Vascular actions of relaxin: nitric oxide and beyond.

The peptide hormone relaxin regulates the essential maternal haemodynamic adaptations in early pregnancy through direct actions on the renal and systemic vasculature. These vascular actions of relaxin occur mainly through endothelium-derived NO-mediated vasodilator pathways and improvements in arterial compliance in small resistance-size arteries. This work catalysed a plethora of studies which revealed quite heterogeneous responses across the different regions of the vasculature, and also uncovered NO-independent mechanisms of relaxin action. In this review, we first describe the role of endogenous relaxin in maintaining normal vascular function, largely referring to work in pregnant and male relaxin-deficient animals. We then discuss the diversity of mechanisms mediating relaxin action in different vascular beds, including the involvement of prostanoids, VEGF, endothelium-derived hyperpolarisation and antioxidant activity in addition to the classic NO-mediated vasodilatory pathway. We conclude the review with current perspectives on the vascular remodelling capabilities of relaxin. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Pharmacokinetic study of saxagliptin in healthy Chinese subjects.

BACKGROUND AND OBJECTIVES: The pharmacokinetics of some medications may be affected by differences in race and ethnicity, which can lead to suboptimal outcomes. The present study was conducted to assess the single- and multiple-dose pharmacokinetics of saxagliptin in healthy Chinese subjects living in China. METHODS: This was an open-label, 9-day study conducted at the Drug Clinical Trial Center, Peking University Third Hospital, Beijing, China. Sixteen healthy Chinese subjects of both sexes between 21 and 33 years of age were administered saxagliptin 5 mg orally on day 1, then once daily on days 3-7. Pharmacokinetic variables for saxagliptin (primary outcome) and its active metabolite, 5-hydroxy saxagliptin (secondary outcome), after single and multiple oral doses of saxagliptin were assessed. Safety was also assessed. RESULTS: Saxagliptin was absorbed rapidly (median time to reach maximum concentration [t(max)]: 0.5 and 1 hour on days 1 and 7, respectively), and its pharmacologically active metabolite, 5-hydroxy saxagliptin, appeared in plasma (median t(max): 1.0 and 1.5 hours, respectively). Plasma exposure to 5-hydroxy saxagliptin was approximately 2- to 3-fold higher than exposure to saxagliptin. Plasma concentration-time profiles for saxagliptin and 5-hydroxy saxagliptin were similar on days 1 and 7, with no evidence of drug accumulation on repeated dosing. The elimination half-lives (t(½)) for saxagliptin and 5-hydroxy saxagliptin were approximately 3 and 4 hours, respectively, with renal excretion as the primary route of elimination. After single and multiple dosing, 54.48% and 52.60%, respectively, of the administered saxagliptin dose was recovered in urine as unchanged drug or 5-hydroxy saxagliptin. Saxagliptin was generally well tolerated. Six (37.5%) subjects experienced an adverse event (AE). All AEs were mild in intensity and judged by the investigator as not related to the study medication. There were no deaths, serious AEs, discontinuations due to AEs, or other clinically significant AEs during this study. CONCLUSION: Saxagliptin 5 mg (single dose and once-daily doses for 5 days) was generally well tolerated; the pharmacokinetics of saxagliptin and 5-hydroxy saxagliptin in healthy Chinese subjects were consistent with previous assessments in the saxagliptin clinical development program. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00770302.

Evaluation of the effect of dapagliflozin on cardiac repolarization: a thorough QT/QTc study.

INTRODUCTION: Dapagliflozin is a first-in-class sodium-glucose transporter 2 (SGLT2) inhibitor under investigation for the treatment of type 2 diabetes mellitus. A thorough QTc study was conducted, according to International Conference on Harmonization E14 guidelines, to characterize the effect of dapagliflozin on cardiac repolarization. METHODS: The present study was a double-blind, four-period, placebo-controlled crossover study at a single-center inpatient clinical pharmacology unit. The study enrolled 50 healthy men who were randomized to receive sequences of single doses of dapagliflozin 150 mg, dapagliflozin 20 mg, moxifloxacin 400 mg, and placebo. The sequences were randomized based on the Williams design for a cross-over study to reduce the "carryover" effects from drug-to-drug even with sufficient washout periods. Digital 12-lead electrocardiograms were recorded at nine time points over 24 hours in each period. QT intervals were corrected for heart rate using a study-specific correction factor (QTcX) and Fridericia's formula. RESULTS: For dapagliflozin, the upper bound of the one-sided 95% confidence interval (CI) for time-matched, placebo-subtracted, baseline adjusted QTc intervals (ΔΔQTc) was <10 ms. ΔΔQTc was independent of dapagliflozin concentrations. No QTc thresholds >450 ms or QTc increases >30 ms were observed. Moxifloxacin increased the mean QTcX interval by 7.7 ms (lower bound 90% CI, 6.2 ms) over 1-4 hours after dosing, confirming assay sensitivity. CONCLUSION: Dapagliflozin, at supratherapeutic doses, does not have a clinically significant effect on the QT interval in healthy subjects.

Altered global gene expression in first trimester placentas of women destined to develop preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. METHODS: Surplus chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. RESULTS: Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. CONCLUSIONS: To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women approximately 6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publisher's online edition.

Proteasomal activity in placentas from women with preeclampsia and intrauterine growth restriction: implications for expression of HIF-alpha proteins.

Hypoxia-inducible transcription factor-1alpha and -2alpha (HIF-alpha) proteins and regulated genes are increased in preeclamptic (PE) placentas. Although placental hypoxia likely stabilizes HIF-alpha proteins, we previously reported that there is also a defect in oxygen-dependent reduction of HIF-alpha proteins in PE relative to normal pregnant (NP) placentas that could contribute to their over-expression. After a 4-h exposure to 2% oxygen, placental villous explants were exposed to 21% oxygen over 90 min. As assessed by Western analysis, the defective oxygen-dependent reduction of HIF-1alpha protein in villous explants from PE placenta was unaffected by the protein synthesis inhibitor, cycloheximide. However, after incubation with the proteasomal inhibitor, clasto-lactacystin, oxygen-dependent reduction of HIF-1alpha protein was markedly and similarly impaired in the villous explants from both normal and PE placentas. Thus, impairment of protein degradation rather than increased synthesis causes inadequate oxygen-dependent reduction of HIF-1alpha protein in PE placentas. Immunoprecipitation studies revealed comparable association of HIF-1alpha with von Hippel Lindau (VHL) protein in placentas from NP and PE women. Furthermore, prolyl hydroxylase-3 protein was appropriately upregulated in the PE placentas as determined by Western analysis paralleling the increases of HIF-alpha proteins. These results suggest that molecular events leading to the formation of the HIF-1alpha:VHL:ubiquitin ligase complex are most likely not impaired in PE placentas. Finally, proteasomal trypsin, chymotrypsin, and peptidyl glutamyl-like activities were significantly reduced by approximately 1/3 in PE placentas by using specific peptide substrates coupled to a fluorescent tag. Unexpectedly, however, they were even further decreased in placentas from normotensive women delivering growth restricted babies >37 weeks gestation-placentas which do not have elevated HIF-alpha proteins. In conclusion, accumulation of HIF-alpha proteins in PE placentas may occur as a consequence of both increased formation secondary to relative ischemia/hypoxia and reduced degradation after reperfusion/oxygenation consequent to proteasomal dysfunction. In contrast, in placentas from normotensive women delivering growth restricted babies >37 weeks gestation, proteasomal activity, albeit markedly reduced, is adequate to cope with degradation of HIF-alpha proteins, which have not been increased by an hypoxic environment.

Expression of von Hippel Lindau (pVHL) protein in placentae from normal pregnant women and women with preeclampsia.

The hypoxia inducible transcription factors, HIF-1alpha and -2alpha proteins, are overexpressed in placentae from women with preeclampsia (Biol Reprod 2001;64:499-506; Biol Reprod 2001;64:1019-1020). Normally, these proteins are regulated in an oxygen-dependent manner being rapidly degraded by the ubiquitin-mediated proteasomal pathway. Recent studies have shown that the tumor suppressor protein, von Hippel Lindau (VHL), targets HIF for ubiquitinylation under nonhypoxic conditions. The objectives of the present work were: (1) to investigate VHL protein expression in normal pregnant (NP), preeclamptic (PE), and preterm (without PE) placentae, (2) to test whether VHL protein is hypoxia inducible in term and first trimester placental villous explants, and (3) to analyze the ontogeny of VHL protein expression in the human placenta. To begin evaluating the potential contribution of VHL to HIF overexpression in preeclamptic placentae, we analyzed the levels of the VHL protein in both normal and preeclamptic placentae (n=7 each). We hypothesized a deficiency of VHL protein in preeclamptic placentae. Eight biopsy sites were tested in each placenta and protein extracts were made. Western analysis was performed using VHL specific antibodies. Human renal adenocarcinoma (ACHN) cell extracts and extracts from COS-7 cells transfected with a VHL expression vector were used as positive controls. In a total of 112 biopsy sites that were analyzed (56 each for normal and preeclamptic placentae), the composite densitometry ratios (PE/NP) for the long (28 kDa) and short (19 kDa) forms of VHL were 1.09+/-0.2 and 1.16+/-0.11, respectively (both p=NS vs 1.0). A ratio of 1.0 indicates equal expression by preeclamptic and normal placentae. The same placentae exhibited composite densitometry (PE/NP) ratios of 1.97+/-0.23 and 1.68+/-0.20 for HIF-1alpha and -2alpha proteins, respectively (both p<0.05 vs 1.0). In a separate analysis, the protein expression of the short form of VHL was also comparable among NP, PE and preterm (n=6) placentae. VHL immunoreactivity was localized to cells within the basal plate and the syncytiotrophoblast. Despite induction of HIF proteins by hypoxia in first and term villous explants, there was no significant upregulation of VHL proteins. Finally, the expression of both the short and long forms of VHL protein decreased with gestational age (both p<0.05 by ANOVA), and in villous tissue from first trimester placentae VHL immunoreactivity was predominantly localized to the cytotrophoblast. These results suggest that (1) deficiency of VHL protein does not account for HIF-alpha overexpression in preeclamptic placentae, (2) VHL protein is not regulated by hypoxia in either first trimester or term placental villous explants, and (3) VHL protein expression in the placenta decreases as a function of gestational age.

Mechanisms of renal vasodilation and hyperfiltration during pregnancy: current perspectives and potential implications for preeclampsia.

A thorough understanding of the renal and cardiovascular adaptations to normal gestation is essential for proper diagnosis and management of hypertensive disorders and renal diseases during pregnancy. Here, we briefly review the renal hemodynamic changes of normal pregnancy. In addition, we present new findings and current concepts related to the underlying hormonal and molecular mechanisms. Finally, we speculate on the potential contribution of these insights from normal pregnancy to the pathogenesis of preeclampsia.

Antihypertensive efficacy of candesartan-lisinopril in combination vs. up-titration of lisinopril: the AMAZE trials.

The AMAZE (A Multicenter Trial Using Atacand and Zestril vs. Zestril to Evaluate the Effects on Lowering Blood Pressure) program included two identical studies sponsored by AstraZeneca LP. The oral form of candesartan is candesartan cilexetil; for simplicity, the term "candesartan" is used throughout this manuscript. Two identical multicenter, randomized, double-blind studies were performed to determine if addition of the angiotensin receptor blocker candesartan was more effective in lowering blood pressure than up-titration of lisinopril. Hypertensive patients (N=1,096) who were uncontrolled on lisinopril 20 mg daily were randomized (1:1) to receive either 8 weeks of high-dose lisinopril (40 mg) or the addition of candesartan (16 mg) for 2 weeks followed by 32 mg for 6 weeks. Study 1 (n=538) demonstrated decreases in trough sitting systolic/diastolic blood pressures at Week 8 by 6.2/5.9 mm Hg, respectively, for the lisinopril up-titration treatment group and by 11.6/8.3 mm Hg, respectively, for the lisinopril plus candesartan treatment group (p<0.01 in comparing both blood pressures reductions between the two treatment groups). Corresponding results for Study 2 (n=558) are reductions of 8.7/6.2 mm Hg and 9.5/7.4 mm Hg, respectively, for each of the two treatment groups. For Study 2, comparisons of systolic/diastolic blood pressures between the two treatment groups were not statistically significantly different (p=0.51/p=0.08, respectively). Post hoc pooled analysis (N=1,096) demonstrated a slightly greater blood pressure reduction with lisinopril plus candesartan compared with lisinopril (3.1/1.7 mm Hg). A 95% confidence interval limit for the difference in least squares mean change from baseline in systolic blood pressure between the two treatment groups is -4.8 to -1.5 and is -2.8 to -0.7 in mm Hg for diastolic blood pressure. The blood pressure control rates (<140/<90 mm Hg) were 42.7% and 36.9%, respectively. Both treatment regimens were well tolerated in all groups. In conclusion, for hypertensive patients not controlled by lisinopril 20 mg once daily, addition of candesartan (32 mg once daily) or doubling the dose of lisinopril provides safe, additional reduction of blood pressure.

Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae.

Placentas from women with preeclampsia overexpress the hypoxia-inducible transcription factor proteins, HIF-1alpha and -2alpha (Rajakumar 2001, Biol Reprod 64; p499-506 and p1019-1020). As a first step in evaluating whether HIF-alpha overexpressed in preeclamptic placentae is capable of transactivation, we tested its ability to bind to the DNA hypoxia response element (HRE). Six pairs of normal and preeclamptic placentae obtained by cesarean section were investigated. Three biopsy sites per placenta were analyzed. We first confirmed HIF-1alpha protein overexpression in the preeclamptic placentae using Western analysis. The ratios of the arbitrary densitometry units for HIF-1alpha protein from the preeclamptic and normal placentae (PE/NP) in the three biopsy sites were: 1.9 +/- 0.3, 1.7 +/- 0.2 and 1.8 +/- 0.2, each p < 0.05 vs 1.0. (A ratio of >1.0 indicates that HIF-1alpha protein expression in placentas of women with PE exceeds that in placentas of NP women.) Conventional methods for extracting nuclear proteins and subsequent analysis by electrophoretic mobility shift assay were not suited for the frozen, archived samples (data not shown). Therefore, we employed DNA affinity chromatography using a biotinylated oligonucleotide representing the HRE of the erythropoietin gene coupled to streptavidin-coated Dynabeads. The HRE-bound proteins were then characterized by Western blot analysis. The PE/NP ratios of HRE-bound HIF-1alpha in the three biopsy sites from the six pairs of normal and preeclamptic placentae were 1.7 +/- 0.2, 2.1 +/- 0.4 and 2.4 +/- 0.5, each p < 0.05 vs 1.0. Having established DNA-binding potential at least in vitro, we subsequently analyzed three proteins that have been shown to be regulated by HIF-alpha as downstream, molecular markers of HIF-1alpha activity in vivo. VEGF receptor Flt-1 and Flk-1 play key roles in angiogenesis. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. All three genes contain functional HRE in their promoter sequences. Total proteins were extracted from the same biopsy samples that were used for total and HRE-bound HIF-1alpha. Using specific antibodies we performed Western analysis and the levels of these three proteins were quantitated. The Flt-1 and tyrosine hydroxylase proteins were significantly higher, and Flk-1 significantly lower in placentae from preeclamptic compared to normal pregnancies. In summary, HIF-1alpha protein overexpressed in preeclamptic placentae is capable of binding to its DNA recognition sequence in vitro, and modulates gene expression in vivo.

Impaired oxygen-dependent reduction of HIF-1alpha and -2alpha proteins in pre-eclamptic placentae.

Pre-eclamptic (PE) placentae overexpress hypoxia inducible transcription factors-1alpha and -2alpha proteins (Biol. Repro. 64: 499-506, 2001; Ibid 1019-1020). Possible explanations include (a) impaired oxygen-dependent reduction, and/or (b) enhanced sensitivity to reduced oxygen. After 18 h equilibration under 21 per cent O(2) atmosphere, we subjected villous explants prepared from placentae of normal pregnant (NP) and pre-eclamptic (PE) women (n=8 each) to 4h of hypoxia (2 per cent oxygen), and then studied the disappearance of HIF-1alpha and -2alpha proteins during subsequent oxygenation over 90 min (21 per cent oxygen). The disappearance of these HIF proteins as assessed by Western analysis was significantly impaired in the pre-eclamptic tissues. Even after 18h equilibration under a 21 per cent O(2) atmosphere, and then a further 4h at 21 per cent O(2), HIF-1alpha and -2alpha protein expression remained increased in villous explants from PE women (both P< 0.04 vs NP). To address whether chronic hypoxia per se (which is believed to exist in the pre-eclamptic placenta) might contribute to these findings, we subjected villous explants from normal placentae (n=6) to 18 h preincubation under 2 per cent or 21 per cent oxygen prior to subsequent incubation for 4h at 2 per cent oxygen and then 90 min at 21 per cent oxygen. The time course of disappearance of HIF proteins during oxygenation was similar irrespective of the 2 per cent or 21 per cent preconditioning. To evaluate oxygen sensitivity, we exposed villous explants from NP and PE women (n=6 each) to different oxygen atmospheres for 4h and measured HIF protein induction. Although the data showed a significant inverse relationship between HIF expression and oxygen concentration, there was no significant difference between the slopes of this relationship for the two groups of women. We conclude that villous explants from PE placentae fail to adequately downregulate HIF protein expression upon oxygenation. This abnormality may contribute to their overexpression in vivo.

Expression of inflammatory cytokines in placentas from women with preeclampsia.

It is postulated that inadequate remodeling of the uterine spiral arteries in preeclampsia leads to focal ischemia and generation of inflammatory cytokines, such as tumor necrosis factor (TNF alpha) and interleukins (ILs), by the placenta. Our objective was to compare TNF alpha, IL-1 alpha, IL-1 beta, and IL-6 levels in placentas from patients with preeclampsia and normal term pregnancies. Because the placenta is a large heterogeneous organ, we analyzed multiple sites per placenta. On the average, there was a 3-fold variation in cytokine protein levels across the eight sites analyzed for each placenta. However, there were no significant overall differences among the normal term, preeclamptic, and preterm placentas from women without preeclampsia. There were also no significant differences in TNF alpha messenger ribonucleic acid between the normal term and preeclamptic placentas, although TNF alpha messenger ribonucleic acid levels were lower in placentas from preterm patients without diagnosis of preeclampsia than in the normal term placentas. In vitro, hypoxia stimulated the production of TNF alpha, IL-1 alpha and IL-1 beta, but not that of IL-6, by placental villous explants from both groups of patients, and this was not exaggerated in preeclampsia. Finally, although peripheral and uterine venous levels of TNF alpha were elevated in preeclamptic women compared with normal term patients, the ratio of uterine to peripheral venous TNF alpha levels was not significantly different from 1.0 for either patient group. Taken together, these results suggest that sources other than the placenta contribute to the elevated concentrations of TNF alpha and IL-6 found in the circulation of preeclamptic women.

Relaxin is essential for renal vasodilation during pregnancy in conscious rats.

Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.

Selective overexpression of the hypoxia-inducible transcription factor, HIF-2alpha, in placentas from women with preeclampsia.

Transcription factors orchestrate the development of extraembryonic tissues. Because placental hypoxia likely plays an important role in both normal and abnormal placentation, we have been investigating the hypoxia-inducible transcription factors (HIFs) in the human placenta. In this report, we focus on the placentas from women with preeclampsia. Because the placenta is a large, heterogeneous organ, we employed a systematic and unbiased approach to placental sampling, and our results are based on the analyses of eight biopsy sites per placenta. We observed no significant differences in HIF-1alpha or -2alpha mRNA expression between normal term and preeclamptic placentas. Nor was HIF protein expression significantly different, with the notable exception of HIF-2alpha, which, on average, was increased by 1.7-fold in the preeclamptic placentas (P: < 0.03 vs. normal term placentas). Considering all 48 paired placental biopsy sites (eight sites each for six normal term and six preeclamptic placentas), HIF-2alpha protein levels in the preeclamptic placentas exceeded those in the normal term placentas in 39, or 81%, of the paired sites (P: < 0.0013). The HIF-2alpha immunoreactivity was mainly located in the nuclei of the syncytiotrophoblast and fetoplacental vascular endothelium in the preeclamptic villous placenta. To control for the earlier gestational age of the preeclamptic placentas, an additional group of placentas from preterm deliveries without preeclampsia were also evaluated. The HIF protein expression was comparable in these preterm specimens and the normal term placentas. We conclude that protein expression of HIF-2alpha, but not of HIF-1alpha or -1beta, is selectively increased in the preeclamptic placenta. The molecular mechanism(s) of this abnormality as well as the genes affected downstream are currently under investigation. To our knowledge, this is the first report of abnormal HIF-2alpha expression in human disease other than cancer.

Endothelin and nitric oxide mediate reduced myogenic reactivity of small renal arteries from pregnant rats.

We tested the hypothesis that endothelin acting through the endothelial ET(B) receptor subtype and the nitric oxide (NO) pathway accounts for reduced myogenic reactivity of the renal resistance vasculature during pregnancy. Small renal arteries (100-200 microm) were isolated from virgin and midterm pregnant rats when gestational renal hyperfiltration and vasodilation are maximal in this species. Myogenic reactivity (the adjustment of arterial diameter in response to a change in transmural pressure) was assessed with a pressurized myograph system. A rapid increase in transmural pressure from 60 to 80 mmHg resulted in a 2.4% diameter increase in vessels from virgin compared with an 8.1% increase in arteries from midgestation rats (n = 8 each, P < 0.05). Thus myogenic reactivity is markedly reduced during pregnancy. Incubation with the NO synthase inhibitors, an ET(B) receptor subtype antagonist (RES-701-1), the nonselective ET(A/B) receptor blocker (SB-209670), or endothelial removal abrogated the reduced myogenic reactivity of vessels from gravid rats without affecting myogenic reactivity in arteries from virgin animals. Thus the endothelium mediates the reduced myogenic reactivity of small renal arteries of midgestation rats most likely through the ET(B) receptor subtype and NO pathway.

Bovine viral diarrhea virus in New World camelids.

A virus known to cause multiple problems in cattle, bovine viral diarrhea virus, was isolated from 3 different cases in New World camelids. Virus isolation, immunoperoxidase staining, and fluorescent antibody staining were used to detect the virus. The herds involved were screened for antibody titers to bovine viral diarrhea and virus isolation from the buffy coat. Bovine viral diarrhea virus should be considered as a cause of death in young and old New World camelids.

Impact of gender and endothelin on renal vasodilation and hyperfiltration induced by relaxin in conscious rats.

Chronic administration of the hormone relaxin elicits renal vasodilation that is dependent on nitric oxide (NO) in both conscious intact and ovariectomized female rats. Our first objective was to test whether the hormone, when administered to approximate serum concentrations found in midterm pregnant rats, induces renal vasodilation in males. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) increased significantly, on average, by 33 and 49% over baseline, respectively, after 5 days of recombinant human relaxin (rhRLX) administration to 12 conscious male rats by subcutaneous osmotic minipump. There were also significant decreases in hematocrit, plasma osmolality, and sodium concentration. Another objective was to determine whether endogenous endothelin (ET; via the endothelial ET(B) receptor) mediates the NO-dependent renal vasodilation produced by relaxin. rhRLX or vehicle was administered to conscious female rats (n = 9 and 8 rats, respectively). On the fifth day, baseline GFR and ERPF were both increased, on average, by 20-30% in the rats administered rhRLX (P < 0.05 vs. vehicle). Next, the specific ET(B)-receptor antagonist RES-701-1 was infused intravenously over 4 h in both groups of rats. In response to RES-701-1, there was a significant decline in both GFR and ERPF in the rats receiving rhRLX such that renal function converged in the two groups of animals. We conclude 1) relaxin induces marked changes in the renal circulation and in osmoregulation regardless of gender and 2) relaxin-induced renal vasodilation and hyperfiltration are mediated by endothelin through the endothelial ET(B) receptor subtype and NO.

Expression, ontogeny, and regulation of hypoxia-inducible transcription factors in the human placenta.

Placental hypoxia likely plays an important role in both normal placental development and pathology. Yet, the molecular mechanisms of hypoxia signaling in this organ are virtually unexplored. Therefore, we investigated the expression of the hypoxia inducible transcription factors (HIF) in normal human placentas spanning the first trimester to term. Several key observations emerged: 1) HIF-1 alpha and -2 alpha mRNA were present in placentas of all gestational ages but with greater variability during early pregnancy; 2) overall, HIF-1 alpha mRNA was expressed at a constant level in all placentas, whereas HIF-2 alpha mRNA increased significantly with gestational age; 3) both HIF-1 alpha and -2 alpha protein decreased significantly with gestational age; and 4) HIF-1 alpha and -2 alpha immunoreactivity were overlapping in cellular distribution being expressed by the syncytiotrophoblast, villous cytotrophoblast, and fetoplacental vasculature with both nuclear and cytoplasmic localization. Next, we studied the regulation of these transcription factors by oxygen using placental villous explants in culture from first-trimester and term placentas. The major findings were 1) HIF-1 alpha and -2 alpha protein, but not mRNA, was induced by hypoxia in the placental villous explants; 2) HIF-1 alpha DNA-binding activity was also stimulated by hypoxia; and 3) glucose transporter-1 mRNA (a known target of HIF) was also increased by hypoxia in placental villous explants. We suggest that physiological hypoxia contributes to the increased expression of HIF-1 alpha and -2 alpha protein in early placentas and that regulation of these transcription factors by hypoxia in the human placenta occurs at the level of protein and not mRNA.

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta.

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.

Plasma and 24-h NO(x) and cGMP during normal pregnancy and preeclampsia in women on a reduced NO(x) diet.

We tested the hypothesis that nitric oxide (NO) biosynthesis increases during normal human pregnancy and decreases in preeclampsia. The major metabolites of NO, nitrate and nitrite (NO(x)), were measured in both the plasma and 24-h urine of women subjected to a reduced NO(x) diet. In this way, the plasma and urinary levels mainly reflected endogenous production rather than dietary intake. Moreover, we assessed cGMP, a second messenger of NO, in the same samples. Both NO(x) and cGMP assays were validated in our laboratory. We first conducted a cross-sectional study of nonpregnant women (n = 15), normal pregnant women in the first (n = 9), second (n = 17) and third (n = 22) trimesters, as well as women with preeclampsia (n = 15) and transient hypertension of pregnancy (n = 7). We also performed a serial study in the same women (n = 9) before, during, and after pregnancy. Taken together, the results of the two investigations suggested marked increases in cGMP production especially during the first trimester when the maternal circulation is rapidly vasodilating. In contrast, whole body NO production as estimated by the plasma level and urinary excretion of NO(x) was not elevated during the first trimester. Finally, unequivocal demonstration of reduced NO biosynthesis in preeclampsia was not forthcoming.

Endothelin mediates renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats.

Profound vasodilation of the kidneys and other nonreproductive organs transpires during early pregnancy. Because nitric oxide (NO) was found to mediate renal vasodilation and hyperfiltration in conscious pregnant rats, and endogenous endothelin (ET) was suggested to be vasodilatory in the renal circulation of nonpregnant rats, we tested whether endothelin mediates the NO-dependent changes in the renal circulation during pregnancy. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious pregnant and virgin rats before and during infusion of 30 micrograms/min RES-701-1 (a selective ETB receptor subtype antagonist). Baseline GFR and ERPF were significantly increased by 35% in gravid rats relative to virgin controls. During infusion of RES-701-1, the pregnant rats responded more robustly, showing a greater decline in both GFR and ERPF such that renal function converged in the two groups of rats. ERPF also converged in pregnant and virgin rats during infusion of SB-209760, a nonselective ETA/B receptor subtype antagonist. Combined infusion of Nomega-nitro-L-arginine methyl ester [L-NAME, an NO synthase (NOS) inhibitor] and RES-701-1 reduced GFR and ERPF to levels comparable to those reached with either agent given alone, suggesting inhibition of a common vasodilatory pathway. RES-701-1 and SB-209670 significantly lowered the cGMP content of small renal arteries from gravid and virgin rats in vitro, strengthening the link between the renal endothelial ETB receptor subtype and NO. Importantly, we showed that RES-701-1 is not a direct inhibitor of NOS. We conclude that endothelin mediates the NO-dependent changes in the renal circulation of conscious rats during pregnancy.