Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Review of Prediabetes and Hypertensive Disorders of Pregnancy.

Obesity and diabetes increase hypertensive disorders of pregnancy (HDP) risk, thus preventive interventions are heavily studied. How pregestational prediabetes and related interventions impact HDP risk is less characterized. Therefore, we searched and reviewed the literature to assess the impact on HDP risk of prediabetes and varied interventions. We identified 297 citations related to pregnancy, prediabetes, and early pregnancy interventions. We also reviewed the references and citations of included articles. We included five studies assessing HDP outcomes in women with first trimester hemoglobin A1c in the prediabetes range (5.7-6.4%). One prospective observational study demonstrated first trimester hemoglobin A1c (5.9-6.4%) is associated with increased HDP risk, while another prospective observational study and one retrospective observational study had similar trends without statistical significance. A small and underpowered randomized controlled trial demonstrated initiating gestational diabetes mellitus treatment (i.e., diet, monitoring, ± insulin) in response to first trimester hemoglobin A1c (5.7-6.4%) did not statistically reduce HDP compared with standard care. One retrospective observational study suggested metformin, when started early, may reduce HDP risk in patients with prediabetes. Pregestational prediabetes appears to increase HDP risk. Interventions (i.e., metformin, diet/glucose monitoring, and/or exercise) to reduce HDP risk require additional study with long-term follow-up.

Catecholestradiol Activation of Adrenergic Receptors Induces Endometrial Cell Survival via p38 MAPK Signaling.

CONTEXT: Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. OBJECTIVE: To investigate ß-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. DESIGN: ß-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2'-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA ß-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±ß-AR antagonist or ±p38 MAPK inhibitor. SETTING: University research institution. PATIENTS: Women with or without endometriosis. INTERVENTIONS: None. MAIN OUTCOME MEASURES: ß-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. RESULTS: Eutopic and ectopic endometrial stromal and epithelial cells displayed ß2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P < 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (P < 0.05), an effect abrogated by ß-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (P < 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (P < 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. CONCLUSIONS: Catecholestradiols increase endometrial cell survival by an ER-independent ß-AR-mediated p38 MAPK activation, suggesting that agents blocking ß-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

Estrogen Receptors and Estrogen-Induced Uterine Vasodilation in Pregnancy.

Normal pregnancy is associated with dramatic increases in uterine blood flow to facilitate the bidirectional maternal-fetal exchanges of respiratory gases and to provide sole nutrient support for fetal growth and survival. The mechanism(s) underlying pregnancy-associated uterine vasodilation remain incompletely understood, but this is associated with elevated estrogens, which stimulate specific estrogen receptor (ER)-dependent vasodilator production in the uterine artery (UA). The classical ERs (ERα and ERß) and the plasma-bound G protein-coupled ER (GPR30/GPER) are expressed in UA endothelial cells and smooth muscle cells, mediating the vasodilatory effects of estrogens through genomic and/or nongenomic pathways that are likely epigenetically modified. The activation of these three ERs by estrogens enhances the endothelial production of nitric oxide (NO), which has been shown to play a key role in uterine vasodilation during pregnancy. However, the local blockade of NO biosynthesis only partially attenuates estrogen-induced and pregnancy-associated uterine vasodilation, suggesting that mechanisms other than NO exist to mediate uterine vasodilation. In this review, we summarize the literature on the role of NO in ER-mediated mechanisms controlling estrogen-induced and pregnancy-associated uterine vasodilation and our recent work on a "new" UA vasodilator hydrogen sulfide (H2S) that has dramatically changed our view of how estrogens regulate uterine vasodilation in pregnancy.

Sexual Dimorphisms of Preeclampsia-Dysregulated Transcriptomic Profiles and Cell Function in Fetal Endothelial Cells.

Preeclampsia impairs fetoplacental vascular function and increases risks of adult-onset cardiovascular disorders in children born to preeclamptic mothers, implicating that preeclampsia programs fetal vasculature in utero. However, the underlying mechanisms remain elusive. We hypothesize that preeclampsia alters fetal endothelial gene expression and disturbs cytokines- and growth factors-induced endothelial responses. RNA sequencing analysis was performed on unpassaged human umbilical vein endothelial cells (HUVECs) from normotensive and preeclamptic pregnancies. Functional assays for endothelial monolayer integrity, proliferation, and migration were conducted on passage 1 HUVECs from normotensive and preeclamptic pregnancies. Compared with normotensive cells, 926 and 172 genes were dysregulated in unpassaged female and male HUVECs from preeclamptic pregnancies, respectively. Many of these preeclampsia-dysregulated genes are associated with cardiovascular diseases (eg, heart failure) and endothelial function (eg, cell migration, calcium signaling, and endothelial nitric oxide synthase signaling). TNF (tumor necrosis factor)-α-, TGF (transforming growth factor)-ß1-, FGF (fibroblast growth factor)-2-, and VEGFA (vascular endothelial growth factor A)-regulated gene networks were differentially disrupted in unpassaged female and male HUVECs from preeclamptic pregnancies. Moreover, preeclampsia decreased endothelial monolayer integrity in responses to TNF-α in both female and male HUVECs. Preeclampsia decreased TGF-ß1-strengthened monolayer integrity in female HUVECs, whereas it enhanced FGF-2-strengthened monolayer integrity in male HUVECs. Preeclampsia promoted TNF-α-, TGF-ß1-, and VEGFA-induced cell proliferation in female, but not in male HUVECs. Preeclampsia inhibited TNF-α-induced cell migration in female HUVECs, but had an opposite effect on male HUVECs. In conclusion, preeclampsia differentially dysregulates cardiovascular diseases- and endothelial function-associated genes/pathways in female and male fetal endothelial cells in association with the sexual dimorphisms of preeclampsia-dysregulated fetal endothelial function.

TNF-alpha inhibits pregnancy-adapted Ca2+ signaling in uterine artery endothelial cells.

Enhancement of vasodilation of uterine arteries during pregnancy occurs through increased connexin (Cx)43 gap junction (GJ) communication supporting more frequent and sustained Ca2+ 'bursts'. Such adaptation is lacking in subjects with preeclampsia (PE). Here we show TNF-alpha, commonly increased in PE subjects, inhibits Cx43 function and Ca2+ bursts in pregnancy-derived ovine uterine artery endothelial cells (P-UAEC) via Src and MEK/ERK phosphorylation of Cx43, and this can be reversed by PP2 or U0126. Of relevance to humans: (1) the nutraceutical Src antagonist t10, c12 CLA also recovers Ca2+ bursting in P-UAEC. (2) TNF-alpha can reduce and PP2 rescue Ca2+ bursting and NO output in human umbilical vein endothelium (HUV Endo) preparations. (3) Treatment of HUV Endo from PE subjects with PP2 alone can rescue bursting and NO output. We conclude TNF-alpha acts via Src more than MEK/ERK to inhibit GJ Cx43 function in PE subjects, and CLA may offer a potential therapy.

Ovine uterine artery hydrogen sulfide biosynthesis in vivo: effects of ovarian cycle and pregnancy†.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P < 0.001). H2S production was greater in P > > > NP UAendo and UAvsm (P < 0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Uteroplacental and Fetal 4D Flow MRI in the Pregnant Rhesus Macaque.

BACKGROUND: Pregnancy complications are often associated with poor uteroplacental vascular adaptation and standard diagnostics are unable to reliably quantify flow in all uteroplacental vessels and have poor sensitivity early in gestation. PURPOSE: To investigate the feasibility of using 4D flow MRI to assess total uteroplacental blood flow in pregnant rhesus macaques as a precursor to human studies. STUDY TYPE: Retrospective feasibility study. ANIMAL MODEL: Fifteen healthy, pregnant rhesus macaques ranging from the 1st trimester to 3rd trimester of gestation. FIELD STRENGTH/SEQUENCE: Abdominal 4D flow MRI was performed on a 3.0T scanner with a radially undersampled phase contrast (PC) sequence. Reference ferumoxytol-enhanced angiograms were acquired with a 3D ultrashort echo time sequence with a center-out radial trajectory. ASSESSMENT: Repeatability of flow measurements was assessed with scans performed same-day and on consecutive days in the uterine arteries and ovarian veins. In-flow was compared against out-flow in the uterus, umbilical cord, and fetal heart with a conservation of mass analysis. Conspicuity of uteroplacental vessels was qualitatively compared between PC angiograms derived from 4D flow data and ferumoxytol-enhanced angiograms. STATISTICAL TESTS: Bland-Altman analysis was used to quantify same-day and consecutive-day repeatability. RESULTS: Same-day flow measurements showed an average difference between scans of 13% in both the uterine arteries and ovarian veins, while consecutive-day measurements showed average differences of 22% and 24%, respectively. Comparisons of in-flow and out-flow showed average differences of 15% in the uterus, 8% in fetal heart, and 15% in the umbilical cord. PC angiograms showed similar depiction of main uteroplacental vessels as high-resolution, ferumoxytol-enhanced angiograms. DATA CONCLUSION: 4D flow MRI could be used in the rhesus macaque for repeatable flow measurements in the uteroplacental and fetal vasculature, setting the stage for future human studies. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:534-545.

Structural analysis of estrogen receptors: interaction between estrogen receptors and cav-1 within the caveolae†.

Pregnancy is a physiologic state of substantially elevated estrogen biosynthesis that maintains vasodilator production by uterine artery endothelial cells (P-UAECs) and thus uterine perfusion. Estrogen receptors (ER-α and ER-ß; ESR1 and ESR2) stimulate nongenomic rapid vasodilatory responses partly through activation of endothelial nitric oxide synthase (eNOS). Rapid estrogenic responses are initiated by the ∼4% ESRs localized to the plasmalemma of endothelial cells. Caveolin-1 (Cav-1) interactions within the caveolae are theorized to influence estrogenic effects mediated by both ESRs. Hypothesis: Both ESR1 and ESR2 display similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using transmission electron microscopy, we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ESR1 and ESR2 in three subcellular locations: membrane, cytosol, and nucleus. Bioinformatics approaches to analyze ESR1 and ESR2 transmembrane domains identified no regions that facilitate ESR interaction with plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns uniquely demonstrated very high protein-protein association only between ESR1, but not ESR2, with Cav-1. These data demonstrate (1) both ESRs localize to the plasmalemma, cytosol and nucleus; (2) neither ESR1 nor ESR2 contain a classic region that crosses the plasmalemma to facilitate attachment; and (3) ESR1, but not ESR2, can be detected in the caveolar subcellular domain demonstrating ESR1 is the only ESR bound in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ESR2 demonstrates a novel independent association of these proteins at the plasmalemma.

Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

Preeclampsia (PE) is a common cause of maternal morbidity, characterized by impaired trophoblast invasion and spiral artery transformation resulting in progressive uteroplacental hypoxia. Given the primary role of LIN28A and LIN28B in modulating cell metabolism, differentiation, and invasion, we hypothesized that LIN28A and/or LIN28B regulates trophoblast differentiation and invasion, and that its dysregulation may contribute to PE. Here we show that LIN28B is expressed ∼1300-fold higher than LIN28A in human term placenta and is the predominant paralog expressed in primary human trophoblast cultures. The expression of LIN28B mRNA and protein levels are significantly reduced in gestational age-matched preeclamptic vs. normal placentas, whereas LIN28A expression is not different. First trimester human placental sections displayed stronger LIN28B immunoreactivity in extravillous (invasive) cytotrophoblasts and syncytial sprouts vs. villous trophoblasts. LIN28B overexpression increased HTR8 cell proliferation, migration, and invasion, whereas LIN28B knockdown in JEG3 cells reduced cell proliferation. Moreover, LIN28B knockdown in JEG3 cells suppressed syncytin 1 (SYN-1), apelin receptor early endogenous ligand (ELABELA), and the chromosome 19 microRNA cluster, and increased mRNA expression of ITGß4 and TNF-α. Incubation of BeWo and JEG3 cells under hypoxia significantly decreased expression of LIN28B and LIN28A, SYN-1, and ELABELA, whereas TNF-α is increased. These results provide the first evidence that LIN28B is the predominant paralog in human placenta and that decreased LIN28B may play a role in PE by reducing trophoblast invasion and syncytialization, and by promoting inflammation.-Canfield, J., Arlier, S., Mong, E. F., Lockhart, J., VanWye, J., Guzeloglu-Kayisli, O., Schatz, F., Magness, R. R., Lockwood, C. J., Tsibris, J. C. M., Kayisli, U. A., Totary-Jain, H. Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration.

E2ß stimulates ovine uterine artery endothelial cell H2S production in vitro by estrogen receptor-dependent upregulation of cystathionine ß-synthase and cystathionine γ-lyase expression†.

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17ß (E2ß) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2ß was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2ß at 48-72 h. E2ß also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2ß stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERß stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2ß. Specific antagonist of either ERα or ERß blocked E2ß-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERß agonists or their antagonists had no additive effects. Thus, E2ß stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERß-dependent CBS and CTH transcription in UAEC in vitro.

Effects of the Catechol and Methoxy Metabolites of 17ß-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2ß, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2ß metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2ß metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2ß metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2ß metabolite-induced NO production. E2ß and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-ß antagonist) inhibited the rises in NOx levels stimulated by E2ß and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (ß2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2ß, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2ß metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy.

Prevention of Diabetes Mellitus in Patients With Prediabetes.

Cardiovascular disease is a leading cause of death in patients with diabetes. Consequently, as antidiabetic medications have demonstrated cardiovascular benefit, cardiologists have been asked to weigh in regarding antidiabetic therapy. The cardiologist's role will continue to grow as antidiabetic agents with cardiovascular benefit are being studied in prediabetes as part of an evolving clinical environment. Still, current guidelines primarily recommend high-intensity lifestyle intervention or metformin for diabetes prevention. Considering that many patients cared for by a cardiologist will have prediabetes, we propose herein that cardiologists can also facilitate diabetes prevention through direct intervention, referring patients to community-based high-intensity lifestyle interventions, and through advocacy, policy, and additional guideline development. The most important messaging for a patient is that avoiding new-onset diabetes can reduce microvascular disease, reduce healthcare cost, and improve health-related quality of life. Moreover, as the mortality risk of patients with a history of myocardial infarction and diabetes is almost double that of patients with a history of myocardial infarction who are free of diabetes, there is even more potential benefit in delaying and/or avoiding diabetes in patients with cardiovascular disease. Despite these important health advantages, the implementation of diabetes prevention strategies is lagging. The under implementation may be exaggerated by published opinions conflicting major guidelines in addition to conflicting guideline recommendations. In conclusion, we propose cardiologists can play a key role in preventing diabetes and aligning practice patterns with guideline recommendations among endocrinology, cardiology, and primary care stake holders.

Metformin's impact on statin-associated muscle symptoms: An analysis of ACCORD study data and research materials from the NHLBI Biologic Specimen and Data Repository Information Coordinating Center.

Statins are widely prescribed, yet statin muscle pain limits their use, leading to increased cardiovascular risk. No validated therapy for statin muscle pain exists. The goal of the study was to assess whether metformin was associated with reduced muscle pain. A secondary analysis of data from the ACCORD trial was performed. An ACCORD sub-study assessed patients for muscle cramps and leg/calve pain while walking, typical non-severe statin muscle pain symptoms. We compared muscle pain between patients using a statin (n = 445) or both a statin and metformin (n = 869) at baseline. Overall patient characteristics were balanced between groups. Unadjusted analysis showed fewer reports of muscle cramps (35%) and leg/calve pain while walking (40%) with statins and metformin compared to statin only (muscle cramps, 42%; leg/calve pain while walking, 47%). Multivariable regression demonstrated a 22% odds reduction for muscle cramps (P = 0.049) and a 29% odds reduction for leg/calve pain while walking (P = 0.01). Metformin appears to reduce the risk of non-severe statin muscle pain and additional research is needed to confirm the findings and assess metformin's impact on statin adherence and related cardiovascular outcomes.

Impact of the ovarian cycle and pregnancy on plasma chemistry values in ewes.

Normative data for plasma chemistry values in pregnant and non-pregnant reproductive age ewes are scant. Availability of data would aid monitoring of ewe health for both research and veterinary medicine. We determined specific plasma chemistry 95% confidence reference intervals (RIs) in non-pregnant and pregnant ewes. Mixed Western-breed ewes were grouped based on phase of ovarian cycle: luteal ( n = 15), follicular ( n = 17), or late-gestation pregnant ( n = 102). Plasma samples were collected for analysis on a commercial biochemical analyzer. For RIs, chemistry panels for the 3 groups of ewes included nutrients and metabolites (glucose, triglycerides, cholesterol, urea, creatinine, total protein, albumin, and bilirubin), enzymes (lactate dehydrogenase, aspartate transaminase, gamma-glutamyl transferase, alanine aminotransferase, and alkaline phosphatase [ALP]), and micronutrients (calcium, phosphorus, iron, sodium, potassium, and chloride). Sample chemistry values for glucose and total protein in pregnant ewes were lower than in follicular ewes; cholesterol was lower in pregnant and luteal ewes than in follicular ewes. In addition, total bilirubin in pregnant ewes differed from that in luteal ewes, and that in follicular ewes also differed from luteal ewes. ALP in pregnant ewes was higher than other groups; phosphorus in pregnant ewes was lower than in luteal ewes. Iron was higher in pregnant ewes than in luteal ewes, with iron in luteal ewes lower than in follicular ewes. These data provide clinical RIs comparing pregnant and non-pregnant ewes for use in monitoring ewe health in both human research and veterinary medicine.

Pleiotropic effects of metformin to rescue statin-induced muscle injury and insulin resistance: A proposed mechanism and potential clinical implications.

The 2013 American Heart Association Blood Cholesterol Guidelines increased the number of patients recommended for statin therapy in the United States to 56million. Two common statin side effects are muscle pain, referred to as "statin-associated muscle symptoms", and increased risk for new onset type-2-diabetes mellitus. Up to 25% of statin users report muscle symptoms resulting in many patients being switched to lower dose or lower potency statins, or refusing statins altogether. The most likely signaling mechanisms for statin-associated muscle symptoms overlaps with the proposed mechanism of statin-induced insulin resistance. Metformin has outstanding utility in reducing insulin resistance and preventing type-2-diabetes mellitus, but has not been studied for statin-associated muscle symptom rescue or prevention. The overlapping mechanisms of statin-associated muscle symptoms, statin-induced insulin resistance, and metformin intervention offers the potential to address two common and detrimental side effects of statins. As statins are the single best medication class for preventing cardiovascular events the potential for clinical benefit is large given metabolic syndrome's growing prevalence in the United States. Herein we hypothesize that metformin will rescue and prevent patients from statin-associated muscle symptoms. This hypothesis can benefit two patient groups: 1) patients at risk for diabetes who are taking a statin and experiencing muscle symptoms; and 2) patients with diabetes taking metformin who are to be started on a statin. Method to test Group 1) Symptom Rescue: randomized control trial of metformin versus placebo in patients with prediabetes who are already taking a statin, and are experiencing mild-to-moderate muscle symptoms. Method to test Group 2) Symptom Prevention: meta-analysis, of statin randomized control trials, with patient level data, comparing patients taking metformin at baseline to patients not taking metformin when a statin is started. An efficient method to simulate both symptom rescue and symptom prevention is a skeletal muscle cell culture model of statin-associated muscle symptom markers. These experiments would identify if metformin reverses (rescues) or prevents markers of statin-associated muscle symptoms. As metformin is recommended by the American Diabetes Association for type-2-diabetes mellitus prevention, yet not frequently used, validating this hypothesis will lead towards research and practice change including: a) decreases in the frequency of statin-associated muscle symptoms; leading to subsequent increases in statin therapy compliance; b) increases in metformin use in prediabetes with subsequent decrease in the incidence of type-2-diabetes mellitus; and c) decreases in complications of both cardiovascular disease and diabetes due to improved statin compliance and type-2-diabetes mellitus prevention.

Preeclampsia Downregulates MicroRNAs in Fetal Endothelial Cells: Roles of miR-29a/c-3p in Endothelial Function.

Context: Preeclampsia is a leading cause of fetal and maternal morbidity and mortality during pregnancy. Although the etiology of preeclampsia is unknown, preeclampsia offspring have increased risks of developing cardiovascular disorders in adulthood, implicating that preeclampsia programs fetal vasculature in utero. Objective: We hypothesize that preeclampsia alters expression profiles of endothelial microRNAs (miRNAs) in fetal endothelial cells and disturbs the vascular endothelial growth factor A (VEGFA)- and fibroblast growth factor 2 (FGF2)-induced endothelial function. Design and Setting: Unpassaged (P0) human umbilical vein endothelial cells (HUVECs) were isolated immediately after cesarean-section delivery from normotensive (NT) and preeclamptic (PE) pregnancies. Differentially expressed miRNAs between P0-HUVECs from NT and PE pregnancies were identified using a miRNA polymerase chain reaction (PCR) array and confirmed using reverse transcription quantitative PCR. To determine the function of these differentially expressed miRNAs, miRNAs of interest were knocked down in NT-HUVECs following by cell functional assays. Results: Sixteen miRNAs, including miR-29a/c-3p, were downregulated in P0-HUVECs from the PE group compared with the NT group. Bioinformatics analysis predicted the PI3K-v-akt murine thymoma viral oncogene homolog 1 (AKT) signaling pathway was dysregulated in P0-HUVECs from the PE group, which was associated with the miR-29a/c-3p downregulation. We further demonstrated that miR-29a/c-3p knockdown inhibited the VEGFA- and FGF2-induced endothelial migration as well as FGF2-induced AKT1 phosphorylation in HUVECs. However, miR-29a/c-3p knockdown did not alter the extracellular signal-regulated kinase 1/2 phosphorylation, cell proliferation, and endothelial monolayer integrity in response to VEGFA and FGF2 in HUVECs. Conclusions: Preeclampsia-downregulated miR-29a/c-3p may impair fetal endothelial function by disturbing the FGF2-activated PI3K-AKT signaling pathway, hence inhibiting endothelial cell migration.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes.

Cell-cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)-dependent Cx43 S365 phosphorylation-associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)-dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (P<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (P<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP-stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (P<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.