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Departments of Obstetrics/Gynecology (S.M.G., J.M.C., S.T., R.R.M., I.M.B.) and Meat/Animal Science (R.R.M.), Perinatal Research Laboratories, University of Wisconsin-Madison, Madison, Wisconsin 53715
Address all correspondence and requests for reprints to: Ian M. Bird, Ph.D., Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: imbird{at}facstaff.wisc.edu.
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Introduction |
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Mounting evidence suggests that dysfunctional endothelial regulation of vasodilator production during pregnancy may result directly in preeclampsia and intrauterine growth retardation (15, 16). Investigating the mechanisms that regulate normal vasodilator production may allow us to understand the pathophysiological etiology of these conditions. To this end, we have developed a cell model that retains functional differences between UAEC from healthy nonpregnant (NP) and pregnant (P) late term ewes (17). After 14–18 d in culture (i.e. by passage 4), the P-UAEC showed increased NO and PGI2 production over the NP-UAEC in response to AII, ATP, bFGF, EGF, and VEGF, although the amount of eNOS and cPLA2 reverted to a similar level (17, 18). In addition, ATP stimulation of P-UAEC caused a more sustained [Ca2+]i response compared with NP-UAEC (18). This retention of pregnancy-specific Ca2+ signaling is consistent with the findings of Mahdy et al. (19) in human hand vein endothelial (HHVE) cells derived from pregnant, nonpregnant, and preeclamptic women. When maintained and expanded for 14–28 d in culture, (a similar time to our fourth passage or beyond for UAEC), HHVE had significantly larger ATP-stimulated increases in [Ca2+]i than occurred in cells isolated from nonpregnant or preeclamptic women. Previous studies on the UAEC model have also shown that although ATP caused an increase in [Ca2+]i (17) that is enhanced by pregnancy, AII, bFGF, EGF, and VEGF all induce NO and PGI2 production without an associated increase in [Ca2+]i. However, ERK 1/2 activation is induced by AII, ATP, bFGF, and VEGF to a higher extent in P-UAEC than in NP-UAEC, and this correlated closely with increased vasodilator production (18). Thus, enhanced coupling to alternate signaling pathways during pregnancy may activate eNOS and cPLA2 independently of an increase in [Ca2+]i.
One limitation of the HHVE or UAEC model is that the data are derived from cells expanded in primary culture. As a consequence, not all of the cells come directly from the in vivo state, and underlying cellular and physiological properties of the cells may have been altered by culture conditions. To determine whether culture conditions altered cellular function, experiments were performed on freshly isolated and passage 4 cell preparations to compare agonist effects on cell signaling pathways. In addition, cDNA microarray and protein screening were used to fingerprint the cellular phenotype of both preparations. Ex vivo results consistent with the passage 4 cells would validate the UAEC model and provide evidence supporting our hypothesis that a cell programming event occurs during pregnancy, which leads to increased coupling of receptor activation to the ERK cascade. Alternatively, if ex vivo cells did not respond similarly to passage 4 UAEC, then the culture conditions may have altered cellular function and the model would be of limited value. Our results indicate that the UAEC model accurately reflects in vivo UA endothelium and pregnancy-associated changes in both Ca2+ and ERK signaling events investigated to date, but the underlying cause remains unclear.
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Materials and Methods |
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, PKCß, A-Raf, and C-Raf were purchased from Transduction Laboratories, Inc. (Lexington, KY). Polyclonal antibodies to PKC and PKC
were purchased from PanVera Corp. (Madison, WI). Polyclonal antibody to phosphorylated ERK (P-ERK) 1/2 was purchased from New England Biolabs, Inc. (Beverly, MA). ATP (disodium salt) was purchased from Sigma-Aldrich Corp. (St. Louis, MO), and thapsigargin was purchased from Calbiochem (San Diego, CA). Unless noted other wise, MEM D-Val and all other cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY), glass-bottom microwell dishes for Ca2+ imaging studies were purchased from MatTek Corporation (Ashland, MA), and eight-well glass chamber slides were purchased from Nunc, Inc. (Naperville, IL).
Isolation of UAEC
UAs were obtained from Polypay and mixed Western breed nonpregnant and pregnant ewes at 120–130 d gestation during nonsurvival surgery. Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison research animal care committees of both the Medical School and the College of Agriculture and Life Sciences, and they follow the recommended American Veterinary Medicine Association guidelines for humane treatment and euthanasia of laboratory farm animals. Briefly, UAs were dissected free of connective tissue, fat, and veins. The arteries were thoroughly rinsed free of blood using medium 199 before tying off arterial branches, clamping off the larger diameter end, and inflating with medium 199 containing 5 mg/ml collagenase B (Roche Applied Science, Indianapolis, IN) and 0.5% BSA through a luerlock three-way tap. Digestion was allowed to proceed for 55 min at 37 C before flushing the collagenase solution and endothelial cell sheets from the inner surface of the vessels. Cells were used immediately for RNA extraction or plated directly to experimental slides or dishes, and incubated overnight for attachment or plated to 35-mm dishes (Falcon Primarea, through Fisher Scientific, Pittsburgh, PA) and expanded by passaging three times before freezing as described (17). Cells were then thawed as needed and plated to similar experimental dishes as for freshly isolated cells.
Fura 2 Ca2+ imaging studies
Groups of freshly isolated cells were incubated in 35-mm glass- bottom dishes overnight to allow attachment before being used for experiments. Immediately before use, the cells were washed twice with 2 ml of prewarmed (37 C) Krebs buffer [125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 6 mM glucose, 25 mM HEPES, 2 mM CaCl2 (pH 7.4)]. The cells were then covered with 1 ml Krebs buffer and loaded with 5 µM of membrane permeable fura 2-AM (Molecular Probes, Eugene, OR) for 45 min. The cells were washed with Krebs buffer as described above, covered in 1 ml of Krebs buffer, and incubated for 30 min to allow complete ester hydrolysis. At the end of the 30-min incubation period, the cells were again washed and covered with 1 ml of Krebs buffer. Fura 2 loading was verified by viewing at 380 nm UV excitation on a Nikon inverted microscope (InCyt Im2, Intracellular Imaging, Inc., Cincinnati, OH). A group of cells was placed in the field of view, and 5-min recordings commenced using alternate excitation at 340 nm and 380 nm at 50-msec intervals and measuring emitted light using a photomultiplier (Intracellular Imaging, Inc.). From the ratio of emission at 510 nm detected at the two excitation wavelengths and by comparison to a standard curve established for the same settings using buffers of known free [Ca2+], the [Ca2+]i was calculated in real time using the InCyt Im2 software. ATP was added at 1, 3, 10, 30, 100, and 300 µM to determine Ca2+ response of the cells to ATP (17, 18). Cells were treated with 10 µM thapsigargin to empty intracellular stores of Ca2+, as required.
Immunocytochemistry (ICC)
After plating to eight chamber slides and incubation overnight, cells were washed and incubated in serum-free medium for 4 h. In some cases, cells were pretreated with U0126 (20 µM) for 20 min before agonist stimulation. Cells were stimulated with the agonists for the reported time at the appropriate doses (AII 100 nM, ATP 100 µM, bFGF 10 ng/ml, EGF 10 ng/ml, and VEGF 10 ng/ml) in a final volume of 400 µl serum-free media. After treatment, cells were washed with 300 µl of ice-cold PBS to terminate the reaction and fixed (4% formaldehyde, 0.1 M sodium cacodylate buffer) for 1 h at room temperature. After fixation, cells were washed in distilled water and stored in 70% ethanol until ICC was performed. Antiphospho p42/p44 MAPK polyclonal antibody (New England Biolabs, Inc.) was used at 1:125 dilution, and secondary antibody [horseradish peroxidase (HRP)-linked antirabbit IgG; Vector Laboratories, Burlingame, CA] was used at the manufacturer’s suggested dilution. Anti-eNOS polyclonal antibody (Transduction Laboratories, Inc.) was used at 0.5 µg/ml, and secondary antibody (HRP-linked antimouse IgG, Vector Laboratories) was used at the manufacturer’s suggested dilution. Cells were briefly counterstained with hematoxylin. After staining, cells were dehydrated, mounted, and scored.
Total RNA isolation
Either freshly dispersed cells were solubilized directly into 1 ml RNAzol B or 70–80% confluent cells in a T75 (passage 4) were rinsed twice in ice-cold PBS and lysed in 2 ml of cold RNAzol B (Tel-Test, Inc., Friendswood, TX). Samples were phase-separated with chloroform, and after brief centrifugation, the upper aqueous phase was removed and extracted twice with phenol/chloroform/isoamyl alcohol using heavy-grade phase lock gel (5Prime-3Prime, Boulder, CO). After isopropanol precipitation of RNA for 1 h at -20 C, pellets were recovered by centrifugation (12,000 x g, 30 min). RNA pellets were washed in 75% ethanol. Total RNA was solubilized in molecular biology grade water (5Prime-3Prime) and quantified by spectrophotometry. Northern analysis verified UAEC RNA quality recovered by this methodology.
Total cellular protein lysate isolation and PowerBlot analysis
At 70% confluency, cells were rinsed twice with ice-cold PBS and lysed in 500 µl phosphoprotein lysis buffer (4 mM sodium pyrophosphate, 50 mM HEPES, 100 mM NaCl, 10 mM EDTA, 10 mM NaF, 2 mM NaVO4, with 1 mM phenylmethylsulfonylfluoride, 1.0% Triton X-100, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Sonicated lysates were centrifuged at 10,000 rpm for 10 min to pellet cellular debris. Protein concentration was determined using the bicinchoninic acid assay. After verification of a small aliquot by SDS-PAGE for eNOS expression in each protein sample, NP-UAEC or P-UAEC lysates were pooled [
8 mg of pooled protein lysates, nonpregnant (n = 4 ewes) and pregnant (n = 4 ewes)] and sent to BD Biosciences (San Jose, CA) for PowerBlot analysis.
cDNA microarray analysis
RNA was pooled from three animals in each group for each microarray analysis. Total cellular RNA (2.5 µg from freshly isolated cells or 5 µg from passage 4 cells) was primed with oligo-dT primer, and cDNA probes were generated in 1x First Strand Buffer, 3.3 µM dithiothreitol, 1 mM deoxy (d)ATP, dGTP, dTTP, 300 U Superscript II reverse transcriptase, in the presence of 100 µCi 33P -dCTP. Probes were purified using Bio-Spin 6 Chromatography Column (Bio-Rad Laboratories, Inc., Hercules, CA). Incorporation of 33P -dCTP was routinely found to be 60–65% per 5 µg. The amount of probe used for hybridization was normalized to the lowest incorporated disintegrations per minute. Human cDNA arrays (GF211; Research Genetics, Inc., Huntsville, AL) were prehybridized in 10 ml of MicroHyb buffer with Cot-1 DNA and Poly dA at 42 C for at least 2 h. After overnight hybridization in fresh MicroHyb/Cot-1/PolydA buffer with radiolabeled probe, membranes were washed twice at 50 C in 2x standard saline citrate, 1% sodium dodecyl sulfate for 20 min, and once at room temperature in 0.5x standard saline citrate, 1% sodium dodecyl sulfate for 15 min. The arrays were analyzed by autoradiography. Exposure times were normalized (24, 48, and 96 h for each 2.5 x 106 dpm/ml of incorporated probe) to allow direct comparison, and images were analyzed using Pathways2.0 (Research Genetics, Inc.), Excel 97 (Microsoft, Redmond, WA) and SigmaPlot 6.0 (SPSS, Inc., Chicago, IL). Data from GF211 arrays presented here were also entirely consistent with that for cultured cells using GF200 arrays run in two separate and independent preliminary experiments.
Validation of cDNA microarray/PowerBlot data
UAEC protein lysates (NP-UAEC, n = 6; and P-UAEC, n = 6), prepared as above, were separated with positive controls and rainbow molecular weight (Amersham Biosciences Inc., Piscataway, NJ) markers using SDS-PAGE (2 h 100V) and electroblotting to Imobillon P polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) using the Mini Protean II electrophoresis system (Bio-Rad Laboratories, Inc.). Blots were blocked in 0.5% fat-free milk as previously described (17) and probed with primary antibodies (2 h, room temperature) and HRP-conjugated Fab2 secondary antibodies (Amersham Biosciences Inc.) for 1 h before final washing and detection of signal using the enhanced chemiluminescence reagent system and Hyper-Film (Amersham Biosciences Inc.). Primary antibody dilutions were as follows: BK B2-R, 1:1000; IP3R3, 1:1000; Jun, 1:2000; PKC (Transduction Laboratories, Inc.), 1:1000; PKC (PanVera Corp.), 1:1000; PKCß, 1:500; PKC, 1:500; A-Raf, 1:1000; and C-Raf, 1:250. Effects of BK on UAEC ERK 1/2 phosphorylation, phospholipase C (PLC) activation, and [Ca2+]i were monitored as previously described (17, 18).
Statistical analysis
The data were analyzed by Student’s t test, split-plot design, and/or ANOVA, as appropriate. Results were considered significant at P < 0.05.
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) in freshly isolated cells from either pregnant or nonpregnant animals. Cells treated with 1–300 µM ATP showed a progressive dose-dependent elevation in [Ca2+]i, with 100–300 µM ATP causing a maximal response for pregnant and nonpregnant derived ex vivo cells (Fig. 2). The [Ca2+]i response observed in cells from pregnant ewes was similar at lower doses (Fig. 1; 30 µM ATP), but the response in cells from pregnant ewes was more consistently sustained above basal for a longer time when higher doses of ATP were used (above 30 µM; Fig. 2, inset). The cells also showed regular oscillations over the 30–300 µM ATP dose range in 12 of 23 observations for cells from pregnant ewes (Fig. 2, inset), whereas oscillations were only seen in 1 of 25 observations at these same doses for cells from nonpregnant ewes. Our previous studies implied a strong dependence of the [Ca2+]i response on intracellular Ca2+ pools in UAEC at passage 4 (18). Thapsigargin (10 µM) pretreatment of freshly isolated cells was able to dramatically impair the subsequent response to ATP in freshly isolated cells from both nonpregnant (n = 3) and pregnant (n = 1) ewes (data not shown). This suggests that the extent to which intracellular Ca2+ stores are involved is similar even if the mechanisms governing Ca2+release and possible oscillations are subtly altered.
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). Random subsets of stained cells were photographed and then scored as positive or negative for P-ERK 1/2. The percentage of positive cells at 5, 10, and 20 min were compared with unstimulated controls. ATP, the most potent agonist tested, robustly increased P-ERK 1/2 immunostaining at 5 min and sustained P-ERK 1/2 staining through 10 and 20 min. Stimulation with bFGF also evoked increased P-ERK 1/2 staining at 5 min, which was sustained to 10 min and then fell to a level below significance at 20 min. VEGF and AII showed a similar pattern to that of ATP with peak stimulation occurring at 5 min. Both VEGF and AII maintained increased P-ERK 1/2 staining at 10 but not 20 min. EGF treatment induced a different pattern of stimulation compared with that for any of the other agonists used. EGF stimulation of P-ERK 1/2 only occurred at 10 min of treatment. In light of these data, the 10-min time point was chosen for subsequent experiments on freshly isolated cells. This time point was also consistent with our previous Western analysis from passage 4 UAEC studies.
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). These data confirmed that the freshly isolated cells and the cultured cells were pure populations of endothelial cells. Second, unstimulated cells were immunostained for P-ERK 1/2 to determine the basal level of P-ERK 1/2 within the cell population. In addition, one chamber of cells was stimulated with bFGF and then stained using IgG to control for antibody specificity. For one further control, cells were pretreated with 20 µM U0126 (an MAPK kinase antagonist) for 20 min before agonist stimulation; U0126 entirely eliminated P-ERK 1/2 staining, further validating the specificity of the P-ERK 1/2 antibody (Fig. 4).
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). In unstimulated cells alone there were no significant differences between the percentage positive for nonpregnant vs. pregnant derived freshly isolated cells or for UAEC at passage 4. In the initial time courses in P-UAEC, AII stimulated a significant increase in P-ERK 1/2 positive cells at 5 and 10 min, but in these experiments (Fig. 5), in which a 10-min stimulation was routinely used, no significant difference was detected, probably because of the choice of a suboptimal stimulation time for AII responsiveness. ATP stimulated a significant response in all cell preparations over the respective unstimulated controls but also achieved a higher level for pregnant compared with nonpregnant in freshly isolated cells and in P-UAEC compared with NP-UAEC. Similar findings were also observed for bFGF. Different results were obtained, however, for EGF and VEGF. For EGF, the responses of nonpregnant freshly isolated and NP-UAEC were significant above the respective unstimulated controls, whereas the pregnant freshly isolated and P-UAEC did not reach significance. In addition, although VEGF consistently showed a response significantly above unstimulated control in all cell preparations, there was no significant difference between pregnant and nonpregnant derived cells for both freshly isolated cells and for UAEC at passage 4. As far as changing responsiveness of cultured cells vs. freshly isolated cells, for ATP and bFGF the responses were comparable, with the exception that ATP responsiveness in NP-UAEC was significantly reduced, but by no means abolished, relative to fresh cells. For EGF, the UAEC at passage 4 responded somewhat more than freshly isolated cells, although the relative nonpregnant vs. pregnant difference was similar in each case. For VEGF, the UAEC showed a significantly bigger response than that for freshly isolated cells, but again the relative change of nonpregnant vs. pregnant was not altered between freshly isolated and UAEC preparations.
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. From this analysis, the combined approach correctly identifies the presence and absence of previously examined proteins and mRNAs. Comparisons of data from freshly isolated cells to passage 4 UAEC revealed no significant differences for either nonpregnant or pregnant ewes (Fig. 6). Likewise, comparison of nonpregnant vs. pregnant freshly isolated endothelium showed few, if any, differences beyond 2-fold (Fig. 6), a level that is also seen when comparing RNA from individual NP-UAEC with NP-UAEC or individual P-UAEC with P-UAEC preparations on GF211 arrays (data not shown). Thus, no differences are seen between nonpregnant and pregnant derived endothelium or UAEC that exceed the level of individual variations between animals in a given nonpregnant or pregnant state.
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). The cDNA array also reported the existence of the type 1 ryanodine receptor, but we have been unable to elicit a Ca2+ response using ryanodine in UAEC (data not shown). Of the PKC isoforms available for detection on the cDNA array, subtypes ßI and 
were clearly detected. A suitable antibody that reacts with ovine PKC is not available; however, the presence of PKCßI was substantiated in UAEC by Western analysis (Fig. 7). PowerBlot analysis demonstrated the presence of A-Raf, and immunoblotting established the presence of both A- and C-Raf isoforms in UAEC (Fig. 7).
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) and functionally confirmed by treating passage 4 NP-UAEC and P-UAEC with BK. BK treatment caused a rise in [Ca2+]i, stimulated phosphoinositol production, and increased ERK 1/2 phosphorylation (Fig. 8). Although the magnitude of the phosphoinositol response was not significantly different between P-UAEC and NP-UAEC, the P-UAEC consistently showed higher phosphoinositol production to all of the agonists tested (Fig. 8). Furthermore, after AII, BK, or ATP stimulation, the ERK 1/2 response as measured by Western analysis was clearly greater in the P-UAEC than in NP-UAEC (Fig. 8).
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). The rapid loss of response by homologous desensitization and receptor internalization is a feature of the BK B2-R (20). We estimate that BK B2-R internalization is complete in less than 5 min in UAEC. In addition, a BK response is not recovered even after as much as 60 min of incubation under basal conditions before the next treatment. Our finding that the subsequent response to ATP was unaffected further implied homologous desensitization. |
Discussion |
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In addition to calcium mobilization, the phosphorylation of ERK 1/2 in cultured and freshly isolated ex vivo cells was examined because both [Ca2+]i and ERK activation can modulate vasodilation production (9, 10, 11, 12, 13, 14, 23, 24, 25, 26, 27). Our previous studies used Western analysis to quantify the extent of ERK 1/2 phosphorylation in response to various agonist treatments; however, acute isolation procedures yield only limited numbers of cells, and therefore ICC was used. Detection of ERK 1/2 phosphorylation differs between ICC and Western analysis because the data collected with ICC reflect the number of cells stained and not a change in staining intensity, whereas Western analysis measures the total amount of P-ERK 1/2 for a population of cells, a function of both individual cell responsiveness and the number of cells responding. When detection of P-ERK 1/2 using ICC on passage 4 P-UAEC was compared with previous Western analysis of the same cells (17), the data from ICC did not achieve the same levels of sensitivity or statistical significance. Use of this assay technique did, however, show that the overall trends between nonpregnant and pregnant derived freshly isolated cell preparations were retained after prolonged culture. Thus, we can now conclude that UAEC are an accurate reflection of the situation observed in vivo. Furthermore, the current investigation also extends this prior finding because the data obtained by cell counting show further that such enhanced responses to many agonists (AII, ATP, bFGF, and VEGF) may in at least some cases (ATP and bFGF) involve recruitment of increasing numbers of responding cells.
Because the pregnancy-specific effect of agonists on Ca2+ and P-ERK signaling pathways observed in freshly isolated cells was largely retained in cultured cells, this allows us to make one further important observation, namely that these changes in signaling are due to a programmed event rather than being due to pregnancy-specific tonic stimulation (shear stress, elevated estrogen, angiotensin or insulin, etc.) because such tonic stimuli would not be differentially retained in culture conditions. It was this finding that led us to attempt microarray analysis in the hope of identifying the mechanistic basis of this programming event.
cDNA array analysis combined with subsequent Western blotting and functional analysis further confirms our findings that the passage 4 UAEC model represents freshly isolated UA endothelium. Analysis of array data can be performed in two ways. The first is to simply identify the data points as a fingerprint of the status of the cells and compare the regression analysis, looking for data points outside predetermined confidence limits as an index of significantly altered expression between groups. A comparison of cDNAs, proteins, and cellular function demonstrates that passage 4 cells maintain the characteristics of freshly isolated endothelium. Although our experiments were successful in providing a detailed fingerprint of the cell state, few differences in the more abundant mRNA species or corresponding protein expression were detected between pregnant and nonpregnant cell preparations for either freshly isolated or passage 4 cells that were beyond that of individual animal variation (2-fold) determined independently (data not shown). This may be due to a lack of sensitivity needed to detect the many less abundant mRNA species or the fact that the relevant cDNAs involved in pregnancy reprogramming of UAEC were absent from the gene filter. An alternative explanation is that altered UA endothelial function is not controlled at the level of mRNA or protein expression, but instead is an alteration of activity of existing proteins, perhaps through subtle changes in intracellular location that may facilitate or impede their participation in signaling events.
A second form of array analysis identifies each spot and considers the role of its gene product in cell signaling. This potentially useful analysis is limited by relative insensitivity, because cDNAs are reverse transcribed but not amplified. A lack of signal for a particular gene may have little meaning because it may simply reflect an expression level below the sensitivity of this reverse Northern analysis. Although only minor differences in mRNA or protein level were detected, the array data nonetheless provide potentially valuable information on the genes expressed in UA endothelium/UAEC. Many of the findings are as expected for endothelium, including the detection of the BK B2-R, endothelin receptors, adenosine receptors, -aminobutyric acidA receptor, insulin receptor, EGF receptor, several FGF receptors, connective tissue growth factor, and VEGF-B. In addition, endothelial cells are known to respond to hypoxia, so the presence of hypoxia-inducible factor-1 was also not surprising. Furthermore, PKC and PKCßI were among the numerous kinases detected in the cells. This may be important for deciphering the mechanism of vasodilator production in these cells because these isoforms may be activated by Ca2+ and be indirect mediators of ERK activation.
Many of the current findings have implications on future studies because endothelial function and signaling are widely published in the literature. For example, cDNA signals for the heterotrimeric G proteins Gs, Gi, Gq, and Gz were detected. Isoform Gq mediates activation of PLCß (also detected) in a number of cell types (28). In endothelial cell models, Gi may mediate activation of ERK 1/2 in response to either heptahelical receptor agonists (29) or nongenomic estrogen receptor activation (30). Furthermore, Gi and Gq are reported to be caveolae-associated proteins, consistent with the finding that BK and ATP stimulation of Ca2+ wave initiation occurs in caveolae-rich regions of the cell (31, 32).
These results were further validated by independently verifying expression levels of key proteins of particular interest to our research goals. Our previous studies demonstrated the presence of C-Raf in UAEC (Sullivan, J., unpublished data), and PowerBlot analysis revealed the additional presence of A-Raf. Western analysis confirmed the presence of similar levels of both isoforms in P-UAEC and NP-UAEC. Raf is likely involved in the activation of MAPK kinase 1 and ERK 1/2 in UAEC and may be a key to the enhancement of this response during pregnancy. Raf signaling has proven to be a complex process (33), and further in-depth studies will be necessary to determine isoform-specific involvement in ERK 1/2 activation in UAEC and how this is altered during pregnancy.
The presence of the BK receptor B2-R, IP3R3, Jun, PKC, PKCß, and PKC was also confirmed by Western blot. The B2-R finding was further substantiated with BK mobilization of intracellular Ca2+ as well as phosphorylation of ERK 1/2 in a pregnancy-specific manner. Homologous desensitization of the B2-R was implied in imaging studies, consistent with the B2-R activities in many other tissues (20). Taken together, these results imply that our metaanalytical approach was valid and suggest that further investigation of mRNA transcripts using more complete arrays may yet identify the pregnancy-specific factors responsible for altered cell signaling and function.
In summary, the extensively characterized UAEC model was compared with freshly isolated UAEC using functional responses as well as cDNA array analyses. The findings support the assertion that our UAEC model and freshly isolated cells function similarly and that pregnancy is associated with increased downstream coupling of extracellular agonists to ERK 1/2 and a more sustained elevation of [Ca2+]i in response to ATP. Our data further support the notion that these changes in cell signaling are not the result of tonic stimulation but are in fact programmed events that are retained in culture. Several molecular components have been identified in UAEC that may couple heptahelical receptors and growth factor receptors to the ERK 1/2 pathway as well as components of Ca2+ signaling pathways. Further studies are still necessary to determine how UAEC are reprogrammed at the level of cell signaling during pregnancy, but the availability of a cell model that retains these in vivo characteristics will clearly facilitate such an investigation.
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Acknowledgments |
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Footnotes |
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Abbreviations: AII, Angiotensin II; B2-R, type 2 receptor; bFGF, basic fibroblast growth factor; BK, bradykinin; [Ca2+]i, intracellular free calcium concentration; cPLA2, cytosolic phospholipase A2; d, deoxy; EGF, epidermal growth factor; eNOS, endothelial NO synthase; HHVE, human hand vein endothelial; HRP, horseradish peroxidase; ICC, immunocytochemistry; IP3R3, inositol 1,4,5-triphosphate receptor 3; NO, nitric oxide; NP-UAEC, nonpregnant UAEC; P-ERK, phosphorylated ERK; PGI2, prostacyclin; PKC, protein kinase C; PLC, phospholipase C; P-UAEC, pregnant UAEC; UA, uterine artery; UAEC, UA endothelial cell(s); VEGF, vascular endothelial growth factor.
Received November 4, 2002.
Accepted for publication April 15, 2003.
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and by mobilization of intracellular Ca2+. Biochem J 353:503–512[CrossRef][Medline]
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, significant differences between the percentages of positive cells from pregnant-freshly isolated cells and nonpregnant freshly isolated cells; 
, significant differences between P-UAEC and NP-UAEC; 
, significant differences between P-ERK 1/2 staining when comparing P-UAEC and freshly isolated cells derived from pregnant animals; 
, significant differences between NP-UAEC and freshly isolated cells derived from nonpregnant animals. P < 0.05.
