMEM contained 400 µM L-arginine and was supplemented with 10% calf serum, 10% fetal bovine serum, and 1% penicillin/streptomycin) was circulated using a pump between the artificial capillary module and the reservoir at a desired flow rate. Shear stress (dynes/cm2) was calculated as 4Q
/
r3, in which Q is flow rate within each capillary, is viscosity of culture media (0.7 x 10-2 poise), and r is the fixed radius of the artificial capillary. The OFPAE cells were grown at a flow rate that provided a pulsatile (60 pulses/min, 8.1 mm Hg) shear stress averaging 3 dynes/cm2 in each capillary in order to adapt the cells to pulsatile flow conditioning and to provide a continuous supply of CO2/O2. Daily lactate levels in culture media were measured spectrophotometrically using lactate kits (Sigma Chemical Co., St. Louis, MO), and the production rate of lactate was calculated to monitor the growth of OFPAE cells cultured in the modules. After 7 days, the production rate of lactate was stabilized, indicating that the cells were viable when they reached confluence. Further shear stress exposure was carried out at Days 11–13 after inoculation. In order to fully exclude the effects of serum on gene activation, the endothelial cells went through 24 h of serum starvation before shear stress was further elevated. Serum-free DMEM was changed into each reservoir bottle, and the entire flow path was also flushed gently with serum-free media. To examine the morphology of OFPAE cells after 12 days of culture inside the system, the capillaries within one module were fixed with 1.25% glutaraldehyde over night at 4°C. Then a randomly selected capillary was longitudinally sliced open, stained with hematoxylin, and viewed under light microscopy.
Shear stress experiments and media sample collection The OFPAE cells were then exposed to higher pulsatile shear stresses, averaging 10 dynes/cm2 (140 pulses/min, 26.4 mm Hg), 15 dynes/cm2 (250 pulses/min, 52.5 mm Hg), or 25 dynes/cm2 (390 pulses/min, 71.7 mm Hg) for 24 h (n = 3–6 for each level of shear stress). Culture media were obtained at 0 (3 dynes/cm2 before elevating pulsatile shear stress), 5, 10, 20, 30, 45, 60, 90 min, and 2, 3, 4, 6, 9 and 24 h of flow stimulation to evaluate both the effluent NOx and affluent NOx of each cartridge. For the eNOS inhibition experiment, 50 µM NG-monomethyl-L-arginine (L-NMMA) was added into the reservoir bottle containing either 5 µM or 400 µM L-arginine in DMEM, 30 min before elevating shear stress (n = 4 for each group). The culture media were obtained 20 min after treatment with either 15 dynes/cm2 or 25 dynes/cm2.
NOX measurement NOX measurement was conducted as described previously [24]. Effluent and affluent culture media (100 µL) at each time point under different shear stress were injected into the NO Analyzer (NOA 280, Siever Incorporation, Boulder, CO). A sodium nitrate standard curve (100 nM–100 µM) was created each day the samples were measured, and all readings were within the range of standard curve. NOX production rate was then calculated as ([NOX]effluent - [NOX]affluent) x Q; Q here is the flow rate that gives each specific shear stress.
Cell recovery In order to study the time course of eNOS protein expression, OFPAE cells were eluted from cartridges after a 7-min exposure to a trypsin (0.05%)/EDTA (0.53 mM) treatment, prior to (0 h) and after various times (2, 6, 12, and 24 h) of shear stress (3, 10, and 25 dynes/cm2) stimulation. To test eNOS mRNA levels, the endothelial cells were exposed to either 3 (n = 4) or 25 (n = 4) dynes/cm2 for 12 h and eluted off the cartridges by trypsin/EDTA. Each cell pellet was split into two parts, one was lysed for protein analysis and the other was subjected to total RNA extraction by RNAzol B.
Western analysis for eNOS protein The cell pellets were lysed by sonication in lysis buffer (50 mM Tris, 0.15 M NaCl, 10 mM EDTA [pH 7.4], 0.1% Tween-20, 0.1% ß-mercaptoethanol, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonylfluoride). Proteins (5 µg/lane) were separated on 7.5% SDS-PAGE gels, electroblotted onto the Immobilon-P membrane (Millipore, Bedford, MA), immunoblotted with mouse monoclonal eNOS antibody (1:750, Transduction Laboratories, San Diego, CA), and visualized by the ECL system as described previously [6, 9, 23]. eNOS protein levels were quantified by scanning densitometry. As a loading control, the same blot was also probed for glyceraldehyde phosphate dehydrogenase (GAPDH) [25].
Total cellular RNA extraction The pellets of OFPAE cells from each experiment were solubilized in 1 ml RNAzol B (Cinna Biotech, Houston, TX). After addition of 150 µl chloroform and phase separation by centrifugation (12 000 x g, 20 min) the upper aqueous phase was removed, extracted twice with phenol/chloroform/isoamyl alcohol using heavy-grade phase lock gel (5-Prime, 3-Prime, Boulder CO) and finally mixed with 110% by volume of isopropanol. RNA was then precipitated by standing at -20°C for 1 h before recovery by centrifugation (12,000 x g, 30 min), and washing of the pellet in 75% ethanol. RNA was finally solubilized in molecular biology-grade water (5-Prime, 3-Prime) and quantified by spectrophotometry [25–27].
eNOS RT-PCR The eNOS mRNA levels were quantified by coupled reverse transcription/polymerase chain reaction (RT/PCR) amplification in single-tube assays using AMV reverse transcriptase and Taq Polymerase as described previously [25, 26]. The forward and reverse primers designed according to the ovine eNOS protein coding region, were 5'-TGTGGCTGTCTGCATGG-3' and 5'-TGGCTGGTAGCGGAAGG-3' [27]. Total cellular RNA of 0.1 µg was examined, data were calculated as copy number of eNOS mRNA per microgram total cellular RNA from the standard curve, which was generated by 104 to 1010 copies of eNOS cDNA plasmid run in each assay. All data were normalized to GAPDH content of each sample, determined by the same RT/PCR procedure using 0.1 µg total cellular RNA [25, 27].
Statistical analysis Data were analyzed by Student t-test or one-way ANOVA. Data are presented as means ± standard error of the means (SEM).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 15 and 25 dynes/cm2, the NOS inhibitor L-NMMA completely inhibited the acute phase (20 min) of NOx production (Fig. 3). In the presence of low concentrations of the eNOS substrate L-arginine (5 µM, based on Km of the enzyme), L-NMMA decreased acute NOx production to nondetectable levels (ND). In contrast, this inhibition by L-NMMA was fully reversed by the excess substrate (400 µM L-arginine), which is normally contained in the DMEM culture media. Because there was no further rise in NOx production in the DMEM containing 400 µM vs. 5 µM L-arginine, these data suggest that substrate availability is not rate limiting for OFPAE cell NO production under shear stress.
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It is possible that both increased expression in eNOS as well as its activity would contribute to the observed prolonged NO production. This supposition was tested by performing time-course studies on protein recovered from separate cartridges with 3, 10, and 25 dynes/cm2 (Fig. 4). We noted that basal shear stress (3 dynes/cm2) throughout the experimental period of 24 h did not alter eNOS protein levels. In contrast, by increasing shear stress from 3 to 10 or 3 to 25 dynes/cm2, there was a tendency for elevations of eNOS protein levels as early as 2 h at 25 dynes/cm2 and 6 h at 10 dynes/cm2. However, increases (P < 0.05) in eNOS levels were observed after the shear force was elevated for 12 h and maintained up to 24 h. Furthermore, the effect of 25 dynes/cm2 to elevate eNOS protein expression were more prominent (P < 0.05) than 10 dynes/cm2, with the differences being observed after 12- and 24-h treatments. In contrast, the expression of the housekeeping gene GAPDH, used as a loading control, was not changed at all the time points and levels of shear forces examined (data not shown). The second more prolonged phase of NOx production described above (Fig. 2) appeared to also be related to this elevation in eNOS expression. In agreement with the increase in eNOS protein, eNOS mRNA levels also tended to be elevated (P < 0.1) after the cells were exposed to 25 dynes/cm2 for 12 h compared with 3 dynes/cm2 (Fig. 5). Moreover, the induction of NOx synthesis increased nearly 8-fold, while eNOS protein expression increased 3.6-fold, and eNOS mRNA was only 1.5-fold greater upon elevating shear stress.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various devices have been developed to study hemodynamic forces (shear stress, circumferential stress, and compressive stress) individually and combined. We are particularly interested in frictional wall shear stress. Although well-defined steady flow has been most investigated [15, 20, 21], we chose to study pulsatile flow because it is more physiologically comparable with the hemodynamic environment in vivo [1]. We adapted the commercially available Cellco CELLMAX artificial capillary module system, developed by the Ballermann group [35–37]. This system provides various irreversible pulsatile shear stresses by establishing graded settings for frequency and amplitude of flow. It has been shown that BAEC grown inside polypropylene hollow fibers perfused with venous or arterial shear stress formed an adherent, confluent monolayer, and aligned themselves in the direction of flow assessed by scanning electron microscopy [37]. This is in total agreement with endothelial morphology addressed by other investigators using a different perfusion system [38]. In the current study, we observed that the OFPAE cells cultured at 3 dynes/cm2 formed a confluent monolayer inside artificial capillaries by the time experimental treatments commenced. Because the reported in vivo systemic physiological arterial shear stress is around 15 dynes/cm2 [39–41], we elected to elevate the flow-adapted OFPAE cells from 3 dynes/cm2 to shear forces of 10, 15, or 25 dynes/cm2 for investigating NOx production and/or eNOS expression. Unfortunately, the in vivo shear stress level in placental vasculature is not yet known.
In the present study, a strength of the current model is that we were able to collect media samples for NOx concentrations from both effluent and affluent flow of the same module at multiple time points after the OFPAE cells were exposed to the various levels of shear stress to calculate the rate of NOx production. We observed that, when shear stress was elevated from 3 to 15, or 3 to 25 dynes/cm2, the NOx production increased significantly and the production rate could be divided into multiple phases. There was an acute induction of NOx production within 30 min with the initial increase in shear stress, followed by a second peak at 2 h and a prolonged phase of NOx production for up to 24 h. Because we calculated NOx production rate by obtaining the difference in NOx concentrations from effluent and affluent flow of the capillary module, it required a sensitive measurement for NOx. Due to the limited sensitivity of the NO analyzer, a severe limitation of this in vitro model lies in the undetectable changes in NOx concentration at any time point with lower shear stresses because we would report this as no significant NOx production. Regardless, our data also demonstrated that the magnitude for both the acute and sustained NOx production phases were dependent on the graded level of shear stress. Responses for NOx production by HUVEC were reported in previous studies, in which endothelial cells were subjected to steady but not pulsatile laminar shear stress, and cumulative NOx levels over time were reported [19]. However, in contrast with our study, although NOx production rates followed a biphasic pattern, their acute rapid burst of NOX within 30 min was completely independent of the level of shear stress. The reason for this discrepancy is likely to be that pulsatile, not steady shear stress, was tested in our experiment. Impulse flow was able to induce NOx at a fairly high production rate even within 1 min [20]. Furthermore, in vivo studies demonstrated that pulsatile flow is more effective in lowering peripheral vascular resistance than nonpulsatile flow and that the difference is related to regulation of endothelium-derived NO [42, 43]. Because both the frequency and the amplitude of pulsatile flow in the systemic circulation are considered as potential stimuli for NO-mediated vasodilation [43], the pulsatility increase in our model, when we elevated shear stress, could be another factor responsible for the observed graded changes in NOx production. In this regard, we must point out that a limitation of the way the current pump system is connected to the cartridges does not allow for differentiation of the NO/eNOS response due to rises in shear rate with elevations in pulse number. Therefore, we cannot dissect the effects of amplitude vs. frequency of pulsatile flow on NO production and eNOS expression. It is noteworthy that the elevations in pulsatility from 60 to 140 pulse/min and 60 to 250 pulse/min (3 to 10 and 15 dynes/cm2, respectively) spans the in vivo range of ovine fetal heart rate (
180–240 pulse/min). However, the most robust rise in NOx production was observed with 25 dynes/cm2 when pulse rate was elevated from 60 to 390 pulses/min. Modifications of these experimental conditions (pulse vs. shear) will need to be made to investigate this issue in future studies.
It is thought that the acute burst of NOx production relates to an increase in eNOS activity, probably via rises in intracellular Ca2+ concentration and/or phosphorylation of eNOS, and that the subsequent NOx production is related to elevations in eNOS activity and expression. We observed that, by increasing shear stress from 3 to 10 or 3 to 25 dynes/cm2, eNOS protein levels began to rise as early as 2–6 h and continued to increase during the 24-h treatment. Compared with 10 dynes/cm2, 25 dynes/cm2 increased eNOS levels at an earlier time point and to a higher level. Moreover, eNOS mRNA tended to be elevated at 12 h under 25 dynes/cm2 compared with 3 dynes/cm2. Ranjan et al. [18] reported that steady shear stress increased eNOS expression at the level of mRNA and protein in HUVEC and BAEC, which are endothelial cells of fetal and adult origin, respectively. Moreover, eNOS mRNA appeared to be increased by unidirectional shear stress due to increased transcription, although it is not clear whether mRNA stability was changed under the same conditions [15, 44]. We observed that elevations in eNOS mRNA level was less than that of protein, suggesting that this could be due to its gene translation efficiency as previously suggested by Xiao et al. [8]. We also observed that shear stress-induced NOx production was elevated acutely before eNOS protein level was increased and that the fold increase in NOx synthesis at 12 h of exposure to 25 dynes/cm2 was substantially more than that of eNOS protein expression. It is unlikely that the greater relative rise in NOx than eNOS expression relates to the activation of additional NOS isoforms (and/or iNOS) because we have previously shown that eNOS is the main if not sole NOS isoform in ovine placental artery endothelium [24]. These data do, however, suggest that eNOS activity of these cells is closely regulated by pulsatile shear stress both during the acute, but also prolonged exposure, to elevated shear stress. Our observations further demonstrated that, in OFPAE cells, pulsatile shear stress-induced eNOS expression was shear-level dependent and the plateaued phase of NOx production described above can also be related to an increased availability in eNOS protein for activation.
Previously, our laboratory has shown that placental artery endothelial eNOS protein levels are increased markedly in vivo during the third trimester [9, 24], when fetal growth and fetoplacental blood flow are elevated [1]. In this study, we demonstrated that one of the major mechanisms for increased eNOS expression in endothelial cells is elevated shear stress or blood flow. In addition, shear stress-mediated vasodilation is augmented during pregnancy [45, 46]. Therefore, these data suggest that endothelium-derived NO leads to vasodilation and placental blood flow increases, which in turn further stimulate eNOS activity and eNOS expression during gestation.
The exact mechanisms by which shear stress induces endothelial NO production through elevations in both eNOS activity and protein expression need further investigation. Because the shear stress response element is present on the 5' upstream promoter region of eNOS, it is believed that this confers direct transcriptional regulation of this gene [47]. Several other endothelial-derived vasoactive factors, which are altered in concert with eNOS expression/NO production during elevations in shear stress (e.g., adrenomedullin and prostacyclin) may interact to further augment the shear stress-induced vasodilation [48, 49]. Moreover, pregnancy is a complicated period of time when not only NO but steroid hormones (estrogen and progesterone) and numerous growth factors such as basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor are all elevated [1, 2, 26]. Therefore, it is very likely that some or all of these factors may synergize with shear stress to modulate endothelial function in pregnancy. Using this novel shear stress system brings us closer to the physiologic environment than conventional static culture to provide us an opportunity to study the interactions of hemodynamic force and other biologically active factors during normal pregnancy adaptation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Ronald R. Magness, Department of Obstetrics and Gynecology, University of Wisconsin, Perinatal Research Laboratories, 7E Meriter Hospital, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; rmagness{at}facstaff.wisc.edu
Received: 18 November 2002.
First decision: 13 December 2002.
Accepted: 8 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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