Reduced uterine perfusion pressure as a model for preeclampsia and fetal growth restriction in murine: a systematic review and meta-analysis.

The reduced uterine perfusion pressure (RUPP) model is frequently used to study preeclampsia and fetal growth restriction. An improved understanding of influential factors might improve reproducibility and reduce animal use considering the variability in RUPP phenotype. We performed a systematic review and meta-analysis by searching Medline and Embase (until 28 March, 2023) for RUPP studies in murine. Primary outcomes included maternal blood pressure (BP) or proteinuria, fetal weight or crown-rump length, fetal reabsorptions, or antiangiogenic factors. We aimed to identify influential factors by meta-regression analysis. We included 155 studies. Our meta-analysis showed that the RUPP procedure results in significantly higher BP (MD = 24.1 mmHg; [22.6; 25.7]; n = 148), proteinuria (SMD = 2.3; [0.9; 3.8]; n = 28), fetal reabsorptions (MD = 50.4%; [45.5; 55.2]; n = 42), circulating soluble FMS-like tyrosine kinase-1 (sFlt-1) (SMD = 2.6; [1.7; 3.4]; n = 34), and lower fetal weight (MD = -0.4 g; [-0.47; -0.34]; n = 113. The heterogeneity (variability between studies) in primary outcomes appeared ≥90%. Our meta-regression identified influential factors in the method and time point of BP measurement, randomization in fetal weight, and type of control group in sFlt-1. The RUPP is a robust model considering the evident differences in maternal and fetal outcomes. The high heterogeneity reflects the observed variability in phenotype. Because of underreporting, we observed reporting bias and a high risk of bias. We recommend standardizing study design by optimal time point and method chosen for readout measures to limit the variability. This contributes to improved reproducibility and thereby eventually improves the translational value of the RUPP model.

Luteolin prevents TNF-α-induced NF-κB activation and ROS production in cultured human placental explants and endothelial cells.

INTRODUCTION: Preeclampsia (PE) is a serious hypertensive pregnancy disorder and a leading cause of maternal and perinatal morbidity and mortality. Despite the prevalence and complications, there are no approved therapeutics to relieve PE symptoms. Inflammation, oxidative stress, and angiogenic imbalance have been shown to contribute to the PE pathophysiology, though there is a lack of understanding in how best to target these pathways in PE. We recently demonstrated that the bioflavonoid luteolin is a potent inhibitor of the anti-angiogenic and pro-hypertensive soluble fms-like tyrosine kinase 1 (sFlt-1), and here we aimed to determine if luteolin was also capable of reducing inflammation and oxidative stress pathways. METHODS: Tumor necrosis factor (TNF)-α, which is upregulated in PE, was utilized to stimulate these pathways in human placental explants and endothelial cells. Endothelin-1 (ET-1) and interleukin (IL)-6 in the media from explants and cells were measured via ELISA, and NF-κB localization and reactive oxygen species were detected via fluorescence microscopy. RESULTS: Pretreatment with luteolin demonstrated significant reductions in NF-κB activation, reactive oxygen species, superoxide, and IL-6 and ET-1 expression in endothelial cells. We also saw a significant reduction in phosphorylation of NF-κB in human placental explants. DISCUSSION: These data demonstrate that luteolin inhibits pathways implicated in the development of PE and should be explored further for its potential as a PE therapeutic.

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta.

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE.

Long-Term Impacts of Preeclampsia on the Cardiovascular System of Mother and Offspring.

Preeclampsia is a pregnancy-specific complication that is associated with an increased postpartum risk of cardiovascular disease (CVD) in both women and their offspring, although the underlying mechanisms have yet to be fully elucidated. Nevertheless, differential methylation of cytosine-phosphate-guanosine islands and alterations in the expression of microRNA, associated with an elevated risk of CVD, have been observed in women and their children following preeclampsia. Among this specific population, genetic and epigenetic factors play crucial roles in the development of CVD in later life. A series of biomolecules involved in inflammation, oxidative stress, and angiogenesis may link pregnancy vascular bed disorders in preeclampsia to the pathogenesis of future CVD and thus could be valuable for the prediction and intervention of long-term CVD in women with a history of preeclampsia and their offspring. Here, we present insights into the cardiovascular structure and functional changes of women with a history of preeclampsia and their offspring. With a focus on various underlying mechanisms, the conclusions from this review are expected to provide more potential diagnostics and treatment strategies for clinical practice.

Epithelial sodium channels in macrophage migration and polarization: role of proinflammatory cytokines TNFα and IFNγ.

Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na+ channels (ENaCs) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH2-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.

Increased NOS coupling by the metabolite tetrahydrobiopterin (BH4) reduces preeclampsia/IUGR consequences.

Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.

Is there a role of proinflammatory cytokines on degenerin-mediated cerebrovascular function in preeclampsia?

Preeclampsia (PE) is associated with adverse cerebrovascular effects during and following parturition including stroke, small vessel disease, and vascular dementia. A potential contributing factor to the cerebrovascular dysfunction is the loss of cerebral blood flow (CBF) autoregulation. Autoregulation is the maintenance of CBF to meet local demands with changes in perfusion pressure. When perfusion pressure rises, vasoconstriction of cerebral arteries and arterioles maintains flow and prevents the transfer of higher systemic pressure to downstream microvasculature. In the face of concurrent hypertension, loss of autoregulatory control exposes small delicate microvessels to injury from elevated systemic blood pressure. While placental ischemia is considered the initiating event in the preeclamptic cascade, the factor(s) mediating cerebrovascular dysfunction are poorly understood. Elevated plasma proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-17 (IL-17), are potential mediators of autoregulatory loss. Impaired CBF responses to increases in systemic pressure are attributed to the impaired pressure-induced (myogenic) constriction of small cerebral arteries and arterioles in PE. Myogenic vasoconstriction is initiated by pressure-induced vascular smooth muscle cell (VSMC) stretch. Recent studies from our laboratory group indicate that proinflammatory cytokines impair the myogenic mechanism of CBF autoregulation via inhibition of vascular degenerin proteins, putative mediators of myogenic constriction in VSMCs. This brief review links studies showing the effect of proinflammatory cytokines on degenerin expression and CBF autoregulation to the pathological cerebral consequences of preeclampsia.

Sustained Elevated Circulating Activin A Impairs Global Longitudinal Strain in Pregnant Rats: A Potential Mechanism for Preeclampsia-Related Cardiac Dysfunction.

Mediators of cardiac injury in preeclampsia are not well understood. Preeclamptic women have decreased cardiac global longitudinal strain (GLS), a sensitive measure of systolic function that indicates fibrosis and tissue injury. GLS is worse in preeclampsia compared to gestational hypertension, despite comparable blood pressure, suggesting that placental factors may be involved. We previously showed that Activin A, a pro-fibrotic factor produced in excess by the placenta in preeclampsia, predicts impaired GLS postpartum. Here, we hypothesized that chronic excess levels of Activin A during pregnancy induces cardiac dysfunction. Rats were assigned to sham or activin A infusion (1.25-6 µg/day) on a gestational day (GD) 14 (n = 6-10/group). All animals underwent blood pressure measurement and comprehensive echocardiography followed by euthanasia and the collection of tissue samples on GD 19. Increased circulating activin A (sham: 0.59 ± 0.05 ng/mL, 6 µg/day: 2.8 ± 0.41 ng/mL, p < 0.01) was associated with impaired GLS (Sham: -22.1 ± 0.8%, 6 µg/day: -14.7 ± 1.14%, p < 0.01). Activin A infusion (6 µg/day) increased beta-myosin heavy chain expression in heart tissue, indicating cardiac injury. In summary, our findings indicate that increasing levels of activin A during pregnancy induces cardiac dysfunction and supports the concept that activin A may serve as a possible mediator of PE-induced cardiac dysfunction.

Animal models of preeclampsia: investigating pathophysiology and therapeutic targets.

Animal models have been critical in investigating the pathogenesis, mediators, and even therapeutic options for a number of diseases, including preeclampsia. Preeclampsia is the leading cause of maternal and fetal morbidity and mortality worldwide. The placenta is thought to play a central role in the pathogenesis of this disease because it releases antiangiogenic and proinflammatory factors into the maternal circulation, resulting in the maternal syndrome. Despite the deleterious effects preeclampsia has been shown to have on the mother and baby during pregnancy and postpartum, there is still no effective treatment for this disease. Although clinical studies in patients are crucial to identify the involvement of pathogenic factors in preeclampsia, there are obvious limitations that prevent detailed investigation of the quantitative importance of time-dependent mechanisms involved in this syndrome. Animal models allow investigators to perform proof-of-concept studies and examine whether certain factors found in women with preeclampsia mediate hypertension and other manifestations of this disease. In this brief review, we summarize some of the more widely studied models used to investigate pathophysiological mechanisms that are thought to be involved in preeclampsia. These include models of placental ischemia, angiogenic imbalance, and maternal immune activation. Infusion of preeclampsia-related factors into animals has been widely studied to understand the specific mediators of this disease. These models have been included, in addition to a number of genetic models involved in overexpression of the renin-angiotensin system, complement activation, and trophoblast differentiation. Together, these models cover multiple mechanisms of preeclampsia from trophoblast dysfunction and impaired placental vascularization to the excess circulating placental factors and clinical manifestation of this disease. Most animal studies have been performed in rats and mice; however, we have also incorporated nonhuman primate models in this review. Preclinical animal models not only have been instrumental in understanding the pathophysiology of preeclampsia but also continue to be important tools in the search for novel therapeutic options for the treatment of this disease.

Placental Ischemia Says "NO" to Proper NOS-Mediated Control of Vascular Tone and Blood Pressure in Preeclampsia.

In this review, we first provide a brief overview of the nitric oxide synthase (NOS) isoforms and biochemistry. This is followed by describing what is known about NOS-mediated blood pressure control during normal pregnancy. Circulating nitric oxide (NO) bioavailability has been assessed by measuring its metabolites, nitrite (NO2) and/or nitrate (NO3), and shown to rise throughout normal pregnancy in humans and rats and decline postpartum. In contrast, placental malperfusion/ischemia leads to systemic reductions in NO bioavailability leading to maternal endothelial and vascular dysfunction with subsequent development of hypertension in PE. We end this article by describing emergent risk factors for placental malperfusion and ischemic disease and discussing strategies to target the NOS system therapeutically to increase NO bioavailability in preeclamptic patients. Throughout this discussion, we highlight the critical importance that experimental animal studies have played in our current understanding of NOS biology in normal pregnancy and their use in finding novel ways to preserve this signaling pathway to prevent the development, treat symptoms, or reduce the severity of PE.

Macula Densa NOS1ß Modulates Renal Hemodynamics and Blood Pressure during Pregnancy: Role in Gestational Hypertension.

BACKGROUND: Regulation of renal hemodynamics and BP via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the ß-splice variant of nitric oxide synthase 1 (NOS1ß) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1ß in the macula densa, thus blunting TGF, allowing the GFR to increase and BP to decrease. METHODS: We used sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radiotelemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1ß/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. RESULTS: Macula densa NOS1ß was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1ß, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1ß expression increased in normotensive pregnancies, but decreased in spontaneous gestational hypertensive pregnancies. CONCLUSIONS: Macula densa NOS1ß plays a critical role in the control of renal hemodynamics and BP during pregnancy.

Expression of Exogenous Epithelial Sodium Channel Beta Subunit in the Mouse Middle Cerebral Artery Increases Pressure-Induced Constriction.

BACKGROUND: Pressure-induced constriction (PIC) is inherent to small arteries and arterioles, in which intraluminal pressure-induced vascular smooth muscle cell stretch elicits vasoconstriction. Degenerin (Deg) proteins, such as beta-epithelial Na+ channel (ßENaC), have been studied in the PIC response because they are evolutionarily linked to known mechanosensors. While loss of Deg function phenotypes are plentiful, a gain-of-function phenotype has not been studied. The aim of this study was to determine if expression of exogenous ßENaC in the isolated middle cerebral artery (MCA) enhances the PIC response. METHODS: Isolated MCA segments from female mice (24 weeks, n = 5) were transfected with enhanced green fluorescent protein-ßENaC (EGFP-ßENaC) or with EGFP alone, incubated overnight at 37 °C, then studied in a pressure myograph. RESULTS: Mechanical/morphological properties and vasoconstrictor responses to KCl and phenylephrine were identical in EGFP-ßENaC and EGFP MCAs. In contrast, PIC responses were greater in EGFP-ßENaC segments with ~2-fold greater peak myogenic tone. CONCLUSIONS: These data confirm previous findings that ßENaC is critical in the PIC response. These data provide proof-of-concept that upregulating ßENaC can enhance PIC responses and lay the foundation to test the hypothesis that inflammation-mediated downregulation of ßENaC contributes to cerebrovascular dysfunction.

Impact of hyperleptinemia during placental ischemia-induced hypertension in pregnant rats.

The prevalence of preeclampsia and obesity have increased. Although obesity is a major risk factor for preeclampsia, the mechanisms linking these morbidities are poorly understood. Circulating leptin levels increase in proportion to fat mass. Infusion of this adipokine elicits hypertension in nonpregnant rats, but less is known about how hyperleptinemia impacts blood pressure during placental ischemia, an initiating event in the pathophysiology of hypertension in preeclampsia. We tested the hypothesis that hyperleptinemia during reduced uterine perfusion pressure (RUPP) exaggerates placental ischemia-induced hypertension. On gestational day (GD) 14, Sprague-Dawley rats were implanted with osmotic mini-pumps delivering recombinant rat leptin (1 µg/kg/min iv) or vehicle concurrently with the RUPP procedure to induce placental ischemia or Sham. On GD 19, plasma leptin was elevated in Sham + Leptin and RUPP + Leptin. Leptin infusion did not significantly impact mean arterial pressure (MAP) in Sham. MAP was increased in RUPP + Vehicle vs. Sham + Vehicle. In contrast to our hypothesis, placental ischemia-induced hypertension was attenuated by leptin infusion. To examine potential mechanisms for attenuation of RUPP-induced hypertension during hyperleptinemia, endothelial-dependent vasorelaxation to acetylcholine was similar between Sham and RUPP; however, endothelial-independent vasorelaxation to the nitric oxide (NO)-donor, sodium nitroprusside, was increased in Sham and RUPP. These findings suggest that NO/cyclic guanosine monophosphate (cGMP) signaling was increased in the presence of hyperleptinemia. Plasma cGMP was elevated in Sham and RUPP hyperleptinemic groups compared with vehicle groups but plasma and vascular NO metabolites were reduced. These data suggest that hyperleptinemia during placental ischemia attenuates hypertension by compensatory increases in NO/cGMP signaling.NEW & NOTEWORTHY Ours is the first study to examine the impact of hyperleptinemia on the development of placental ischemia-induced hypertension using an experimental animal model.

Interleukin-17 induces hypertension but does not impair cerebrovascular function in pregnant rats.

Preeclampsia affects 5-8% of pregnancies and is characterized by hypertension, placental ischemia, neurological impairment, and an increase in circulating inflammatory cytokines, including Interleukin-17 (IL17). While placental ischemia has also been shown to impair cerebrovascular function, it is not known which placental-associated factor(s) drive this effect. The purpose of this study was to examine the effects of IL17 on cerebrovascular function during pregnancy. To achieve this goal, pregnant rats were infused with either IL17 (150 pg/day, 5 days, osmotic minipump), or vehicle (saline/0.7% BSA osmotic minipump) starting at gestational day (GD) 14. On GD 19, the cerebral blood flow (CBF) response to increases in mean arterial pressure (MAP) was measured in vivo, and myogenic constrictor responses of the middle cerebral artery (MCA) were assessed ex vivo. IL17 increased MAP but impaired CBF responses only at the highest arterial pressure measured (190 mmHg). Myogenic constrictor responses overall were mostly unaffected by IL17 infusion; however, the intraluminal pressure at which peak myogenic tone was generated was lower in the IL17 infused group (120 vs 165 mm Hg), suggesting maximal tone is exerted at lower intraluminal pressures in IL17-treated pregnant rats. Consistent with the lack of substantial change in overall myogenic responsiveness, there was no difference in cerebral vessel expression of putative mechanosensitive protein ßENaC, but a tendency towards a decrease in ASIC2 (p = 0.067) in IL17 rats. This study suggests that infusion of IL17 independent of other placental ischemia-associated factors is insufficient to recapitulate the features of impaired cerebrovascular function during placental ischemia. Further studies to examine of the role of other pro-inflammatory cytokines, individually or a combination, are necessary to determine mechanisms of cerebral vascular dysfunction during preeclampsia.

Long-Term Postpartum Cardiac Function and Its Association With Preeclampsia.

Background Preeclampsia is a prominent risk factor for long-term development of cardiovascular disease. Although existing studies report a strong correlation between preeclampsia and heart failure, the underlying mechanisms are poorly understood. One possibility is the glycoprotein growth factor activin A. During pregnancy, elevated activin A levels are associated with impaired cardiac global longitudinal strain at 1 year, but whether these changes persist beyond 1 year is not known. We hypothesized that activin A levels would remain increased more than 1 year after a preeclamptic pregnancy and correlate with impaired cardiac function. Methods and Results To test our hypothesis, we performed echocardiograms and measured activin A levels in women approximately 10 years after an uncomplicated pregnancy (n=25) or a pregnancy complicated by preeclampsia (n=21). Compared with women with a previously normal pregnancy, women with preeclampsia had worse global longitudinal strain (-18.3% versus -21.3%, P=0.001), left ventricular posterior wall thickness (0.91 mm versus 0.80 mm, P=0.003), and interventricular septal thickness (0.96 mm versus 0.81 mm, P=0.0002). Women with preeclampsia also had higher levels of activin A (0.52 versus 0.37 ng/mL, P=0.02) and activin/follistatin-like 3 ratio (0.03 versus 0.02, P=0.04). In a multivariable model, the relationship between activin A levels and worsening global longitudinal strain persisted after adjusting for age at enrollment, mean arterial pressure, race, and body mass index (P=0.003). Conclusions Our findings suggest that both activin A levels and global longitudinal strain are elevated 10 years after a pregnancy complicated by preeclampsia. Future studies are needed to better understand the relationship between preeclampsia, activin A, and long-term cardiac function.

Luteolin-induced vasorelaxation in uterine arteries from normal pregnant rats.

BACKGROUND: The flavonoid, luteolin, promotes vasorelaxation in various arteries through endothelial-dependent and independent mechanisms. Although there is growing interest in the vasoactive effects of flavonoids on maternal vascular function during pregnancy, it is unknown whether luteolin elicits vasorelaxation in the uterine circulation. We tested the hypothesis that luteolin induces vasorelaxation via endothelial-dependent mechanisms in uterine arteries from normal pregnant rats during late gestation. METHODS: Uterine arteries and aortas were isolated from Sprague-Dawley rats at gestational day 19 and prepared for wire myography. RESULTS: The potency of luteolin-induced vasorelaxation was examined between uterine arteries and the aortas. By 50 µM of luteolin, there was complete relaxation (100.5 ± 5.2%) in uterine arteries as compared to aortas (27.5 ± 10.0%). Even the highest concentration of 100 µM luteolin produced less than half relaxation (43.6 ± 8.6%) in aortas compared to uterine arteries. We then explored if luteolin-induced vasorelaxation in uterine arteries from pregnant rats was mediated by endothelial-dependent vasorelaxation pathways, including nitric oxide synthase (NOS), cyclooxygenase (COX), or potassium (K+) channels. Blocking these pathways with N(G)-Nitro-l-arginine methyl ester hydrochloride (L-NAME), indomethacin, or tetraethylammonium (TEA)/high potassium chloride (KCl), respectively, did not alter luteolin responses in uterine arteries from pregnant rats. These findings suggested that endothelial factors may not mediate luteolin-induced vasorelaxation in uterine arteries during pregnancy. Indeed, experiments where the endothelium was removed did not alter luteolin-induced vasorelaxation in uterine arteries during pregnancy. CONCLUSIONS: Luteolin directly promotes vasorelaxation in the medial smooth muscle layer of uterine arteries during normal pregnancy.

Preeclampsia: Linking Placental Ischemia with Maternal Endothelial and Vascular Dysfunction.

Preeclampsia (PE), a hypertensive disorder, occurs in 3% to 8% of pregnancies in the United States and affects over 200,000 women and newborns per year. The United States has seen a 25% increase in the incidence of PE, largely owing to increases in risk factors, including obesity and cardiovascular disease. Although the etiology of PE is not clear, it is believed that impaired spiral artery remodeling of the placenta reduces perfusion, leading to placental ischemia. Subsequently, the ischemic placenta releases antiangiogenic and pro-inflammatory factors, such as cytokines, reactive oxygen species, and the angiotensin II type 1 receptor autoantibody (AT1-AA), among others, into the maternal circulation. These factors cause widespread endothelial activation, upregulation of the endothelin system, and vasoconstriction. In turn, these changes affect the function of multiple organ systems including the kidneys, brain, liver, and heart. Despite extensive research into the pathophysiology of PE, the only treatment option remains early delivery of the baby and importantly, the placenta. While premature delivery is effective in ameliorating immediate risk to the mother, mounting evidence suggests that PE increases risk of cardiovascular disease later in life for both mother and baby. Notably, these women are at increased risk of hypertension, heart disease, and stroke, while offspring are at risk of obesity, hypertension, and neurological disease, among other complications, later in life. This article aims to discuss the current understanding of the diagnosis and pathophysiology of PE, as well as associated organ damage, maternal and fetal outcomes, and potential therapeutic avenues. © 2021 American Physiological Society. Compr Physiol 11:1315-1349, 2021.

Antepartum Aspirin Administration Reduces Activin A and Cardiac Global Longitudinal Strain in Preeclamptic Women.

Background Approximately 60% of women have Stage B heart failure 1 year after a preeclamptic delivery. Emerging evidence suggests that the profibrotic growth factor activin A, which has been shown to induce cardiac fibrosis and hypertrophy, is elevated in preeclampsia and may be inhibited by aspirin therapy. We hypothesized that preeclamptic women receiving aspirin would have lower activin A levels and reduced global longitudinal strain (GLS), a sensitive measure of cardiac dysfunction, than women who do not receive aspirin. To test our hypothesis, we performed a cohort study of women with preeclampsia or superimposed preeclampsia and compared activin A levels and GLS in parturients who did or did not receive aspirin. Methods and Results Ninety-two parturients were enrolled, of whom 25 (27%) received aspirin (81 mg/day) therapy. GLS, plasma activin A, and follistatin, which inactivates activin A, were measured. Women receiving aspirin therapy had lower median (interquartile range) levels of activin A (8.17 [3.70, 10.36] versus 12.77 [8.37, 31.25] ng/mL; P=0.001) and lower activin/follistatin ratio (0.59 [0.31, 0.93] versus 1.01 [0.64, 2.60] P=0.002) than women who did not receive aspirin, which also remained significant after multivariable analysis. Furthermore, GLS was worse in patients who did not receive aspirin (-19.84±2.50 versus -17.77±2.60%; P=0.03) despite no differences in blood pressure between groups. Conclusions Our study suggests that antepartum aspirin therapy reduced serum activin A levels and improved GLS in preeclamptic patients, suggesting that aspirin may mitigate the postpartum cardiac dysfunction seen in women with preeclampsia.

Pressure-induced constriction of the middle cerebral artery is abolished in TrpC6 knockout mice.

Pressure-induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. While TrpC6 has been suggested to contribute to PIC signaling, direct supporting evidence is contradictory. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20-90 mmHg), depolarization (4-80 mM KCl)-, and adrenergic receptor (phenylephrine; PE 10-7-10-4 M)-mediated constriction of isolated middle cerebral artery (MCA) segments from 9-wk-old male wild-type (TrpC6+/+, n = 7) and homozygous null (TrpC6-/-, n = 9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). Vasoconstrictor responses to KCl and PE were identical in TrpC6-/- and TrpC6+/+ mice. In contrast, PIC responses were totally abolished in TrpC6-/- mice. At 90 mmHg, the calculated myogenic tone was -0.8 ± 0.5 vs. 10.7 ± 1.7%, P = 0.0002 in TrpC6-/- and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain and stress or morphological properties of wall thickness and wall-to-lumen ratio at 50 mmHg between TrpPC6-/- and TrpC6+/+ mice. Although these results demonstrate that TrpC6 is critical for the integrated PIC response, they do not identify whether TrpC6 acts as a mechanosensor or a downstream signaling component.NEW & NOTEWORTHY Pressure-induced, but not agonist-induced, vasoconstriction is abolished in the middle cerebral artery (MCA) of TrpC6 null mice. TrpC6 localization in dissociated cerebral vascular smooth muscle cells is primarily cytoplasmic and not associated with the surface membrane where a mechanoelectrical coupler might be expected. These findings suggest that TrpC6 is required for transduction of pressure-induced constriction in the MCA; however, its role as a mechanoelectrical coupler or downstream signal amplifier remains unresolved.

Interleukin-17 Reduces ßENaC via MAPK Signaling in Vascular Smooth Muscle Cells.

Degenerin proteins, such as the beta epithelial Na+ channel (ßENaC), are essential in the intracellular signaling of pressure-induced constriction, an important vascular smooth muscle cell (VSMC) function. While certain cytokines reduce ENaC protein in epithelial tissue, it is unknown if interleukin-17 (IL-17), a potent pro-inflammatory cytokine, directly mediates changes in membrane-associated ßENaC in VSMCs. Therefore, we tested the hypothesis that exposure to IL-17 reduces ßENaC in VSMCs through canonical mitogen-activated protein kinase (MAPK) signaling pathways. We treated cultured rat VSMCs (A10 cell line) with IL-17 (1-100 ng/mL) for 15 min to 16 h and measured expression of ßENaC, p38MAPK, c-jun kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). IL-17 reduced ßENaC protein expression in a concentration-dependent fashion and increased phosphorylation of p38MAPK by 15 min and JNK by 8 h. NFκB was unaffected by IL-17 in VSMCs. IL-17 treatment reduced VSMC viability but had no effect on cell death. To determine the underlying signaling pathway involved in this response, VSMCs were treated before and during IL-17 exposure with p38MAPK or JNK inhibitors. We found that JNK blockade prevented IL-17-mediated ßENaC protein suppression. These data demonstrate that the pro-inflammatory cytokine IL-17 regulates VSMC ßENaC via canonical MAPK signaling pathways, raising the possibility that ßENaC-mediated loss of VSMC function may occur in inflammatory disorders.