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The epidermal growth factor receptor (EGFR) is expressed robustly in the placenta, and critical processes of pregnancy such as placental growth and trophoblast fusion are dependent on EGFR function. However, the role that aberrant EGFR signaling might play in the etiology and/or maintenance of preeclampsia (PE) remains largely unexplored. Recently, we have shown that overexpression of EGFR in cultured uterine artery endothelial cells (UAEC), which express little endogenous EGFR, remaps responsiveness away from vascular endothelial growth factor receptor (VEGFR) signaling and toward EGFR, suggesting that endothelial EGFR expression may be kept low to preserve VEGFR control of angiogenesis. Here we will consider the evidence for the possibility that the endothelial dysfunction observed in PE might in some cases result from elevation of endothelial EGFR. During pregnancy, trophoblasts are known to synthesize large amounts of EGFR protein, and the placenta regularly releases syncytiotrophoblast-derived exosomes and microparticles into the maternal circulation. Although there are no reports of elevated EGFR gene expression in preeclamptic endothelial cells, the ongoing shedding of placental vesicles into the vascular system raises the possibility that EGFR-rich vesicles might fuse with endothelium, thereby contributing to the symptoms of PE by interrupting angiogenesis and blocking pregnancy-adapted vasodilatory function. [ABSTRACT FROM AUTHOR]

Given prostate cancer is driven, in part, by its responsiveness to androgens, treatments historically employ methods for their removal from circulation. Approaches as crude as castration, and more recently blockade of androgen synthesis or receptor binding, are still of limited use long term, since other steroids of adrenal origin or tumor origin can supersede that role as the ‘castration resistant’ tumor re-emerges. Broader inhibition of steroidogenesis using relatively nonselective P450 inhibitors such as ketoconazole is not an alternative since a general disruption of steroid biosynthesis is neither safe nor effective. The recent emergence of drugs more selectively targeting CYP17 have been more effective, and yet extension of life has been on the scale of months rather than years. It is now becoming clear this shortcoming arises from the adaptive capabilities of many tumors to initiate local steroid synthesis and/or become responsive to novel early pathway adrenal steroids that are synthesized when lyase activity is not selectively blocked, and ACTH rises in the face of declining cortisol feedback. Abiraterone has been described as a lyase selective inhibitor, yet its use still requires co-administration of prednisone to suppress such a rise of ACTH and fall in cortisol. So is creation of a selective lyase inhibitor even possible? Can C19 steroid production be achieved without a prominent decline in cortisol and corresponding rise in ACTH? Decades of scientific study of CYP17 in humans and nonhuman primates, as well as nature’s own experiments of gene mutations in humans, reveal ‘true’ or ‘isolated’ 17,20 lyase deficiency does quite selectively prevent C19 steroid biosynthesis whereas simple 17 hydroxylase deficiency also suppresses cortisol. We propose these known outcomes of natural mutations should be used to guide analysis of clinical trials and long term outcomes of CYP17 targeted drugs. In this review, we use that framework to re-evaluate the basic and clinical outcomes of many compounds being used or in development for treatment of castration resistant prostate cancer. Specifically, we include the nonselective drug ketoconazole, and then the CYP17 targeted drugs abiraterone, orteronel (TAK-700), galaterone (TOK-001), and seviteronel (VT-464). Using this framework, we can fully discriminate the clinical outcomes for ketoconazole, a drug with broad specificity, yet clinically ineffective, from that of abiraterone, the first CYP17 targeted therapy that is limited by its need for prednisone co-therapy. We also can identify potential next generation CYP17 targeted drugs now emerging that show signs of being far more 17,20 lyase selective. We conclude that a future for improved therapy without substantial cortisol decline, thus avoiding prednisone co-administration, seems possible at long last. [ABSTRACT FROM AUTHOR]

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca2+ and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S 1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S 1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation. [ABSTRACT FROM AUTHOR]

During normal pregnancy, elevated angiotensin II (Ang II) concentrations in the maternal and fetal circulations are associated with dramatic increases in placental angiogenesis and blood flow. Much is known about a local renin–angiotensin system within the uteroplacental vasculature. However, the roles of Ang II in regulating fetoplacental vascular functions are less well defined. In the fetal placenta, the overall in vivo vasoconstrictor responses of the blood vessels to Ang II infusion is thought to be less than that in its maternal counterpart, even though infused Ang II induces vasoconstriction. Recent data from our laboratories suggest that Ang II stimulates cell proliferation and increases endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) in ovine fetoplacental artery endothelial cells. These data imply that elevations of the known vasoconstrictor Ang II in the fetal circulation may indeed play a role in the marked increases in fetoplacental angiogenesis and that Ang II-elevated endothelial NO production may partly attenuate Ang II-induced vasoconstriction on vascular smooth muscle. Together with both of these processes, the high levels of Ang II in the fetal circulation may serve to modulate overall fetoplacental vascular resistance. In this article, we review currently available data on the expression of Ang II receptors in the ovine fetal placenta with particular emphasis on the effects of Ang II on ovine fetoplacental endothelium. The potential cellular mechanisms underlying the regulation of Ang II on endothelial growth and vasodilator production are discussed. [ABSTRACT FROM AUTHOR]

The marmoset adrenal is interesting as it is developmentally similar to humans and other primates, but in adulthood 17,20-lyase activity is very low. One possible explanation is an altered regulation of P450c17 expression by AT1-R. We investigated the expression and zonal distribution of the AT1-R in marmoset adrenal glands and adrenocortical cells in culture since it is known that AT1-R is a regulator of P450c17 expression and activity. AT1-R was expressed strongly in the ZG and to diminishing degrees through the remainder of the cortex. There was negative expression in a putative ZI. Dispersed adrenocortical cells retained AT1-R protein as detected by ICC. Cells cultured for several days and treated in serum-free media for 48 h maintained AT1-R expression, as measured by western. However, at that 48 h time point, treatment with Forsk, AII, TPA, or the combination of A + F or T + F did not appear to effect AT1-R expression. We conclude that marmoset adrenals express AT1-R similarly to humans and other higher mammals, adrenocortical cells retain AT1-R expression in culture, and consistent with other adrenal cell culture models, AT1-R expression is not lost in response to agonists long term. [ABSTRACT FROM AUTHOR]

Territorial aggression is influenced by many social and environmental factors. Since aggression is a costly behavior, individuals should account for multiple factors such as population density or reproductive status before engaging in aggression. Previous work has shown that male California mice (Peromyscus californicus) respond to winning aggressive encounters by initiating aggression more quickly in future encounters, and we investigated the physiological basis for this effect. We found that injections that produced a transient increase in testosterone (T) following an aggressive encounter caused males to behave more aggressively in an encounter the following day. Experience alone was not enough to change aggression, as males treated with saline injections showed no change in aggression. The effect of T injections on aggression was androgen-based, as the inhibition of aromatase did not block the T injections from increasing aggression. Aromatase inhibition did, however, increase aggression in the initial aggression tests (before application of T or saline injections), and aromatase activity in the bed nucleus of the stria terminalis (BNST) was negatively correlated with aggression. A previous study suggested that aromatase activity in the BNST decreases after males become fathers. Thus, distinct neuroendocrine mechanisms allow male California mice to adjust aggressive behavior in response to changes in social and reproductive status. [Copyright &y& Elsevier]

The effects of aromatase within the brain on sexual behavior have been studied in a wide variety of species. Relatively few non-mating behaviors have been considered, despite evidence that estrogen affects many social behaviors. Testosterone promotes paternal behavior in California mouse (Peromyscus californicus) fathers, acting primarily via aromatization to estradiol. Virgin male California mice rarely exhibit paternal behavior, so we investigated whether aromatase in the brain changed with the onset of paternal behavior in California mouse fathers. In the medial preoptic area (MPOA), a brain area known to regulate parental behavior in rodents, we found that fathers had significantly more aromatase activity than mated males without pups, suggesting that an increase in estrogen production in this brain area contributes to the onset of paternal behavior. We also found that progesterone (P[sub 4] ) levels were lower in fathers compared to sexually inexperienced males and that P[sub 4] was negatively correlated with aromatase activity in the MPOA. These P[sub 4] findings agree with a recent study that found an inhibitory effect of P[sub 4] on paternal behavior. Overall, we found that aromatase activity and P[sub 4] levels change in association with an important life history transition, and may provide a mechanistic basis for plasticity in paternal behavior.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI[sub 2]) may be as much a consequence of cellular reprogramruing at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI[sub 2] biosynthetic pathway (cPLA[sub 2], COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that chronic high-altitude hypoxia during pregnancy is associated with the upregulation of endothelial nitric oxide synthase (eNOS) gene expression and messenger ribonucleic acid (mRNA) in ovine uterine arteries. Increase of eNOS and mRNA levels; Effect of chronic hypoxia on basal endothelial activity; Modulating effect of endothelial NO in vascular activity.

Deals with a study which tested the hypothesis that chronic high-altitude hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide. Methods; Results; Discussion.

Examines the principles and clinical applications of high performance liquid chromatography (HPLC). Monitoring of disease through the study of molecules; Separation of molecules through HPLC; Adaptability of the method for investigating a range of molecules.

Two monoclonal antibodies, T1/7 and T1/61, have been prepared which are specific for chicken fast muscle troponin T. Both are of the IgGλ1 subclass. Both antibodies cross-react strongly with human fast and chicken cardiac troponin T, but while T1/7 reacts weakly with rabbit fast troponin T, T1/61 does not. The antibodies can be used for fibre typing of both chicken and human muscle. The antibodies have been used to identify fast troponin T on two-dimensional maps of proteins from a variety of chicken muscles by electrophoretic transfer to nitrocellulose followed by immunoperoxidase staining. Using this technique live variant forms of fast troponin T, have been identified. Two variants, fBT1 and fBT2, are expressed in breast muscle, while the other three, fLT1, fLT2 and fLT3 are expressed in leg muscle. Of the leg muscle variants, fLT1 and fLT2 correspond to the two forms described previously [Wilkinson, J. M., Moir, A. J. G. and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47 - 56]. The third variant, fLT3, has not been described before and is expressed in muscles which have a high content of slow fibres. In addition to these clearly defined variant forms immunostaining reveals multiple minor variants of troponin T present in leg muscle which may reflect complex RNA processing of the troponin T gene transcript. [ABSTRACT FROM AUTHOR]

The human adrenal cortex comprises three distinct zones with unique steroid products, namely the zona glomerulosa, which secretes the mineralocorticoids, the zona fasciculate, which secretes the glucocorticoids and the zona reticularis (ZR), which at adrenarche, begins to produce the so-called adrenal androgens. Of all the adrenal zones, we still understand control of ZR emergence the least, and yet the consequences of such dysregulation can be devastating. Premature adrenarche is a growing problem and the correspondingly inappropriate emergence of ZR function can negatively influence puberty and lead to adult infertility. Our understanding is limited and more needs to be done. The purpose of these three reviews is to provide a survey of where we are in our current understanding of what adrenarche is, and indeed if it is unique to humans at all. Furthermore, these reviews describe what is also known of how the functional ZR emerges during adrenarche and what steroids of physiologic relevance result beyond the widely known DHEA and DHEAS elevated at this time. Such advances in human, primate and indeed stem-cell biology are clearly laying the foundation for new directions in the hunt for the factors involved in the regulation and functional emergence of a ZR at the appropriate time, as well as insight into how they may fail. Given support for these new directions, considerable progress can clearly be made. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology. [ABSTRACT FROM AUTHOR]

Discusses topics and issues about receptors and signal transduction. Insights into the role of exercise in bypassing the morbid effects of MC4R knockout in mice models; Elucidation on the use of modern protein and cloning technology to address the issue of the function of the now cloned MC2R receptor; Discussion on issues of fatty acid metabolites in the control of steroidogenesis.

As in women with polycystic ovary syndrome (PCOS), hyperinsulinemia is associated with anovulation in PCOS-like female rhesus monkeys. Insulin sensitizers ameliorate hyperinsulinemia and stimulate ovulatory menstrual cycles in PCOS-like monkeys. To determine whether hyperinsulinemia (>694 pmol/L), alone, induces PCOS-like traits, five PCOS-like female rhesus monkeys with minimal PCOS-like traits, and four control females of similar mid-to-late reproductive years and body mass index, received daily subcutaneous injections of recombinant human insulin or diluent for 6–7 months. A cross-over experimental design enabled use of the same monkeys in each treatment phase. Insulin treatment unexpectedly normalized follicular phase duration in PCOS-like, but not control, females. In response to an intramuscular injection of 200 IU hCG, neither prenatally androgenized nor control females demonstrated ovarian hyperandrogenic responses while receiving insulin. An intravenous GnRH (100 ng/kg) injection also did not reveal evidence of hypergonadotropism. Taken together, these results suggest that experimentally induced adult hyperinsulinemia, alone, is insufficient to induce PCOS-like traits in female rhesus monkeys and to amplify intrinsic PCOS-like pathophysiology. [ABSTRACT FROM AUTHOR]

Abstract: Purpose of the study: To determine whether pioglitazone will improve menstrual cyclicity in a fetal programming model for polycystic ovary syndrome. Basic procedures: Eight prenatally androgenized (PA) and 5 control female rhesus monkeys of similar age, body weight and body mass index received an oral placebo daily for 6–7 months followed, after at least 90 days, by daily oral dosing with pioglitazone (3mg/kg) for an additional 6–7 months. Blood was sampled thrice weekly to monitor ovulatory function, and a variety of endocrine challenges were performed to quantify changes in ovarian, gonadotropin and glucoregulatory function. Most important findings: Pioglitazone normalized menstrual cycles in 5 out of 8 (62%) PA females (pioglitazone responsive; PioRESP). Pioglitazone increased serum 17α-hydroxyprogesterone responses to an hCG injection in PioRESP PA females, while diminishing serum progesterone, and increasing DHEA and estradiol responses to hCG in PioRESP PA and all normal females. Principal conclusions: Insulin resistance plays a mechanistic role in maintaining anovulation in a majority of PA female monkeys. [Copyright &y& Elsevier]

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca2+ sensitive and be subject to regulation by Ca2+-independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca2+ concentration ([Ca2+]i) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca2+ pump inhibitor) were used to maximally elevate [Ca2+]i and fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 μM) and TG (10 μM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 μM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca2+]i response but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca2+]i-insensitive pathways as well as through a greater eNOS sensitivity to [Ca2+]i. [ABSTRACT FROM AUTHOR]

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm 2 , then were exposed to 10, 15, or 25 dynes/cm 2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p de novo ; i.e., inhibited by L-NMMA (N G -monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm 2 (8-fold) exceeded (p [ABSTRACT FROM AUTHOR]

Marmosets express a fetal zone during adrenal development but fail to produce significant amounts of C19 steroids in adulthood. It is not clear however if P450c17 regulation is different from that in humans/rhesus or the primary sequence is altered. To this end we isolated marmoset and rhesus adrenocortical cells, and treated the cells with known regulators of P450c17 expression for 48 h. P450c17 protein increased with Forskolin(F) treatment, but was marginally inhibited by AII(A) and TPA(T) alone. Combined A + F and T + F dramatically ablated the F response. Cortisol levels(EIA) increased upon F treatment and were inhibited by A and T. Combination of treatments partially inhibited the F-induced response. The protein-coding region of marmoset and rhesus P450c17 cDNAs were then isolated from adrenals using RT-PCR/TA cloning. Marmoset P450c17 shows one amino acid deletion but otherwise shares 90.6% and 91.4% homologies with the human and rhesus cDNA sequences, and 82.4% and 85% homologies with the human and rhesus predicted AA sequences, respectively. Since marmoset adrenocortical cells exhibit similar endocrine function to rhesus, impaired 17,20-lyase activity in the adult marmoset adrenal may in part be due to differences in the primary sequence. [ABSTRACT FROM AUTHOR]

: ObjectiveThird-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis.: MethodsPlacentas from four normal pregnancies and four pregnancies complicated by preeclampsia were studied. In a preliminary effort to identify possible differences between the two groups, complementary DNA (cDNA) probes were prepared from pooled total RNA by reverse transcription in the presence of 33P-dCTP. After hybridization to human GeneFilter cDNA microarrays (GF211; Research Genetics, Huntsville, AL), 319 positive signals were detected above background out of a possible 4131 human cDNAs spotted on the filters.: ResultsTen most highly expressed mRNA species, ten most up-regulated, and ten most down-regulated genes in placentas from both groups of patients were identified for future studies. Of the 319 positive hybridizations, one transcript was clearly elevated in preeclamptic pregnancy. This cDNA encodes the muscle subtype of glycogen phosphorylase (GP-M) and was increased more than 2.8-fold (P < .05) in the preeclamptic placentas. In contrast, cDNA for glycogen synthase (muscle and liver isoforms) was not significantly increased, being near the limits of detection. The preeclampsia-induced increase of placental GP-M mRNA expression (approximately 3.5-fold) was confirmed by northern blot analysis.: ConclusionWe conclude that microarray analysis can detect trends in mRNA and gene expression in placentas from normal and preeclamptic pregnancies that may be further studied in a more targeted fashion. We found that placental GP-M mRNA level is up-regulated in preeclampsia, which is consistent with previous reports of increased glycogen phosphorylase activity, and we suggest that it may be largely regulated at the level of transcription. Further studies may determine whether such up-regulation might be a response to hypoxia. [Copyright &y& Elsevier]

Millions of individuals who have recovered from SARS-CoV-2 infection may be eligible to participate in convalescent plasma donor programs, yet the optimal window for donating high neutralizing titer convalescent plasma for COVID-19 immunotherapy remains unknown. Here we studied the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers (VN) in 175 convalescent donors longitudinally sampled for up to 142 days post onset of symptoms (DPO). We observed robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 that persist, in the aggregate, for at least 100 DPO. However, there is a notable decline in VN titers ≥160 for convalescent plasma therapy, starting 60 DPO. The results also show that individuals 30 years of age or younger have significantly lower VN, IgG and IgM antibody titers than those in the older age groups; and individuals with greater disease severity also have significantly higher IgM and IgG antibody titers. Taken together, these findings define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor. Gontu et al. conducted a longitudinal study in COVID patients in which they assessed the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers. Their measurements demonstrate the presence of an optimum window for convalescent plasma donation as well as predictions of the most suitable donor type. [ABSTRACT FROM AUTHOR]

To assess the feasibility of ferumoxytol-enhanced MRI in pregnancy with a nonhuman primate model. In this prospective study, eleven pregnant rhesus macaques at day 98 ± 5 of gestation were divided into three groups, untreated control (UC) (n = 3), saline control (SC) (n = 4) and interleukin 1 beta (IL-1β) treated (IT) (n = 4), which were administered with either saline or IL-1β into the amniotic fluid. All animals were imaged at multiple time points before and after ferumoxytol administration (4 mg/kg). Longitudinal R2* and susceptibility of tissues were obtained using region-of-interest analysis and the longitudinal changes were assessed using linear mixed models and Student's t -test. In fetuses, a slope of 0.3 s−1/day (P = 0.008), 0.00 ppm/day (P = 0.699) and − 0.2 s−1/day (P = 0.023) was observed in liver R2*, liver susceptibility, and lung R2*, respectively. In placentas, R2* and susceptibility increased immediately after ferumoxytol administration (P < 0.001) and decreased to baseline within two days. The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points. In maternal livers, R2* increased immediately after ferumoxytol administration, further increased at one-day, and then decreased but remained elevated (P < 0.001). The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points. This work demonstrates the feasibility of quantitative ferumoxytol-enhanced MRI to measure dynamics of ferumoxytol delivery and washout in the placenta. Stable MRI measurements indicated no evidence of iron deposition in fetal tissues of nonhuman primates after maternal ferumoxytol exposure. [ABSTRACT FROM AUTHOR]

Ultraviolet germicidal irradiation (UVGI) is a highly effective means of inactivating many bacteria, viruses, and fungi. UVGI is an attractive viral mitigation strategy against coronaviruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-2019 (COVID-19) pandemic. This investigation measures the susceptibility of two human coronaviruses to inactivation by 254 nm UV-C radiation. Human coronavirus NL63 and SARS-CoV-2 were irradiated in a collimated, dual-beam, aqueous UV reactor. By measuring fluence and integrating it in real-time, this reactor accounts for the lamp output transients during UVGI exposures. The inactivation rate constants of a one-stage exponential decay model were determined to be 2.050 cm2/mJ and 2.098 cm2/mJ for the NL63 and SARS-CoV-2 viruses, respectively. The inactivation rate constant for SARS-CoV-2 is within 2% of that of NL63, indicating that in identical inactivation environments, very similar UV 254 nm deactivation susceptibilities for these two coronaviruses would be achieved. Given the inactivation rate constant obtained in this study, doses of 1.1 mJ/cm2, 2.2 mJ/cm2, and 3.3 mJ/cm2 would result in a 90%, 99%, and 99.9% inactivation of the SARS-CoV-2 virus, respectively. The inactivation rate constant obtained in this study is significantly higher than values reported from many 254 nm studies, which suggests greater UV susceptibility to the UV-C than what was believed. Overall, results from this study indicate that 254 nm UV-C is effective for inactivation of human coronaviruses, including SARS-CoV-2. • 254 nm UV-C effectively inactivates human coronaviruses, including SARS-CoV-2. • Inactivation rate constants for SARS-CoV-2 and NL63 are identical. • NL63 can be used as a suitable surrogate for SARS-CoV-2 for inactivation studies. • 254 nm UV-C could be effective against a broad range of coronaviruses. [ABSTRACT FROM AUTHOR]

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre‐eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2‐ and VEGFA‐induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O2) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad‐GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2‐stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA‐stimulated cell migration, VEGFA‐weakened cell monolayer integrity, and intracellular Ca++ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2‐ or VEGFA‐induced phosphorylation of ERK1/2. However, GNA14 overexpression time‐dependently elevates (p < 0.05) phosphorylation of phospholipase C‐β3 (PLCβ3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCβ3 under physiological chronic low oxygen. Overexpression of G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily has been implicated to play a role in several hypertensive disorders. However, little is known about roles of GNA14 in mediating placental endothelial function. Herein, we show GNA14 overexpression dysregulate FGF2‐stimulated placental endothelial function, possibly in part by altering activation of PLCβ3. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Pregnancy is associated with enhanced Ca2+ signaling in UA endothelial cells, mediated by enhanced Cx43 connectivity. • We now show TNF-alpha inhibits Cx43 function and so periodic Ca2+ bursts in pregnancy-derived UA endothelial cells. • TNF acts via Src to inhibit Cx43 by causing phosphorylation directly at positions 265 and indirectly at position 279/282. • TNF action is reversed by the Src inhibitor PP2 or the nutraceutical Src antagonist t10,c12 CLA or MEK/ERK inhibitor U0126. • In umbilical vessels from preeclamptic subjects, Src inhibitors promotes rescue of otherwise impaired Ca2+ and NO responses. [ABSTRACT FROM AUTHOR]

Pregnancy-derived uterine artery endothelial cells (P-UAEC) express P2Y2 receptors and at high cell density show sustained and synchronous [Ca2+]i burst responses in response to ATP. Bursts in turn require coupling of transient receptor potential canonical type3 channel (TRPC3) and inositol 1,4,5-triphosphate receptor type 2 (IP3R2), which is upregulated in P-UAEC in a manner dependent on connexin 43 (Cx43) gap junctions. While there is no known direct interaction of TRPC3 with Cx43, early descriptions of TRPC3 function showed it may also be influenced by altered membrane potential (Vm). Herein, we ask if enhanced TRPC3 Ca2+ bursting due to enhanced Cx43 coupling may be coupled via dynamic alterations in Vm in P-UAEC, as reported in some (HUVEC) but not all endothelial cells. Following basic electrical characterization of UAEC, we employed a high sensitivity cell imaging system to simultaneously monitor cell Vm and [Ca2+]i in real time in continuous monolayers of UAEC. Our findings show that while acute and sustained phase [Ca2+]i bursting occur dose-dependently in response to ATP, Vm is not coregulated with any periodicity related to [Ca2+]i bursting. Only a small but significant progressive change in Vm is seen, and this is more closely related to overall mobilization of Ca2+. Surprisingly, this is also most apparent in NP-UAEC > P-UAEC. In contrast [Ca2+]i bursting is more synchronous in P-UAEC and even achieves [Ca2+]i waves passing through the P-UAEC monolayer. The relevance of these findings to mechanisms of pregnancy adaptation and its failure in hypertensive pregnancy are discussed. [ABSTRACT FROM AUTHOR]

Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G-protein coupled receptor. Our localization of OXTR to the retinal pigment epithelium (RPE), in close proximity to OXT in the adjacent photoreceptor neurons, leads us to propose that OXT plays an important role in RPE-retinal communication. An increase of RPE [Ca 2 + ] i in response to OXT stimulation implies that the RPE may utilize oxytocinergic signaling as a mechanism by which it accomplishes some of its many roles. In this study, we used an established human RPE cell line, a HEK293 heterologous OXTR expression system, and pharmacological inhibitors of Ca 2 + signaling to demonstrate that OXTR utilizes capacitative Ca 2 + entry (CCE) mechanisms to sustain an increase in cytoplasmic Ca 2 + . These findings demonstrate how multiple functional outcomes of OXT-OXTR signaling could be integrated via a single pathway. In addition, the activated OXTR was able to inhibit the Kir7.1 channel, an important mediator of sub retinal waste transport and K + homeostasis. [ABSTRACT FROM AUTHOR]

Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild-type (WT) C57Bl/6 mice were cross-bred: female hAGT X male hREN for preeclampsia (PRE) model and female WT X male WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1, and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; P = 0.02), and placental necrosis (60% vs. 0%; P < 0.01). PRE group demonstrated declining BPs with advancing gestation. Plasma sFlt1 increased across pregnancy in PRE; VEGF did not vary. IHC demonstrated the presence of sFlt1 in glomeruli, lymphatics, and collecting tubules of PRE kidneys, suggesting excretion. VEGF immunostaining was increased specifically in the glomeruli of PRE kidneys. Placenta in PRE showed marked immunostaining for sFlt1. We conclude that this transgenic model of preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to acetylcholine. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling, and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF. [ABSTRACT FROM AUTHOR]

Long-chain polyunsaturated fatty acids like conjugated linoleic acids (CLA) are required for normal neural development and cognitive function and have been ascribed various beneficial functions. Recently, oral CLA also has been shown to increase testosterone (T) biosynthesis, which is known to diminish traumatic brain injury (TBI)-induced neuropathology and reduce deficits induced by stroke in adult rats. To test the impact of CLA on cognitive recovery following a TBI, 5–6 month old male Sprague Dawley rats received a focal injury (craniectomy + controlled cortical impact (CCI; n = 17)) or Sham injury (craniectomy alone; n = 12) and were injected with 25 mg/kg body weight of Clarinol® G-80 (80% CLA in safflower oil; n = 16) or saline (n = 13) every 48 h for 4 weeks. Sham surgery decreased baseline plasma progesterone (P4) by 64.2% (from 9.5 ± 3.4 ng/mL to 3.4 ± 0.5 ng/mL; p = 0.068), T by 74.6% (from 5.9 ± 1.2 ng/mL to 1.5 ± 0.3 ng/mL; p < 0.05), 11-deoxycorticosterone (11-DOC) by 37.5% (from 289.3 ± 42.0 ng/mL to 180.7 ± 3.3 ng/mL), and corticosterone by 50.8% (from 195.1 ± 22.4 ng/mL to 95.9 ± 2.2 ng/mL), by post-surgery day 1. CCI injury induced similar declines in P4, T, 11-DOC and corticosterone (58.9%, 74.6%, 39.4% and 24.6%, respectively) by post-surgery day 1. These results suggest that both Sham surgery and CCI injury induce hypogonadism and hypoadrenalism in adult male rats. CLA treatment did not reverse hypogonadism in Sham (P4: 2.5 ± 1.0 ng/mL; T: 0.9 ± 0.2 ng/mL) or CCI-injured (P4: 2.2 ± 0.9 ng/mL; T: 1.0 ± 0.2 ng/mL, p > 0.05) animals by post-injury day 29, but rapidly reversed by post-injury day 1 the hypoadrenalism in Sham (11-DOC: 372.6 ± 36.6 ng/mL; corticosterone: 202.6 ± 15.6 ng/mL) and CCI-injured (11-DOC: 384.2 ± 101.3 ng/mL; corticosterone: 234.6 ± 43.8 ng/mL) animals. In Sham surgery animals, CLA did not alter body weight, but did markedly increase latency to find the hidden Morris Water Maze platform (40.3 ± 13.0 s) compared to saline treated Sham animals (8.8 ± 1.7 s). In CCI injured animals, CLA did not alter CCI-induced body weight loss, CCI-induced cystic infarct size, or deficits in rotarod performance. However, like Sham animals, CLA injections exacerbated the latency of CCI-injured rats to find the hidden MWM platform (66.8 ± 10.6 s) compared to CCI-injured rats treated with saline (30.7 ± 5.5 s, p < 0.05). These results indicate that chronic treatment of CLA at a dose of 25 mg/kg body weight in adult male rats over 1-month 1) does not reverse craniectomy- and craniectomy + CCI-induced hypogonadism, but does reverse craniectomy- and craniectomy + CCI-induced hypoadrenalism, 2) is detrimental to medium- and long-term spatial learning and memory in craniectomized uninjured rats, 3) limits cognitive recovery following a moderate-severe CCI injury, and 4) does not alter body weight. [ABSTRACT FROM AUTHOR]

The role increased vascular endothelial growth factor (VEGF) plays in vascular function during normal vs. preeclamptic pregnancy has been a source of some controversy of late. In this study, we seek to understand how VEGF165 influences vasodilator production via Ca2+ signaling mechanisms in human endothelial cells. We utilize human umbilical vein endothelial cells (HUVEC) as well as intact ex vivo human umbilical vein (HUV Endo) to address direct stimulation of Ca2+ and NO by VEGF165 alone, as well as the effect of VEGF165 on subsequent ATP-stimulated Ca2+ signaling and NO production. We show that VEGF165 stimulates Ca2+ responses in both HUVEC and HUV Endo, which results in a corresponding increase in NO production in HUV Endo. Longerterm VEGF165 pretreatment then inhibits sustained Ca2+ burst responses to ATP in HUVEC and HUV Endo. This is paralleled by a corresponding drop in ATP-stimulated NO production in HUV Endo, likely through inhibition of Cx43 gap-junction function. Thus, although VEGF165 makes a small initial positive impact on vasodilator production via direct stimulation of Ca2+ responses, this is outweighed by the greater subsequent negative impact on Ca2+ bursts and vasodilator production promoted by more potent agonists such as ATP. Overall, elevated levels of VEGF165 associated with preeclampsia could contribute to the endothelial dysfunction by preventing Ca2+ bursts to other agonists including but not limited to ATP. [ABSTRACT FROM AUTHOR]

Normal pregnancy requires increased uterine endothelial cell driven vasodilation that is related to increases in sustained Ca2+ signaling via increased connexin 43 (Cx43) gap junction function. Preeclampsia, a hypertensive disorder of pregnancy associated with endothelial dysfunction, is also linked with down regulation of Ca2+ driven vasodilator production and increased levels of vascular endothelial growth factor (VEGF). Cx43 function can be acutely down-regulated by phosphorylation of multiple inhibitory residues and VEGF is known to promote phosphorylation of Cx43. Herein, we show that VEGF-165 promotes Cx43 phosphorylation at Ser-279/282 and Tyr-265 residues and blocks pregnancy-adapted Ca2+ signaling in ovine uterine artery endothelial cells (UAEC). Pharmacological Src and ERK kinase pathway inhibitors (PP2 and U0126) reverse these phosphorylations and rescue Ca2+ signaling. We also report a nutraceutical Src inhibitor, t10,c12 conjugated linoleic acid (10,12 CLA), rescues Ca2+ signaling in UAEC and therefore may have therapeutic potential for preeclampsia. [ABSTRACT FROM AUTHOR]

In pregnancy, the uterine vasculature undergoes dramatic vasodilatory adaptations. Previously, vascular endothelial growth factor (VEGF) has been shown to stimulate endothelial nitric oxide synthase (eNOS) in uterine artery endothelial cells (UAECs) derived from pregnant ewes to a greater extent than those from non-pregnant ewes in a manner not fully explained by changes in the phosphorylation of eNOS. In this study, we used Fura-2 Ca2+ imaging and arginine-to-citrulline conversion eNOS activity assays to assess the importance of VEGF-stimulated Ca2+ responses in pregnancy-related changes in NO production in UAEC. In this study, we show that pregnancy-induced changes in VEGF-stimulated Ca2+ responses could account in part for the greater capacity of VEGF to stimulate eNOS in UAECs from pregnant versus non-pregnant animals. VEGF-stimulated Ca2+ responses in UAECs from pregnant and non-pregnant animals were mediated through VEGF receptor 2 and were detected in roughly 15% of all cells. There were no pregnancyspecific differences in area under the curve or peak height. UAECs from pregnant animals were more consistent in the time to response initiation, had a larger component of extracellular Ca2+ entry, and were more sensitive to a submaximal dose of VEGF. In UAECs from pregnant and non-pregnant animals Ca2+ responses and eNOS activation were sensitive to the phospholipase C/inositol 1,4,5-trisphosphate pathway inhibitors 2-aminoethoxydiphenylborane and U73122. Thus, changes in VEGF-stimulated [Ca2 + ]i are necessary for eNOS activation in UAECs, and pregnancy-induced changes in Ca2+ responses could also in part explain the pregnancy-specific adaptive increase in eNOS activity in UAECs. [ABSTRACT FROM AUTHOR]

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca2+ concentration ([Ca2+]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca2+]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca2+]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca2+]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible. [ABSTRACT FROM AUTHOR]

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca2+]i) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca2+]i bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca2+]i cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF165. While treatment of luteal NP-UA Endo and P-UA Endo with VEGF165 acutely stimulates a very modest [Ca2+]i and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca2+]i and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia. [ABSTRACT FROM AUTHOR]

Uterine artery endothelium undergoes a form of functional adaptation during pregnancy because of an increase in Cx43 communication, resulting in increased Ca2+/IP3 exchange and more synchronous and sustained vasodilator production. We have shown previously that acute exposure to growth factors and TNF can block this adaptation through ERK and/or Src-mediated Cx43 phosphorylation. In preeclampsia such adapted function is already missing, but while elevated TNF is associated with this condition, particularly after 28 weeks (late PE), elevated circulating VEGF165 is not. Given PE is a long term condition emerging in the second half of pregnancy, and is often associated with added edema, we now compare the chronic effects of these two factors on the cell monolayer in order to establish if the breakdown of junctional adherens and tight junctional assemblies in which Cx43 resides could also explain loss of vasodilatory function. We report that while TNF can degrade monolayer integrity even in the 0.1–1 ng/ml physiologic range, VEGF up to 10 ng/ml does not. In addition, the progressive action of TNF is mediated through Src and ERK signaling to promote internalization and destruction of VE-Cadherin (VE-Cad) and ZO-1, as well as the expression and secretion of a variety of proteases. At least one protein degraded from the extracellular space is VE-Cad, resulting in release of a shed VE-Cad protein product, and consistent with monolayer breakdown being sensitive to both Src and MEK/ERK kinase inhibitors and the general protease inhibitor GM6001. We conclude that the greater association of TNF with 'late' PE is as much due to its longer term destabilizing effects on junctional assemblies as it is to acute closure of Cx43 channels themselves. New therapies aimed at stabilizing these junctional assemblies may help treat this hypertensive condition. • TNF but not VEGF damages junctional assemblies at physiologic levels in uterine artery endothelial cells. • TNF acts through multiple mechanisms inside and outside the cell to cause this damage. • Our data supports the argument cytokines are more damaging than growth factors in loss of vasodilation in the preeclampsia. [ABSTRACT FROM AUTHOR]

Abstract: Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p <0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p <0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive. [Copyright &y& Elsevier]

Basal plasma ACTH1–39 concentrations are elevated in long-term hypoxic (LTH) fetal sheep. This study was designed to determine whether the expression of genes regulating cortisol biosynthesis was altered after LTH. Pregnant ewes were maintained at high altitude (3,820 m) from day 30 of gestation to near term, when the animals were transported to the laboratory. Reduced Po2 was maintained by nitrogen infusion through a maternal tracheal catheter. On days 137–141, fetal adrenal glands were collected from LTH and normoxic control fetuses. Real-time PCR was used to quantify mRNA for steroidogenic acute regulatory protein, 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), cholesterol side-chain cleavage (CYP 11A1), 3β-hydroxysteroid dehydrogenase type II (HSD3B2), and the ACTH receptor. We analyzed mRNA by slot-blot hybridization and also quantified mRNA for transcription factors necessary for adrenocortical development by quantitative real-time PCR: steroidogenic factor 1 and dosage-sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome (DAX-1). Protein was quantified by Western blot analysis. Adrenal mRNAs for CYP17, CYP11A1, and the ACTH receptor were significantly reduced in LTH fetal sheep compared with levels shown in controls. Similarly, CYP11A1 protein and CYP17 protein were reduced in the LTH group. CYP21, steroidogenic acute regulatory protein, HSD3B2, steroidogenic factor 1, and DAX-1 expressions were not altered in response to LTH. We conclude that expression of two key steroidogenic enzymes (CYP17, CYP11A1) regulating cortisol biosynthesis and the ACTH receptor is lower in response to LTH. This likely represents an adaptive response to LTH, to prevent excessive cortisol production that would restrict fetal growth and potentially induce preterm delivery. [ABSTRACT FROM AUTHOR]

While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca2+/calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca2+]i). The sheep, however, is widely used as a model for cardiovascular adaptation to pregnancy and ovine uterine artery endothelial cell (UAEC) eNOS undergoes pregnancy-specific (P) enhancement of activity associated with increased Ca2+ and protein kinase signaling in response to a number of agonists, including adenosine triphosphate (ATP). The degree of homology between the ovine and human full-length cDNAs was not previously known and yet is necessary to determine the validity in using an ovine model to study human physiology. The objectives of this study were to isolate and validate the clone of ovine eNOS cDNA and investigate ovine eNOS activation when expressed in COS-7 cells. The ovine eNOS cDNA has high homology to published human and bovine sequences and shares identity with the bovine amino acid sequence. When ovine eNOS was transiently expressed in COS-7 cells (COS-7/oeNOS), A23187 increased specific catalytic activity in a dose- and time-dependent manner. A23187-stimulated activation of eNOS was, however, also accompanied by phosphorylation of eNOS S1179 and dephosphorylation of T497, demonstrating that an increase in [Ca2+]i may not be the sole mechanism of activation. The physiologic relevance of this was further underscored by the finding that ATP dose-dependently increased peak [Ca2+]i and eNOS activity in COS-7/oeNOS, but also increased eNOS p-S1179 and decreased p-T497. This finding was similar to those in ovine P-UAEC treated with the Ca2+-mobilizing agonist ATP, wherein activation of eNOS was again concomitant with a rise p-S1179 as well as a slight decrease in p-T497. In conclusion, we describe the full-length ovine eNOS cDNA sequence and show that both physiologic and nonphysiologic calcium-mobilizing agents, which activate ovine eNOS in COS-7 and P-UAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca2+ elevation and specific phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction. [Copyright &y& Elsevier]

Although pregnancy is clearly associated with refractoriness to infused angiotensin II (AII) in the uteroplacental unit, there is still dispute over the mechanism by which angiotensin type 1 and type 2 receptors (AT 1 R and AT 2 R) may mediate this response in the uterine artery. This is in large part due to incomplete knowledge of levels of AT 1 R and AT 2 R expression and function in uterine artery endothelium (UA Endo) in the nonpregnant (NP) and pregnant (P) states, combined with the disagreement on whether AII may act through release of adrenomedullary catecholamines. The authors have previously described an increase in AT 1 R in UA Endo but not UA vascular smooth muscle (VSM) during pregnancy as compared to the nonpregnant intact ewe. Herein they report that the pregnancy-associated increase in AT 1 R expression in UA Endo is regulated by ovarian steroids. Using a recently developed antibody to AT 2 R, the authors now show there is no change in AT 2 R in UA Endo or VSM associated with ovarian function, and although AT 2 R is not changed in UA Endo by pregnancy, there is a significant decrease observed in UA VSM at that time. The authors also examined changes in receptors in UA Endo and VSM in estrogen (E2ß)-primed ewes in view of the common use of this model as a control for physiologic studies. In contrast to their findings in nonprimed nonpregnant or pregnant animals, the authors observed a significant increase in both AT 1 R and AT 2 R in UA Endo in response to the supraphysiologic priming with E2ß. In order to address the possible functionality of AT 1 R or AT 2 R in UA Endo, the authors used the uterine artery endothelial cell (UAEC) model of UA endothelial cells maintained in culture to passage 4. Differences in expression of AT 1 R or AT 2 R were normalized at passage 4 in P-UAECs and NP-UAECs. Treatment with AII activated phospholipase C (PLC) in both NP- and P-UAECs but signaling through the extracellular signal–regulated kinase (ERK) pathway was dramatically enhanced in P-UAECs compared to NP-UAECs. Surprisingly, both phosphoinositol turnover and ERK2 phosphorylation responses failed to display the expected dose-responses. Inhibition of AII-stimulated ERK2 phosphorylation with antagonists DUP 753 (AT 1 R, 10 µM) and PD 123319 (AT 2 R, 10 µM) failed to selectively inhibit ERK2 phosphorylation. The authors conclude that (a) the net effect of pregnancy may be an increase in the AT 1 R/AT 2 R ratio in both UA Endo and VSM but through apparently distinct mechanisms, (b) the ovariectomized animal model is similar to the luteal state for AT 1 R and AT 2 R expression, while the E2ß-primed model does not resemble the nonpregnant or pregnant state, and (c) there is a real possibility that AII may mediate its effects either through a complex AT 1 R-AT 2 R interaction or via an as-yet unidentified non-AT 1 , non-AT 2 receptor. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that prolonged treatment with progesterone would have little or no effect on endothelial nitric oxide synthase (eNOS) protein expression in the uterine or systemic artery endothelium. Protein expression for eNOS in endothelial-isolated proteins and vascular smooth muscle of uterine and systemic arteries.

Tests the hypothesis that pregnancy and the follicular phase are associated with increases in endothelial nitric oxide synthase (eNOS) mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide levels. Effect of ovariectomy, ovarian cycle, and pregnancy on eNOS expression.

Sustained Ca2+ burst signaling is crucial for endothelial vasodilator production and is disrupted by growth factors and cytokines. Conjugated linoleic acid (CLA), a Src inhibitor in certain preparations, is generally regarded as safe during pregnancy by the FDA. Multiple CLA preparations; t10, c12 or c9, t11 CLA, or a 1:1 mixture of the two were administered before growth factor or cytokine treatment. Growth factors and cytokines caused a significant decrease in Ca2+ burst numbers in response to ATP stimulation. Both t10, c12 CLA and the 1:1 mixture rescued VEGF 165 or TNFα inhibited Ca2+ bursts and correlated with Src-specific phosphorylation of connexin 43. VEGF 165 , TNFα, and IL-6 in combination at physiologic concentrations revealed IL-6 amplified the inhibitory effects of lower dose of VEGF 165 and TNFα. Again, the 1:1 CLA mixture was most effective at rescue of function. Therefore, CLA formulations may be a promising treatment for endothelial dysfunction in diseases such as preeclampsia. • GF/Cyt, alone or in combination, promote endothelial dysfunction and are rescued by the nutraceutical CLA. • GF/Cyt coupled directly to Src mediate this damage, and is rescued by t10, c12 CLA or a 1:1 mix of c9, t11 and t10, c12 CLA. • Interleukins are less damaging than GFs, and are more resistant to CLA rescue. [ABSTRACT FROM AUTHOR]

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE. • We have previously shown VEGF can promote endothelial dysfunction by Src and Erk kinase-mediated closure of Cx43 channels. • The role of VEGFR2 in this response is clear, but the role of VEGFR1 in this response is unclear. • Addition of EGFR can make endothelium newly responsive to EGF and fully reproduce Cx43 phosphorylation and closure. • Addition of EGFR also blocks VEGFR coupling to Cx43 phosphorylation and closure. • Physiologic relevance to preeclampsia is supported by placental exosomes being EGFR positive. [ABSTRACT FROM AUTHOR]