Peroxisome proliferator‐activated receptor gamma blunts endothelin‐1‐mediated contraction of the uterine artery in a murine model of high‐altitude pregnancy
2020; Wiley; Volume: 34; Issue: 3 Linguagem: Inglês
10.1096/fj.201902264rr
ISSN1530-6860
AutoresSydney L. Lane, Alexandrea S. Doyle, Elise S. Bales, Julie A. Houck, Ramón A. Lorca, Lorna G. Moore, Colleen G. Julian,
Tópico(s)Cardiovascular Issues in Pregnancy
ResumoThe FASEB JournalVolume 34, Issue 3 p. 4283-4292 RESEARCH ARTICLE Free Access Peroxisome proliferator-activated receptor gamma blunts endothelin-1-mediated contraction of the uterine artery in a murine model of high-altitude pregnancy Sydney L. Lane, Integrated Physiology Program, University of Colorado Graduate School, Aurora, CO, USA Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorAlexandrea S. Doyle, Department of Biochemistry, Colorado Mesa University, Grand Junction, CO, USASearch for more papers by this authorElise S. Bales, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorJulie A. Houck, Department of Biochemistry, Colorado Mesa University, Grand Junction, CO, USASearch for more papers by this authorRamón A. Lorca, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorLorna G. Moore, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorColleen G. Julian, Corresponding Author colleen.julian@cuanschutz.edu Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA Correspondence Colleen G. Julian, Department of Medicine, University of Colorado School of Medicine, 12700 E 19th Avenue, Mailstop 8611, 3rd Floor Research Complex 2, P15-3122, Aurora, CO 80045, USA. Email: colleen.julian@cuanschutz.eduSearch for more papers by this author Sydney L. Lane, Integrated Physiology Program, University of Colorado Graduate School, Aurora, CO, USA Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorAlexandrea S. Doyle, Department of Biochemistry, Colorado Mesa University, Grand Junction, CO, USASearch for more papers by this authorElise S. Bales, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorJulie A. Houck, Department of Biochemistry, Colorado Mesa University, Grand Junction, CO, USASearch for more papers by this authorRamón A. Lorca, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorLorna G. Moore, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USASearch for more papers by this authorColleen G. Julian, Corresponding Author colleen.julian@cuanschutz.edu Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA Correspondence Colleen G. Julian, Department of Medicine, University of Colorado School of Medicine, 12700 E 19th Avenue, Mailstop 8611, 3rd Floor Research Complex 2, P15-3122, Aurora, CO 80045, USA. Email: colleen.julian@cuanschutz.eduSearch for more papers by this author First published: 23 January 2020 https://doi.org/10.1096/fj.201902264RR Funding information: This work was supported by the American Heart Association Pre-Doctoral Award 7PRE33410652 (SLL), NIH R01 HL138181 (CGJ), and NIH R01 HD088590 (LGM, CGJ) AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The environmental hypoxia of high altitude (HA) increases the incidence of intrauterine growth restriction (IUGR) approximately threefold. The peroxisome proliferator-activated receptor γ (PPAR-γ), a ligand-activated nuclear receptor that promotes vasorelaxation by increasing nitric oxide and downregulating endothelin-1 (ET-1) production, has been implicated in IUGR. Based on our prior work indicating that pharmacologic activation of the PPARγ pathway protects against hypoxia-associated IUGR, we used an experimental murine model to determine whether such effects may be attributed to vasodilatory effects in the uteroplacental circulation. Using wire myography, ex vivo vasoreactivity studies were conducted in uterine arteries (UtA) isolated from pregnant mice exposed to hypoxia or normoxia from gestational day 14.5 to 18.5. Exposure to troglitazone, a high-affinity PPARγ agonist-induced vasorelaxation in UtA preconstricted with phenylephrine, with HA-UtA showing increased sensitivity. Troglitazone blunted ET-1-induced contraction of UtA in hypoxic and normoxic dams equivalently. Immunohistological analysis revealed enhanced staining for ET-1 receptors in the placental labyrinthine zone in hypoxic compared to normoxic dams. Our results suggest that pharmacologic PPAR-γ activation, via its vasoactive properties, may protect the fetal growth under hypoxic conditions by improving uteroplacental perfusion and thereby justify further investigation into PPARγ as a therapeutic target for IUGR in pregnancies complicated by hypoxia. Abbreviations ET-1 endothelin-1 IUGR intrauterine growth restriction NO nitric oxide PPARγ peroxisome proliferator-activated receptor gamma TGZ troglitazone 1 INTRODUCTION Intrauterine growth restriction (IUGR) raises the risk of neonatal death up to 20-fold and is associated with increased morbidity and mortality rates across the lifespan.1-3 Despite its public health importance, the pathophysiology of IUGR remains unclear and, as a result, no effective therapies or preventative strategies exist. Impaired maternal oxygenation, such as that resulting from the chronic hypoxia of high-altitude (>2500 m) residence, reduces the birth weight and increases the incidence of IUGR nearly threefold.4-7 For this reason, high-altitude research models provide a unique opportunity to identify the physiologic and molecular mechanisms by which hypoxia contributes to reduced fetal growth without the confounding effects of other pathologic changes present in IUGR. In healthy pregnancy, widespread maternal vascular adaptations, including the extensive remodeling of the uterine spiral arteries and altered uterine artery vasoreactivity, collectively serve to reduce the uteroplacental vascular resistance, enhance uterine artery blood flow, and thereby maintain adequate uteroplacental perfusion for fetal development.8, 9 Such effects are due, in part, to changes in the production of or sensitivity to vasodilators (eg, nitric oxide [NO]) and vasoconstrictors (eg, endothelin 1 [ET-1]).8 In contrast, high-altitude pregnancy and IUGR are marked by a reduction in the normal pregnancy-associated rise in the uteroplacental blood flow, an effect that not only precedes slowed fetal growth but also directly corresponds to birth weight.10-13 Peroxisome proliferator-activated receptor gamma (PPARγ), a hypoxia-sensitive, ligand-activated transcription factor of the nuclear hormone receptor superfamily, is predominantly recognized for its role in the regulation of metabolic homeostasis and inflammation.14 However, PPARγ is also vital for establishing the uteroplacental villous circulation15, 16 and maintaining uterine vascular function.17, 18 PPARγ also exhibits the vasoprotective properties, lowering blood pressure in hypertensive disease, and improving endothelium-dependent vasorelaxation19 by suppressing ET-1 synthesis and prepro-ET-1 expression in human vascular endothelial cells,20 and increasing NO production.21, 22 In line with these functions, PPARγ is abundantly expressed in the rodent placental labyrinthine zone,23 human trophoblast,15, 24 and human vascular smooth muscle cells.25 Experimental animal studies also strongly demonstrate vascular sites of action for PPARγ. In rats, for instance, antagonizing PPARγ decreases fetal growth, reduces vasodilation of the radial UtA and induces endothelial dysfunction.17, 18 In mice, PPARγ activation reduces vascular sensitivity to angiotensin II, lowers blood pressure and improves fetal growth in heterozygote Rgs5± mice, known to otherwise be susceptible to IUGR.26 An abundant literature implicates PPARγ for the development of vascular disorders of pregnancy. 17, 27-29 In humans, placental PPARγ mRNA expression is twofold lower in IUGR compared to controls and directly related to fetal and placental weight.29 Our prior work demonstrates that high-altitude pregnancy also suppresses the PPARγ pathway gene expression in maternal peripheral blood cells in parallel with reduced birth weight.30 Experimental animal studies further support the involvement of the PPARγ pathway for IUGR. In recent work, for example, we show that dietary supplementation with pioglitazone, a pharmacologic PPARγ agonist, partially prevented hypoxia-associated fetal growth restriction in mice.31 Here, we propose that one mechanism by which thiazolidinediones, a class of PPARγ agonists used clinically to treat diabetes and polycystic ovarian syndrome,32, 33 may protect fetal growth under hypoxic conditions is by promoting uterine artery vasorelaxation and, in turn, enhancing uteroplacental perfusion. Using a murine model of hypoxia-associated IUGR, we hypothesized that (i) PPARγ activation ex vivo induces vasorelaxation in UtA preconstricted with phenylephrine (PE) and that these effects are augmented in UtA isolated from dams exposed to hypoxia during late gestation and, (ii) hypoxia enhances vasocontractility to ET-1 and that this response is blunted by PPARγ activation. 2 MATERIALS AND METHODS All animal procedures and protocols were completed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals34 and approved by the Institutional Animal Care and Use Committee at the University of Colorado (IACUC, Protocol Number: 113016(08)1E). 2.1 Mice and late-gestation hypoxia exposure Female C57Bl/6 mice (Charles River, Wilmington, MA) were mated at 8-14 weeks of age. A copulatory plug was considered evidence of successful breeding and considered to be gestational day (GD) 0.5. From GD14.5 to GD18.5, pregnant mice were placed in hyperbaric (PB ~ 760 mm Hg, sea level) or hypobaric (PB ~ 385 mm Hg, 5300 m) chambers to simulate normoxia or hypoxia, respectively. Given the moderate altitude of Denver, Colorado (1609 m), a hyperbaric chamber had to be used for a true normoxic (ie, sea level) exposure. On GD18.5, mice were euthanized by carbon dioxide asphyxiation and cervical dislocation. Directly after euthanasia, the main UtA were excised, cleaned of surrounding connective tissue placed in an ice-cold PBS bath and immediately prepared for wire myography studies. Fetuses and placentas were excised, cleaned of fetal membranes, and weighed. Placentas were fixed in 4% paraformaldehyde overnight then embedded in paraffin or flash frozen in liquid nitrogen for homogenization and protein extraction. Our previous study utilizing the same model of hypoxia-associated fetal growth restriction reported that there was no effect of hypoxia on litter size, maternal food intake, or resorption number, indicating that dams likely tolerated the normoxic and hypoxic chambers equally well and fetal growth restriction was not due to maternal stress.31 2.2 Small vessel wire myography For each dam, segments of each main UtA were mounted in a four-chamber, small vessel wire myograph (DMT 610, Copenhagen, Denmark) with two wires (40 μm) threaded through the vessel lumen and connected to either a tension transducer or micrometer. Each vessel was normalized to a resting tension equivalent to 13.3 kPa for a minimum of 40 minutes in warm (37°C) Krebs solution (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 11 mmol/L glucose, 2.5 mmol/L CaCl2) continuously bubbled with 95% O2, 5% CO2. To establish viability, UtAs were (i) constricted with 60 and 120 mmol/L KCl (Sigma Aldrich, St. Louis, MO), (ii) a sub-maximal concentration of phenylephrine (PE, 10 μmol/L) and (iii) exposed to the endothelial-dependent vasodilator, acetylcholine (1 μmol/L) to test for an intact endothelium. For our experimental protocol, vessels were preconstricted with 10 μmol/L PE (Sigma-Aldrich; % PEmax) and the vasodilator response to the high-affinity PPARγ agonist troglitazone (1-100 μmol/L) was established. Our rationale for using troglitazone is based on recent calls for revisiting the therapeutic value of thiazolidinediones for vascular disorders of pregnancy,35 and on-going human clinical trials for a wide array of vascular diseases.36 To assess the contribution of NO production for the vasodilatory effects of troglitazone, UtAs were incubated for 20 minutes with and without the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 10 μmol/L) prior to PE constriction and the application of troglitazone. Given that ET-1 is a potent vasoconstrictor implicated in hypoxic and hypertensive disorders of pregnancy37, 38 and is downregulated by PPARγ,20 we also determined the contractile response of the UtA to ET-1 (0.1-10 nmol/L) prior to and after a 20-minute incubation with troglitazone (1 μmol/L). 2.3 Placental immunohistochemistry Placental paraffin sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol (100%-80%) and de-ionized water. For antigen retrieval, sections were submerged in 10 mmol/L, pH 6.0 citrate buffer, and boiled in a pressure cooker for 30 minutes at 45°C. Sections were blocked in 1% bovine serum albumin (BSA) in 1x Tris-Buffered Saline (TBS) for 30 minutes. Primary antibodies against ET-1 receptor A and B (Thermo Fisher Scientific, Rockford, IL) were diluted 1:1000 in 1% BSA in 1x TBS and applied to sections overnight at 4°C. Secondary anti-HRP antibody (Dako, Santa Clara, CA) was applied for 1 hour at room temperature. DAB substrate (Dako) was then added and allowed to develop for 6 minutes. Sections were then counterstained with hematoxylin for 1 minute, dehydrated with increasing concentrations of ethanol (70%-100%) and xylene, and then covered with cytoseal and cover slips. 2.4 Placental protein expression Placenta total protein quantification was carried out using the Wes System (ProteinSimple, San Jose, CA). Placentas from dams at E18.5 were cleaned of fetal membranes and flash frozen. Placentas corresponding to the fetus closest to the litter mean fetal weight were homogenized using a sonicator in RIPA buffer (50 mmol/L Tris (pH 7.4), 2 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl, 50 mM NaF, 5 mmol/L sodium vanadate) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein levels were determined by capillary electrophoresis size-based separation via the WES System (ProteinSimple) according to manufacturer's instructions. Data were analyzed with Compass software (ProteinSimple). Primary antibodies against ET-1 Receptor A and ET-1 Receptor B (Thermo Fisher Scientific, PA3-065 and PA3-066, 1:100 dilution for both) were used and normalized to vinculin (Sigma-Aldrich). 2.5 Analytical strategy Fetoplacental weights were averaged per dam and are expressed as the group mean ± SEM with N representing the number of dams per group. Fetoplacental weights were compared between normoxic and hypoxic dams using Student's unpaired t tests (Prism v.8; GraphPad Software, San Diego, CA). Concentration-response curves were generated, and sigmoidal curves fitted to the data using the least squares method (Prism v.8; GraphPad Software). Uterine artery relaxation evoked by troglitazone is expressed as the % PEmax. The contractile force elicited by ET1 is normalized to that evoked by 60 mM KCl (Kmax). Half-maximal effective concentration (EC50), maximal force and area under the curve (AUC) were compared using two-way analysis of variance (ANOVA) with Sidak's multiple comparisons to determine the main effects of exposure during pregnancy (normoxia vs hypoxia) and ex vivo treatment (eg, control vs troglitazone), or Student's unpaired t tests, as appropriate. Two-way ANOVA with repeated measures was performed for vascular dose–response curves. If significant interactions were observed, then individual group means were compared, with the level of significance adjusted by Bonferroni's method to account for multiple comparisons, or Student's unpaired t test were performed for post hoc comparisons. Significance was defined as a two-tailed P < .05. 3 RESULTS 3.1 Effectiveness of hypoxic protocol to reduce fetal weight Maternal exposure to hypoxia from GD 14.5 to 18.5 reduced the fetal weight by 38% (Figure 1A; P < .0001). Compared to normoxic controls, mean placental weight tended to be higher (Figure 1B; P = .08) and placental efficiency, expressed as the ratio of fetal to placental weight, lower in the hypoxic group (Figure 1C; P < .0001). Figure 1Open in figure viewer Effects of late-gestation hypoxia on E18.5 feto-placental weights. Hypoxia (HX) resulted in A, smaller fetal weights, B, similar placental weights, and C, reduced placental efficiency (ratio of fetal to placental weights) compared to normoxic (NORM) dams. Symbols are individual dams, bars are means ± SEM and compared by Student's t test; n = 8 NORM dams, 8 HX dams 3.2 Vasodilatory effects of pharmacologic PPARγ activation in normoxic and hypoxic uterine artery In normoxic and hypoxic dams, ex vivo exposure to the PPARγ agonist TGZ vasodilated UtA preconstricted with PE (Figure 2A). Although maximal uterine artery vasorelaxation in response to TGZ was equivalent between normoxic and hypoxic mice (Figure 2B), the overall sensitivity to TGZ (expressed by the AUC) was greater in the hypoxic group (Figure 2C; 2-way ANOVA for hypoxic effect, P < .05) with an accompanying significant effect on the EC50 (12.1 ± 2.0 vs 12.9 ± 0.8 μmol/L for normoxic and hypoxic vessels, respectively; P < .001). Figure 2Open in figure viewer Effects of in vivo hypoxia and ex vivo L-NAME incubation on uterine artery troglitazone (TGZ)-induced vasorelaxation. Uterine arteries were preconstricted with PE prior to TGZ or L-NAME exposure. A, Concentration response curves to troglitazone (TGZ; 0.1-100 μmol) for UtA with preincubation with L-NAME (30 min; 10 μmol) or control (CON) in UtA preconstricted with PE from NORM or HX mice. HX and NORM dams are represented by open squares and solid circles, respectively. Curves generated without or with L-NAME are represented by solid or dashed lines, respectively. B, The maximum vasodilator response to TGZ (expressed as the % PE-induced constriction achieved by TGZ or TGZ + L-NAME) was blunted by L-NAME in normoxic dams. C, Overall sensitivity to TGZ as represented by area under the curve. Data are presented as means ± SEM and compared by two-way ANOVA with differences between groups identified by Sidak's multiple comparisons; n = 10 NORM CON, 10 NORM + L-NAME, 9 HX CON, 9 HX + L-NAME Inhibition of NO synthase by L-NAME raised the EC50 in normoxic and hypoxic groups (34.3 ± 13.1 vs 37.8 ± 18.9 μmol/L, respectively; P < .05 compared to controls), indicating that NO production contributed to the vasodilatory effects of TGZ in both groups. In normoxic mice, L-NAME reduced the maximum vasodilator response to TGZ by 33% (Figure 2B; P < .05). In hypoxic mice, however, L-NAME had no effect on maximum vasodilator response to TGZ (Figure 2B). These findings indicate a lesser contribution of NO to the effects of TGZ in hypoxic mice and suggest that NO production may be reduced by hypoxia. 3.3 Effects of pharmacologic PPARγ activation on ET1-induced contraction of the uterine artery The UtA from normoxic and hypoxic mice contracted in response to ET-1 (Figure 3A). The maximum contractile response to ET-1 was augmented by hypoxia (Figure 3B; P < .05), but the overall sensitivity remained the same as that in normoxic vessels (Figure 3C). Preincubating vessels with TGZ to activate PPARγ reduced the maximal response to ET-1 in normoxic and hypoxic groups (Figure 3B; P < .05 or P < .0001, respectively). It is notable that vessels obtained from the hypoxic dams had a comparatively blunted, albeit not statistically significant, maximal ET-1 response after TGZ incubation compared to controls. Overall sensitivity to ET-1 was reduced by TGZ in both the normoxic and hypoxic groups (Figure 3C; 2-way ANOVA for TGZ effect, P < .05). The contractile response to KCl was similar in normoxic and hypoxic mice (6.36 ± 0.48 vs 6.13 ± 0.38 mN, respectively, NS), indicating that the mechanical integrity of the UtA was not affected by hypoxia. Figure 3Open in figure viewer Effects of in vivo hypoxia and ex vivo troglitazone on the uterine artery contractile response to endothlelin-1 (ET-1). A, Concentration response curves to ET-1 (0.1-30 nmol/L) with 20 min preincubation with troglitazone (TGZ; 1 μmol/L) or control (CON) in UtA from NORM or HX mice. B, Maximum contractile response to ET-1 was higher in HX dams, and TGZ blunted the maximal response in NORM and HX dams. C, Overall sensitivity to ET-1 as represented by area under the curve. Data are presented as means ± SEM and compared by two-way ANOVA with differences between groups identified by Sidak's multiple comparisons; n = 11 NORM CON, 11 NORM + TGZ, 15 HX CON, 14 HX + TGZ 3.4 Effect of hypoxia on the expression of ET1 receptors in the placenta and uterine artery Given evidence suggesting that ET1 may be important for regulating the uteroplacental blood flow,37, 39 we evaluated the effect of late-gestation hypoxia on the expression of ET1 receptors A and B in the placenta and uterine artery. In normoxic and hypoxic animals, placental ET-1 receptors A and B were detected (Figure 4A,B). Compared to normoxic controls, placental ET-1 receptor A expression was greater in hypoxic dams (Figure 4A). Using immunohistochemistry to localize the ET-1 receptor expression in the placenta, we found that, in normoxic mice, the ET1 expression was predominantly localized to the junctional zone (Figure 4Ci,ii). Qualitatively, ET-1 receptor staining was enhanced in placentas from hypoxic mice, and apparent in both the junctional zone and the labyrinthine zone (Figure 4Ciii,iv). Figure 4Open in figure viewer Effects of in vivo hypoxia on the expression of ET-1 receptors in the murine placenta. A, ET-1 receptor A protein expression is higher in HX compared to NORM mouse placental homogenates. B, ET-1 receptor B protein expression is unaffected by HX. C, Representative immunohistochemical staining of placentas from NORM (i and ii) and HX (iii and iv) mice reveal qualitatively higher expression of both ET-1 receptors A and B with HX. N = 6 NORM, 6 HX dams for each receptor in placenta 4 DISCUSSION Abundant evidence supports involvement of the PPARγ pathway for vascular disorders of pregnancy, including IUGR and preeclampsia. Our prior human and experimental animal studies suggest that impaired PPARγ signaling may also be important for hypoxia-associated fetal growth restriction. Dietary supplementation with a selective PPARγ agonist, for instance, appears to protect against hypoxia-associated fetal growth restriction in mice.31 In the present study, we have shown that ex vivo PPARγ activation evokes profound relaxation of the uterine arteries, and that vessels obtained from hypoxia-exposed dams are more sensitive to vasorelaxation in response to PPARγ activation compared to normoxic controls. In support of our observation, in an interventional experimental animal study, treatment with the PPARγ agonist troglitazone abolished hypertensive responses to angiotensin II infusion during pregnancy and normalized fetal growth in a genetic mouse model (Rgs5±) that is otherwise known to be susceptible to hypertension and IUGR.26 Our observation that UtA from hypoxia-exposed dams are more sensitive to the vasodilatory effects of PPARγ activation may reflect increased sensitization to pharmacologic PPARγ agonist exposure due, in part, to reduced PPARγ expression under hypoxic conditions.27, 29-31 Our data indicate that inhibition of NO by L-NAME blunted vasorelaxation in response to TGZ incubation to a lesser extent in hypoxic compared to normoxic mice at higher TGZ concentrations (100 µM), whereas at lower TGZ concentrations inhibition of NO had a greater effect on inhibiting TGZ-induced vasorelaxation. PPARγ activation increases NO production via PPARγ-dependent mechanisms21 and increases eNOSser1177 phosphorylation via non-genomic mechanisms.22 Therefore, one possible explanation for our observations is that hypoxia increased sensitization of PPARγ activation-induced NO signaling, synthesis or bioavailability.40 An alternative explanation may be related to augmented vasocontractility to ET-1 in the UtA of hypoxic dams. Circulating ET-1 is elevated in human HA pregnancy compared to low-altitude pregnancy,39 and has been associated with lower birth weight in HA pregnancy.37 ET-1 is also elevated in pregnancies complicated by preeclampsia or small-for-gestational-age births,41, 42 and when its vasoconstrictor actions are inhibited with the use of ET-1 antagonists, hypoxia-associated reductions in uteroplacental blood flow, and fetal growth in rat models are abolished.43 In the context of hypoxic pregnancy, therefore, it is plausible that PPARγ activation has an enhanced vasodilatory effect by reducing ET-1 signaling. In support of this hypothesis, PPARγ pathway activation enhances endothelium-dependent vasorelaxation (as previously reviewed),19 an effect that may be due to the suppression of ET-1 secretion,20, 44 ET-1 synthesis and prepro-ET-1 expression.20 While the brief troglitazone incubation implemented in our study likely excludes the possibility that changes in ET-1 synthesis or prepro-ET-1 expression affected vasoreactivity, significant changes in ET-1 secretion, as determined by circulating levels in the plasma, have been observed after only 5-6 minutes of low-grade exercise (recumbent cycling), indicating that ET-1 secretion shifts may occur rapidly.45 Additional studies are needed, however, to determine whether a 20-minute troglitazone exposure is sufficient to suppress ET-1 secretion. Chronic PPARγ activation, which we did not address in this experiment, may also have non-genomic effects in reducing ET-1 signaling through microRNA regulation, as it does in models of pulmonary hypertension.46 Our findings further indicate that pharmacologic activation of the PPARγ pathway blunts ET-1 induced contraction of the uterine artery in normoxic controls and hypoxia-exposed dams. From a therapeutic perspective, this effect may be considered advantageous. Most prominently, since ET-1 signaling may also be elevated in vascular disorders of pregnancy that are unrelated to maternal hypoxic exposure, our findings suggest that targeting PPARγ to improve uteroplacental blood flow is likely not limited to hypoxia-related disease. However, indicating that the effect of PPARγ activation to reduce ET-1-induced contraction of the UtA may be of particular importance for the vascular dysfunction characteristic of hypoxia-associated complications of pregnancy, the maximum contractile response to ET-1 was enhanced in UtA of hypoxia-exposed dams. Inhibition of NO production with L-NAME blunted the vasodilatory effects of troglitazone in UtA from hypoxic and normoxic mice, indicating that elevated NO signaling contributed to the vasodilatory effects of troglitazone in both groups. However, the maximal vasorelaxation was blunted by L-NAME only in normoxic mice, indicating less NO production in hypoxic mice. This is consistent with previous findings of reduced NO signaling in UtA from hypoxic animal models47-49 and in myometrial arteries from our recent HA human studies.50 In addition to enhanced NO signaling and blunted contractility as described above, troglitazone may be acting through endothelial-derived hyperpolarizing factor, cyclooxygenase-dependent mechanisms in the vascular endothelium to cause relaxation, or other non-genomic mechanisms including the regulation of calcium influx in vascular smooth muscle cells by, for instance, suppressing increased basal intracellular free calcium concentration or store-operated calcium entry as has been observed in pulmonary artery vascular smooth muscle cells51; these mechanisms require further investigation. Placental blood flow is also a major determinant of nutrient and oxygen delivery to the fetus.52 Placental vasomotor activity is largely dependent on the production of vasoactive compounds including ET-1 and expression of receptors including ET-1 receptors, allowing for autocrine and paracrine control of vascular function within the placenta.53-55 Upregulation of ET-1 and its receptors in the placenta is associated with preeclampsia,56, 57 a hypertensive disorder of pregnancy that often results in fetal growth restriction. For this reason, we assessed placental expression of ET-1 receptors A and B in normoxic and hypoxic mice with the hypothesis that increased placental ET-1 receptor expression would enhance ET-1 signaling, resulting in reduced placental perfusion and restricted nutrient delivery to the fetus. Indeed, compared to normoxic controls, we observed increased placental expression of both the ET-1 receptors A and B in the junctional and labyrinthine zones of hypoxic mice. This observation supports the possibility that hypoxia may increase contractility in the portion of the placenta import
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