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Endocrinology, doi:10.1210/en.2007-0568
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Endocrinology Vol. 149, No. 2 626-633
Copyright © 2008 by The Endocrine Society

Expression and Biological Activity of Parathyroid Hormone-Related Peptide in Pregnant Rat Uterine Artery: Any Role for 8-Iso-Prostaglandin F2{alpha}?

Ferhat Meziani, Angela Tesse, Sandra Welsch, Hélène Kremer, Mariette Barthelmebs, Ramaroson Andriantsitohaina, Francis Schneider and Alexis Gairard

Université Louis Pasteur-Strasbourg I, Institut Gilbert-Laustriat and Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 7175 (F.M., H.K., A.G.), Département Pharmacologie et Physicochimie, Faculté de Pharmacie, 67401 Illkirch, France; Institut National de la Santé et de la Recherche Médicale (INSERM) UMR 771 (F.M., A.T., R.A.); CNRS UMR 6214; Faculté de Médecine, Université d’Angers, 49045 Angers, France; INSERM Unité 727 (S.W., M.B.), Université Louis Pasteur (Strasbourg I), 67070 Strasbourg, France; Département de Réanimation médicale et de médecine hyperbare (F.M.), Centre Hospitalier Universitaire, 49933 Angers, France; and Réanimation Médicale (H.K., F.S.), Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France

Address all correspondence and requests for reprints to: Pr. Alexis Gairard, Institut Gilbert-Laustriat, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7175, Faculté de Pharmacie, 74, route du Rhin, 67401 Illkirch, France. E-mail: alexis.gairard{at}pharma.u-strasbg.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTHrP is produced in vessels and acts as a local modulator of tone. We recently reported that PTHrP(1–34) is able to induce vasorelaxation in rat uterine arteries, but in pregnancy, this response is blunted and becomes strictly endothelium dependent. The present study aimed to get insights into the mechanisms involved in these changes because the adaptation of uterine blood flow is essential for fetal development. On d 20 of gestation, RT-PCR analysis of uterine arteries showed that PTH/PTHrP receptor (PTH1R) mRNA expression was decreased, whereas that of PTHrP mRNA was increased. This was associated with a redistribution of the PTHrP/PTH1R system, with both PTH1R protein and PTHrP peptide becoming concentrated in the intimal layer of arteries from pregnant rats. On the other hand, the blunted vasorelaxation induced by PTHrP(1–34) in uterine arteries from pregnant rats was specifically restored by indomethacin and a specific cyclooxygenase-2 inhibitor, NS 398. This was associated with an increase in cyclooxygenase-2 expression and in 8-iso-prostaglandin F2{alpha} release when uterine arteries from pregnant rats were exposed to high levels of PTHrP(1–34). Most interestingly, 8-iso-prostaglandin F2{alpha} itself was able to increase PTHrP expression and reduce PTH1R expression in cultured rat aortic smooth muscle cells. These results suggest a local regulation of uterine artery functions by PTHrP during pregnancy resulting from PTH1R redistribution. Moreover, they shed light on a potential role of 8-iso-prostaglandin F2{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PTHrP WAS FIRST identified as a factor causing malignant humoral hypercalcemia. In contrast to PTH, the expression of PTHrP is observed in a wide range of tissues where it is involved in the control of calcium homeostasis, cartilage resorption, and cell proliferation/apoptosis and in vasodilatation (1). PTHrP is considered as a regulator of vascular tone through both paracrine and autocrine pathways (2). PTHrP has also been reported to play a key role in the pathophysiology of several diseases (3, 4). In addition, the expression of PTHrP and its cognate receptor, the PTH/PTHrP receptor (PTH1R), are known to be differentially regulated during development (5) and inflammatory states (6).

In a previous study, we reported that exogenous PTHrP(1–34) acts on vascular smooth muscle via the cAMP pathway to promote relaxation in uterine artery from nonpregnant rat, whereas the same agonist induces almost exclusively an endothelium-dependent relaxation during pregnancy acting via nitric oxide and endothelial-derived hyperpolarizing factor pathways (7). The reasons for these changes still remain unknown. Furthermore, we reported that the relaxation in response to PTHrP(1–34) is significantly reduced in uterine artery from pregnant rats. Vasoconstrictor metabolites released from vascular cells, originating namely from cyclooxygenases (COXs) are known to participate in the impairment of agonist-induced relaxation. The COX metabolites involved are not only those that can activate the thromboxane A2/endoperoxide receptor such as prostaglandin (PG)E2 or thromboxane A2 but also 8-iso-PGF2{alpha}. On the other hand, changes in the uterine vascular reactivity to PTHrP could also be linked to a change in PTH1R expression. Indeed, PTHrP-induced renal vasodilatation was shown to closely correlate with PTH1R expression under various experimental settings (8, 9).

The aim of this study was to better understand why PTHrP-induced relaxation of uterine arteries became endothelium dependent and was reduced in late pregnant rats. In this context, we investigated both 1) the expression of the PTHrP/PTH1R system and 2) the modulation of the vascular response to PTHrP during pregnancy by COX-1/COX-2 metabolites, with particular interest in 8-iso-PGF2{alpha}. The most original finding of our study is that PTHrP expression is enhanced in late pregnant uterine arteries, although PTH1R expression is decreased, but both concentrate to endothelial and subendothelial layers of the vascular wall. Moreover, exogenous PTHrP(1–34) is able to overexpress COX-2 in the same layers and enhance 8-iso-PGF2{alpha} release when the prostanoid itself amplifies PTHrP expression in cultured vascular smooth muscle cells. These data provide novel evidence for a possible contribution of the PTHrP/PTH1R system in uterine artery physiology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The experiments were conducted in compliance with statutory requirements by accredited research scientists (Authorization No. 67-125 from the French Ministry of Agriculture).

Animals
Female Wistar rats (Nico Iffa strain), 12–15 wk of age, supplied by Charles River (L’Arbresle, France) were housed separately in temperature- and humidity-controlled quarters with constant 12-h light, 12-h dark cycles and were provided with standard food (A04 diet; Safe, Villemoisson, France) and water ad libitum. The rats were mated, and the day on which the sperm plug was observed was denoted as d 1 of gestation. Nonpregnant rats (n = 18) or rats on d 20 of pregnancy (n = 15) were anesthetized with pentobarbital (60 mg/kg ip; Sanofi, Paris, France) and killed by cervical dislocation. After laparotomy, the uterus together with both ovaries and uterine arteries were excised and immediately placed in cold (4 C) physiological salt solution (PSS; see below for composition).

Aortic smooth muscle cells (AoSMC) in culture
Primary cultures of rat AoSMC were obtained by the explant method as reported previously (10) and used at passages 15–17. AoSMC were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37 C in 10% CO2. Cells at 80% confluence were grown for a 24-h period of quiescence in serum-free medium supplemented with 0.1% BSA, before being exposed for 8 h to 8-iso-PGF2{alpha} (10 µM; Cayman Chemical, SPI-BIO, Montigny le Bretonneux, France), or angiotensin II (Ang II, 100 nM; Sigma-Aldrich, Saint Quentin Fallavier, France).

RNA extraction and RT-PCR analysis in arteries and cells
The uterine arteries from pregnant and nonpregnant rats were dissected under a magnifier. Endothelial cells were removed from some preparations as described previously (7), by a bolus injection of 0.3% CHAPS,3-[(3-cholamidopropyl) dimethyl- ammonio]-1-propanesulfonate, so that arteries with (E+) and without (E–) endothelium could be analyzed. Total RNA was rapidly extracted from homogenized uterine arteries with the QIAGEN kit for fibrous tissue (QIAGEN S.A., Courtaboeuf, France) or from cells in culture by TRIzol according to the manufacturer’s protocol (Invitrogen, Cergy-Pontoise, France) and quantified by absorbance at 260 nm. RT was performed on 5 µg RNA in the presence of 400 U reverse transcriptase (leukemia virus reverse transcriptase, Fermentas, St. Leon-Rot, Germany). Quantitative real-time PCR analysis was used to amplify cDNA with the LightCycler-FastStart DNA Master SYBR Green kit (Roche Diagnostics, Meylan, France) as described previously (11). A standard curve was obtained by serial dilutions of mixed cDNA samples. Amplifications were performed in a 20-µl mix, containing 4 mM MgCl2, 0.5 µM of each primer set, reaction buffer (Taq DNA polymerase, dNTP mix, 0.2 µM SYBR Green I dye) and 50 ng cDNA sample. The following sets of primers were used: rat PTH1R, sense 5'-GGG CAC AAG AAG TGG ATC AT-3' and antisense 5'-GGC CAT GAA GAC GGT GTA GT-3'; rat PTHrP, sense 5'-CAG CCG AAA TCA GAG CTA CC-3' and antisense 5'-CTC CTG TTC TCT GCG TTT CC-3'; and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-CAT GGA GAA GGC TGG GGC TC-3' and antisense 5'-AAC GGA TAC ATT GGG GGT AG-3'. GAPDH was used as a housekeeping gene to normalize the expression of other genes. PCR were run at 95 C for 10 min, followed by 45 cycles of 10 sec at 95 C, 5 sec at 60 C, and 16 sec at 72 C (PTH1R and PTHrP) or 20 sec at 72 C (GAPDH). As a negative control, cDNA was replaced by PCR-grade water. Each sample was run twice. Data were quantified with the LightCycler analysis software (Roche Diagnostics) and mRNA signals normalized for GAPDH (PTH1R or PTHrP/GAPDH mRNA ratio). PCR products were identified on 1.5% of agarose gels containing 0.5 µg/ml ethidium bromide by their expected size of 210 bp (PTH1R), 206 bp (PTHrP), and 415 bp (GAPDH) (not shown).

In vitro vascular reactivity
The uterine arteries from pregnant and nonpregnant rats were dissected under a magnifier, and uterine arterial rings were mounted on a wire myograph (EMKA Technologies, Paris, France) for the in vitro study of vascular reactivity exactly as previously described (7). Briefly, experiments were performed in prewarmed (37 C), oxygenated (95% O2 and 5% CO2) PSS medium of the following composition (in mM): 119 NaCl; 4.7 KCl; 14.9 NaHCO3; 1.2 MgSO47H2O; 2.5 CaCl2; 1.18 KH2PO4; 5.5 glucose. The force of vessels was recorded with an isometric force transducer, and data acquisition was performed using Datanalyst version 1.4 and playback software (EMKA Technologies). After an equilibration period under an optimal passive tension for at least 20 min, the phenylephrine concentration giving 80% of the maximal contraction (PE80) was determined on each vessel and chosen for preconstriction in the following relaxation experiments. All the studies were performed in arteries with functional endothelium as assessed by the ability of acetylcholine to induce relaxation. The effects of PTHrP on the vessels were quantified as the percentage of relaxation in preparations previously contracted with phenylephrine. Concentration-response curves were constructed by cumulative application of PTHrP(1–34) (1–100 nM; Neosystem, Strasbourg, France) in the absence or the presence of COX inhibitors indomethacin (10 µM/liter), the selective COX-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398, 10 µM/liter), and the selective COX-1 inhibitor valeryl salicylate (3 mM/liter). The inhibitors were added in the bath 30 min before addition of PTHrP(1–34). The drugs used in the experiments were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

In vitro prostanoids release by uterine arteries
Uterine arteries were dissected from pregnant and nonpregnant rats as described above, put in plate wells with 5 ml PSS, and incubated at 37 C for 4 h in the absence or the presence of 100 nM PTHrP(1–34). Thromboxane B2, 8-iso-PGF2{alpha}, and PGE2 metabolites were measured in the incubation medium by enzymatic immunoassay kits (Cayman Chemical).

Staining and imaging by confocal microscopy
Freshly dissected uterine arteries from pregnant and nonpregnant rats were used for the double immunostaining with endothelial anti-PECAM-1 (C-20) antibody (1:50; Santa Cruz Biotechnology, Heidelberg, Germany) and either anti-PTH1R or anti-PTHrP antibodies. Vessels were frozen and cut in 10-µm sections, fixed (5 min at –20 C) in 100% methanol, and treated (1 h at room temperature) in blocking buffer (5% nonfat dry milk in PBS and 0.05% Tween 20) to block nonspecific binding sites. Fixed and blocked tissue sections were coincubated (overnight at 4 C) with a polyclonal goat anti-PECAM-1 antibody and a monoclonal mouse anti-PTH1R antibody (1:50; Acris, Hiddenhausen, Germany) or a rabbit anti-PTHrP(1–34) antibody (1:20; Bachem, Voisins-le-Bretonneux, France). After three washes, tissue sections were incubated (1 h at room temperature) with Alexa fluor-488-labeled antigoat IgG (1:100) to stain endothelial PECAM-1, Alexa fluor-546-labeled antimouse IgG (1:100) to stain PTH1R or Alexa fluor-546-labeled antirabbit IgG (1:100) to stain PTHrP (all from Invitrogen Molecular Probes, Leiden, The Netherlands). After final washes, vessel sections were mounted on glass slides.

In another set of experiments, freshly dissected uterine arteries from pregnant and nonpregnant rats exposed to vehicle or 100 nM PTHrP(1–34) for 4 h were used. Vessels were frozen, cut in 10-µm sections, and processed as described above. Fixed and blocked tissue sections were incubated (overnight at 4 C) for the double immunostaining with a polyclonal rabbit anti-COX-2 antibody (1:10; Transduction Laboratories, Heidelberg, Germany) and a polyclonal goat anti-PECAM-1 antibody to stain COX-2 and the endothelium, respectively. After three washes, tissue sections were incubated (1 h at room temperature) with Alexa fluor-546-labeled antirabbit IgG (1:100; Invitrogen Molecular Probes) and Alexa fluor-488-labeled antigoat IgG (1:100; Invitrogen Molecular probes).

Slides were examined with an Olympus light microscope Fluoview FU 300 Laser Scanning Confocal Imaging System (Paris, France), equipped with an argon ion laser (EM 488 nm) and helium-neon ion laser (excitation wavelength 543 nm). Pictures were taken with a x40 objective (oil immersion). The laser was adjusted in the green/red fluorescence mode, which yielded two excitation/emission wavelengths at 488/520 nm for Alexa 488 and at 543/572 nm for Alexa 546. Z-series were collected in 1-µm steps, and final images were obtained after stacking. Green and red images were obtained from two separated channels and a third superposition of green and red pictures. Red staining corresponded to COX-2, PTHrP, or PTH1R detections, whereas green staining corresponded to endothelial PECAM-1 detection in the same section.

Data analysis
One-way ANOVA, Kruskal-Wallis, and Mann-Whitney U tests or two-way ANOVA for repeated measurements and subsequent post hoc tests were performed with the Statview version 5.0 software (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. All values are presented as mean ± SEM for n experiments, and n represents the number of animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pregnancy decreases PTH1R mRNA but increases PTHrP mRNA in uterine arteries
The expression of PTH1R and PTHrP were analyzed by quantitative real-time RT-PCR on uterine arteries with (E+) and without (E–) endothelium, freshly dissected from pregnant and nonpregnant rats. The expression of PTH1R mRNA is reduced by 50% in the E+ arteries from pregnant rats (Fig. 1AGo) compared with those from nonpregnant rats. In endothelium-denuded (E–) pregnant arteries, this decrease becomes even more pronounced (75%) (Fig. 1BGo). In contrast, PTHrP mRNA expression seems to be increased overall by a factor of three in uterine E+ arteries from pregnant rats (Fig. 1CGo). However, in endothelium-denuded pregnant arteries, PTHrP expression is no longer significantly increased in the remaining medial layer (Fig. 1DGo). These results show that changes in PTHrP and PTH1R mRNA expression are heterogeneous within the vascular wall of pregnant arteries.


Figure 1
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  		FIG. 1. PTH1R and PTHrP mRNA expression in uterine arteries from pregnant and nonpregnant rats. Quantitative real-time RT-PCR analysis was performed for the evaluation of PTH1R (A and B) and PTHrP (C and D) mRNA in uterine arteries freshly prepared from pregnant (P) and nonpregnant (NP) rats with (E+) and without (E–) endothelium. Expression of target transcripts was compared with the expression of GAPDH used as a housekeeping gene. Normalized ratios were calculated (PTH1R/GAPDH mRNA and PTHrP/GAPDH mRNA) and set at 1 for nonpregnant rats. Note that PTHrP expression is spontaneously up-regulated and PTH1R expression down-regulated in pregnant uterine arteries. Of particular interest, in the E– uterine arteries from pregnant rats, the down-regulation of PTH1R expression becomes more pronounced, whereas PTHrP expression is no longer significantly increased. Results are given as mean ± SEM for three to six independent experiments.

 
Uterine artery expression of PTH1R and PTHrP is shifted from medial layer to intimal layer during pregnancy
To analyze the influence of pregnancy on the expression and distribution of PTH1R and PTHrP in the vascular wall of uterine arteries, double staining was performed for endothelial PECAM-1 and either PTH1R protein or PTHrP peptide. Representative immunographs of uterine arteries from one pregnant and one nonpregnant rat are given on Fig. 2Go, with similar scale showing the increase in size of arteries from pregnant rats. The most remarkable observation is the changes in the distribution of PTH1R and PTHrP in arteries from pregnant and nonpregnant rats. Indeed, PTH1R and PTHrP immunostaining is uniformly distributed in the vascular wall of uterine arteries from nonpregnant rats (Fig. 2Go, E and K), whereas only some PTH1R colocalization with endothelial PECAM-1 is assessed by the yellow staining in the merged figure (Fig. 2FGo). In contrast, in pregnant uterine arteries, marked labeling for PTH1R or PTHrP is found in the intima (i.e. endothelial and subendothelial layers), whereas labeling is very weak in the medial layer of the arteries (Fig. 2Go, B and H). Colocalization of PTH1R or PTHrP and endothelial PECAM-1 is observed in endothelial layer of pregnant arteries (Fig. 2Go, C and I, respectively). Negative controls, obtained by incubation of arteries only with the secondary fluorescence-labeled IgG, display no staining (not shown). Taken together, these results show that pregnancy is associated with a redistribution of PTH1R protein and PTHrP peptide within the vascular wall, staining being markedly increased in endothelial and subendothelial layers as demonstrated by the colocalized staining of PTH1R or PTHrP with the endothelial protein PECAM-1.


Figure 2
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  		FIG. 2. Immunohistochemical staining for PTH1R and PTHrP in pregnant and nonpregnant uterine arteries. Representative pictures are shown of immunohistochemical red staining for PTH1R in uterine arteries from pregnant (P) (B) or nonpregnant rats (NP) (E) and green staining for endothelial PECAM-1 in uterine arteries from pregnant (A) and nonpregnant rats (D). Colocalization of PTH1R and PECAM-1 (yellow staining) is pronounced in vessels from pregnant rats (C) but weak in vessels from nonpregnant rats (F). Representative pictures are shown of immunohistochemical red staining for PTHrP in uterine arteries from pregnant (P) (H) or nonpregnant rats (NP) (K) and green staining for endothelial PECAM-1 in uterine arteries from pregnant (G) and nonpregnant rats (J). Colocalization of PTHrP and PECAM-1 (yellow staining) is pronounced in vessels from pregnant rats (I) but very weak in vessels from nonpregnant rats (L). Note that the diffuse staining present in nonpregnant rats for both PTH1R and PTHrP becomes restricted to the intimal layer in pregnant rats. Bars, 50 µm.

 
COX-2 inhibitors enhance relaxation to PTHrP in arteries from pregnant rat
As previously reported (7), PTHrP(1–34) produces a concentration-dependent relaxation in uterine arteries that was blunted in arteries from pregnant rats (Fig. 3Go, A and B). Indeed, the maximal relaxation to PTHrP(1–34) is then decreased by about half (28 ± 6% in pregnant vs. 46 ± 10% in nonpregnant; n = 6; P < 0.05). To investigate whether COX derivatives could explain this difference, we evaluated PTHrP(1–34)-induced uterine relaxation in the presence of COX inhibitors with specificity for COX-1 (valeryl salicylate) or COX-2 (NS-398) as well as in the presence of a nonspecific COX inhibitor (indomethacin).


Figure 3
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  		FIG. 3. Effects of COX inhibitors on PTHrP(1–34)-induced relaxation in pregnant and nonpregnant uterine arteries. A, Concentration-response curves on uterine arteries from pregnant rats (n = 6) exposed to increasing concentrations of PTHrP(1–34) in the absence ({square}) or in the presence of indomethacin ({blacksquare}), NS-398 ({diamondsuit}), or valeryl salicylate ({blacktriangleup}). B, Concentration-response curves on uterine arteries from nonpregnant rats (n = 6) exposed to increasing concentrations of PTHrP in the absence ({square}) or in the presence of indomethacin ({blacksquare}), NS-398 ({diamondsuit}), or valeryl salicylate ({blacktriangleup}). Please note that PTHrP(1–34) produces a vasorelaxant effect that is more pronounced in vessels from pregnant rats when indomethacin or a COX-2 inhibitor is added but not when the study is performed in the presence of valeryl salicylate, a COX-1 inhibitor. Results are given as means ± SEM and were compared by a two-way ANOVA on repeated measurements, followed by Tukey test for multiple comparisons.*, P < 0.05 vs. control (absence of inhibitor).

 
Indomethacin does not modify the relaxation induced by PTHrP(1–34) in arteries from nonpregnant rats (Fig. 3BGo) but significantly enhances the response in arteries from pregnant rats (Fig. 3AGo). The selective COX-1 inhibitor, valeryl salicylate, does not affect the relaxation to PTHrP(1–34) whatever the origin of the arteries is. In contrast, the selective COX-2 inhibitor, NS-398, markedly enhances PTHrP-induced vasodilatation in uterine arteries from pregnant rats (Fig. 3AGo). Taken together, these results show that vasoconstrictor metabolite(s) derived from COX-2 partially inhibit PTHrP-induced uterine relaxation in pregnant rats only.

PTHrP(1–34) enhances COX-2 expression in uterine arteries from pregnant rats
To assess whether COX-2 expression is changed during pregnancy and to localize the protein within the vascular wall, we performed double immunohistochemical detection of both endothelial PECAM-1 and COX-2 protein. No staining or weak staining of COX-2 is found in naive vessels from nonpregnant rats (Fig. 4HGo), whereas weak staining is found on the adventitial layer of vessels from pregnant rats (Fig. 4BGo). However, marked COX-2 labeling is observed in the endothelial, subendothelial, and adventitial layers of uterine arteries from pregnant rats exposed for 4 h to 100 nM PTHrP(1–34) (Fig. 4EGo). Colocalization with endothelial PECAM-1 is assessed by the yellow staining in the merged figure (Fig. 4FGo). In contrast, no labeling or only weak labeling of COX-2 is found after PTHrP treatment of arteries from nonpregnant rats (Fig. 4KGo). Negative controls, obtained by incubation of arteries only with the secondary fluorescence-labeled IgG, display no staining (not shown).


Figure 4
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  		FIG. 4. PTHrP(1–34)-enhanced COX-2 expression in uterine arteries from pregnant rats. A, D, G, and J, Representative pictures of green immunohistochemical staining for endothelial PECAM-1 in vessels from pregnant (A and D) or nonpregnant rats (G and J); B and H, representative pictures of red immunohistochemical staining for COX-2 in uterine arteries from pregnant (B) and nonpregnant rats (H) treated with vehicle; E and K, representative pictures of immunohistochemical staining for COX-2 after exposure of uterine arteries from pregnant (E) or nonpregnant rats (K) to PTHrP(1–34) (100 nM for 4 h). F, Note that colocalization of COX-2 and PECAM-1 (yellow staining) in vessels from pregnant rats incubated with PTHrP(1–34) (100 nM for 4 h). Bars, 50 µm. Note also that the COX-2 labeling is localized in endothelial and subendothelial layers of vessels from pregnant rats.

 
PTHrP(1–34) stimulates the release of 8-iso-PGF2{alpha} only in uterine arteries from pregnant rats
To further document the identity of COX-2 vasoconstrictor products released by uterine arteries, we measure thromboxane B2, the stable metabolite of thromboxane A2, as well as PGE2 and 8-iso-PGF2{alpha} in arteries freshly dissected from both pregnant and nonpregnant rats. As shown on Fig. 5Go, the basal release of PGE2 is 2.5-fold higher in arteries from pregnant rats (Fig. 5AGo), whereas there is no difference in the basal release of 8-iso-PGF2{alpha} (Fig. 5BGo) or thromboxane B2 (Fig. 5CGo). In contrast, when arteries are in vitro exposed for 4 h to 100 nM PTHrP(1–34), no change in the release of prostanoids is observed in arteries from nonpregnant rats, whereas the release of 8-iso-PGF2{alpha} is enhanced by a factor of two in uterine arteries from pregnant rats (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of pregnancy and PTHrP(1–34) on prostanoids production by pregnant and nonpregnant uterine arteries. Arachidonic acid derivatives related to COX-2 pathway released from pregnant (P) or nonpregnant (NP) uterine arteries, exposed or not to PTHrP(1–34) (100 nM/liter for 4 h), was assessed. A, Basal PGE2 release is higher from pregnant vs. nonpregnant arteries (#, P < 0.05) but is not modified by exposure to PTHrP(1–34); B, basal 8-iso-PGF2{alpha} release is not different between pregnant and nonpregnant arteries but is enhanced from pregnant arteries exposed to PTHrP(1–34) (*, P < 0.05); C, thromboxane B2 release is no different among the four groups. Results are given as means ± SEM (n = 3).

 
8-Iso-PGF2{alpha} regulates PTHrP and PTH1R mRNA expression in AoSMC
Because Ang II, as well as other vasoconstrictors, have been reported to modify PTHrP and PTH1R expressions in vascular smooth muscle cells (12, 13), we tested whether 8-iso-PGF2{alpha} may be an additional factor able to modulate PTHrP and/or PTH1R expression in vessels. For this study, we used AoSMC that were incubated for 8 h with 8-iso-PGF2{alpha} (10 µM) or Ang II (100 nM), which is used here as a reference drug. As expected, the RT-PCR analysis shows that Ang II induces a 2-fold increase in PTHrP mRNA expression (Fig. 6BGo) and markedly down-regulates the expression of PTH1R mRNA (Fig. 6AGo). More interestingly, incubation of the cells in the presence of 8-iso-PGF2{alpha} elicits a quite similar effect, inducing overexpression of PTHrP and down-regulation of PTH1R expression (Fig. 6Go, B and A, respectively). These results establish now that 8-iso-PGF2{alpha} belongs to the extensive family of factors regulating the vascular expression of the PTHrP/PTH1R system.


Figure 6
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  		FIG. 6. Modulation by 8-iso-PGF2{alpha} of PTHrP and PTH1R mRNA expression in rat aortic vascular smooth muscle cells. Quantitative real-time RT-PCR analysis was performed for the evaluation of PTH1R (A) and PTHrP (B) mRNA in aortic vascular smooth muscle cells, under control conditions and after incubation for 8 h with 8-iso-PGF2{alpha} (10 µM) or Ang II (100 nM). Expression of target transcripts was compared with the expression of GAPDH used as a housekeeping gene. Normalized ratios were calculated (PTHrP/GAPDH mRNA and PTH1R/GAPDH mRNA) and set at 1 for control. Of note, 8-iso-PGF2{alpha} induces down-regulation of PTH1R mRNA and up-regulation of PTHrP mRNA (*, P < 0.05) exactly as Ang II does (#, P < 0.05). Results are given as mean ± SEM for three to six independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We recently reported that PTHrP(1–34) is able to induce vasorelaxation in rat uterine arteries, but this response was blunted in arteries from pregnant rats and became strictly endothelium dependent. The present study was performed to better understand the mechanisms involved in PTHrP(1–34)-induced uterine vasodilatation changes during pregnancy. The main findings were that the overall expression of PTH1R was decreased, whereas that of PTHrP was increased in uterine arteries from pregnant rats. This was associated with a redistribution of the PTHrP/PTH1R system in these vessels. Thus, both PTH1R protein and PTHrP peptide expression became concentrated in the intimal layer of arteries from pregnant rats. These data were in agreement with RT-PCR results in E+ and E– arteries, suggesting that PTH1R and PTHrP mRNA expression are high in the endothelium of pregnant arteries. Besides, relaxation in response to PTHrP(1–34) was restored in the presence of COX-2 inhibitors in uterine arteries from pregnant rats. This was associated with an increased COX-2 expression within the vessel wall and an enhanced 8-iso-PGF2{alpha} release. Most interestingly, 8-iso-PGF2{alpha} was able to reduce PTH1R expression and increased PTHrP expression in cultured vascular smooth muscle cells.

PTH1R and PTHrP are widely expressed in the cardiovascular system, including the heart, vascular smooth muscle, and endothelial cells. The effectiveness of the endogenous PTHrP/PTH1R system as a modulator of regional hemodynamics mainly depends on local expression of both PTH1R and PTHrP (2, 9). In the present study, we clearly document the presence of a local PTHrP/PTH1R system in uterine arteries. Indeed, PTH1R and PTHrP transcripts as well as immunoreactive proteins were detected. Of interest, the vascular uterine PTHrP/PTH1R system adapts during pregnancy with a down-regulation of PTH1R and an overall overexpression of PTHrP as shown by our results from RT-PCR studies. However, PTHrP-induced vasorelaxation persists in arteries from pregnant rats, although at a lower level, and becomes strongly endothelium dependent as we reported previously (7). One of the novel findings of the present study is the redistribution of the PTHrP/PTH1R system within the uterine arteries during pregnancy that may explain the obligatory role of the endothelium in the response to PTHrP in vessels from pregnant rats. Indeed, PTH1R mRNA expression is strongly reduced in endothelium-denuded uterine arteries from pregnant rats, and immunostaining is almost absent in the medial layer of pregnant arteries, whereas strong expression of PTH1R protein is found in the endothelium. To our knowledge, no such vascular redistribution of PTH1R has been described before. Taken together, our results suggest that PTHrP can be considered as one of the factors regulating uterine hemodynamics, even during pregnancy, inasmuch as the local expression of PTHrP itself is increased in the endothelium.

The blood supply to the uterus is provided in major part by the uterine arteries. During pregnancy, they are transformed into large dilated vessels undergoing dramatic structural changes in their wall to maximize the delivery of maternal blood to the intervillous space. Their arterial lumen becomes wider and responsiveness to vasoconstrictors is reduced (14). Hyporesponsiveness to Ang II is at least partially mediated by prostacyclin, the potent vasodilator whose synthesis is enhanced during pregnancy (15). PTHrP may also play a role in conjunction with other well-known factors such as NO (16, 17) and PGI2 (18). Direct impairment of uterine blood flow results in fetoplacental abnormalities (19).

The present results highlight the interplay between PTHrP and COX-2 in uterine arteries from pregnant rats. To some extent, COX-2 derivatives blunt PTHrP-induced uterine relaxation as shown by the increased response in the presence of NS-398 or indomethacin. It is, however, not clear what kind of COX-2 derivative is involved in this blunting. PGE2 may account for this blunting, because it is the only one whose release is enhanced in naive pregnant uterine arteries. On the other hand, 8-iso-PGF2{alpha} constitutes an alternative possibility because it is the only one whose release is increased in uterine arteries after induction of COX-2 expression. Overexpression of COX-2 is specifically observed in the present study in pregnant uterine arteries exposed for a period of 4 h to high concentration of PTHrP(1–34). Of interest, COX-2 expression is then restricted to the endothelial and subendothelial layer where the PTHrP/PTH1R system is expressed in the pregnant uterine arteries. We cannot exclude that such an effect occurs in vitro during the course of the construction of the concentration-response curve to PTHrP(1–34) in the uterine arteries. 8-iso-PGF2{alpha} can then contribute to blunting PTHrP-induced vasorelaxation by eliciting vasoconstrictor and functional antagonism. However, 8-iso-PGF2{alpha} is probably not responsible for the redistribution of the PTH1R during normal pregnancy. Indeed, COX-2 is not expressed in naive pregnant arteries, and 8-iso-PGF2{alpha} release is not enhanced in the same arteries. Because basal PGE2 release was increased in pregnant arteries, we looked at the effects of PGE2 on the expression of the PTHrP/PTH1R system in AoSMC but found it inactive (Barthelmebs, M., personal results). The mechanism of the PTHrP/PTH1R redistribution in uterine arteries during pregnancy remains therefore unknown and needs further investigation, as is also the case for the specific effect of PTHrP on COX-2 overexpression in pregnant uterine arteries.

Although the mechanism of this particular effect of PTHrP remains to be determined, its physiopathological significance may be of particular value. PTHrP is considered as a member of the cascade of cytokines induced during the inflammatory response (6). PTHrP expression is increased in a number of vascular diseases characterized by inflammatory processes and oxidative stress, such as atherosclerotic lesions (3, 20), hypertension (2), and ischemic cardiac injury (21). The possibility of COX-2 induction by high local concentrations of PTHrP adds further support for an active contribution of this peptide in these inflammatory processes. Moreover, in the same experimental settings, PTH1R expression is usually found to be down-regulated. Our data now add 8-iso-PGF2{alpha} as a possible factor responsible for both PTHrP up-regulation and PTH1R down-regulation, a profile shared with Ang II (13). In this context, it is interesting to note that AngII-induced PTHrP overexpression in AoSMC was inhibited by indomethacin (12). During normal rat pregnancy, deleterious vascular effects of 8-iso-PGF2{alpha} are probably blunted by endothelial NO (7). 8-Iso-PGF2{alpha} is considered as a valuable marker for vascular oxidative stress (22, 23, 24).

Furthermore, it was negatively implicated in trophoblast invasion where it reduces metalloproteinase activity (25). Besides, preeclampsia has been associated with increased oxidative stress and subsequent increase in 8-iso-PGF2{alpha} levels in fetoplacental membranes and amniotic fluids (26). In a recent review, Maioli et al. (27) suggested that reduced local production of PTHrP could be a major causative factor in preeclampsia for defective placentation. Our present data shed a different light on the possible implication of PTHrP, suggesting that overexpression of PTHrP during pathological states might be a harmful factor because it can further increase vascular 8-iso-PGF2{alpha} release and local oxidative stress. Of interest, preeclampsia occurred in a pregnant woman with a PTHrP-secreting pancreatic neuroendocrine tumor and high circulating levels of PTHrP (28).

In conclusion, the present results suggest that PTHrP participates in the regulation of local uterine hemodynamics during gestation. Besides, the present study points out a local feedback control loop involving the interplay between PTHrP/PTH1R and 8-iso-PGF2{alpha} release from COX-2. Because the above pathway could have implications in uteroplacental perfusion abnormalities in pathological pregnancy, the quantitative importance of PTHrP in maternal physiology needs to be further clarified.


   Acknowledgments
 
We appreciate the valuable technical assistance of J. F. Poirier and V. Roques (Université Louis Pasteur de Strasbourg), L. Preisser from IFR132, M. H. Guilleux from Service Commun de Cytométrie et d’Analyses Nucléotidiques, and R. Mallet of the Service commun d’imageries et d’analyses microscopiques (Univesité d’Angers). Finally, we thank A. Bernard for careful review of the English editing of the manuscript.


   Footnotes
 
This work was supported by the Medical School of Louis Pasteur University from Strasbourg (Grant 2002-5196) for financial support.

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 29, 2007

Abbreviations: Ang II, Angiotensin II; AoSMC, aortic smooth muscle cells; COX, cyclooxygenase; PG, prostaglandin; PSS, physiological salt solution; PTH1R, PTH/PTHrP receptor.

Received April 30, 2007.

Accepted for publication November 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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