Efficacy and safety of phospholipid nanoparticles (VBI-S) in reversing intractable hypotension in patients with septic shock: a multicentre, open-label, repeated measures, phase 2a clinical pilot trial.

Background: Since the 1990's attempts to favorably modulate nitric oxide (NO) have been unsuccessful. We hypothesized that because NO is lipophilic it would preferentially localize into intravascularly infused hydrophobic nanoparticles, thereby reducing its bioavailability and adverse effects without inhibiting its production. We aimed to determine the efficacy and safety of intravenous infusion of a fluid comprised of hydrophobic phospholipid nanoparticles (VBI-S) that reversibly absorb NO in the treatment of hypotension of patients in severe septic shock. Methods: This is a multicentre, open-label, repeated measures, phase 2a clinical pilot trial done at six hospital centers in the USA. Patients in severe septic shock were enrolled after intravenous fluid therapy had failed to raise mean arterial blood pressure (MAP) to at least the generally accepted level of 65 mmHg, requiring the use of vasopressors. The primary endpoint of this study is the proportion of patients in whom MAP increased by at least 10 mmHg. VBI-S was administered intravenously to patients as boluses of 100 ml, 200 ml, 400 ml, and 800 ml at 999 ml/min until the blood pressure goal was reached after which the infusion was stopped, and the MAP was recorded. All patients who received any volume of VBI-S were included in the primary and safety analysis. The study is registered with ClinicalTrials.gov, NCT04257136. Findings: Between February 17, 2020 and January 3, 2023, 20 eligible patients were enrolled in the study. In all 20 (100%) patients, the goal of increasing MAP by at least 10 mmHg using VBI-S was achieved (p = 0.0087, effect size = 0.654). Mean VBI-S volume required to meet the primary goal was 561.0 ± 372.3 ml. The goal of lowering vasopressor dose was also achieved (p = 0.0017). Within 48 h or less after VBI-S, there was a statistically significant improvement in oxygenation, serum creatinine, clotting variables, procalcitonin, lactic acid, and the sequential organ failure assessment (SOFA) score. At 24 h and 48 h following administration of VBI-S, 12/15 (80%) and 9/12 (75%) patients developed hyperlipidemia, respectively. No severe adverse events of VBI-S were observed, and there were no treatment-related deaths. Interpretation: These preliminary findings suggest the safety and efficacy of VBI-S in treating hypotension in patients with septic shock. However, a definitive mortality benefit cannot be demonstrated without a randomized controlled study. Funding: The Naval Medical Research Command-Naval Advanced Medical Development program via the Medical Technology Enterprise Consortium.

Classical Estrogen Signaling in Ciliated Epithelial Cells of the Oviduct Is Nonessential for Fertility in Female Mice.

Ciliary action performs a critical role in the oviduct (Fallopian tube) during pregnancy establishment through sperm and egg transport. The disruption of normal ciliary function in the oviduct affects oocyte pick-up and is a contributing factor to female infertility. Estrogen is an important regulator of ciliary action in the oviduct and promotes ciliogenesis in several species. Global loss of estrogen receptor α (ESR1) leads to infertility. We have previously shown that ESR1 in the oviductal epithelial cell layer is required for female fertility. Here, we assessed the role of estrogen on transcriptional regulation of ciliated epithelial cells of the oviduct using single-cell RNA-sequencing analysis. We observed minor variations in ciliated cell genes in the proximal region (isthmus and uterotubal junction) of the oviduct. However, 17ß-estradiol treatment had little impact on the gene expression profile of ciliated epithelial cells. We also conditionally ablated Esr1 from ciliated epithelial cells of the oviduct (called ciliated Esr1d/d mice). Our studies showed that ciliated Esr1d/d females had fertility rates comparable to control females, did not display any disruptions in preimplantation embryo development or embryo transport to the uterus, and had comparable cilia formation to control females. However, we observed some incomplete deletion of Esr1 in the ciliated epithelial cells, especially in the ampulla region. Nevertheless, our data suggest that ESR1 expression in ciliated cells of the oviduct is dispensable for ciliogenesis and nonessential for female fertility in mice.

Variable Cre Recombination Efficiency in Placentas of Cyp19-Cre ROSAmT/mG Transgenic Mice.

The aromatase-Cre recombinase (Cyp19-Cre) transgenic mouse model has been extensively used for placenta-specific gene inactivation. In a pilot study, we observed unexpected phenotypes using this mouse strain, which prompted an extensive characterization of Cyp19-Cre placental phenotypes using ROSAmT/mG transgenic reporter mice. The two strains were mated to generate bi-transgenic Cyp19-Cre;ROSAmT/mG mice following a standard transgenic breeding scheme, and placental and fetal tissues were analyzed on embryonic day 17.5. Both maternal and paternal Cre inheritance were analyzed by mating the respective Cyp19-Cre and ROSAmT/mG males and females. The genotype results showed the expected percentage of Cyp19-Cre;ROSAmT/mG fetuses (73%) and Cre mRNA was expressed in all of the Cyp19-Cre placentas. However, surprisingly, only about 50% of the Cyp19-Cre;ROSAmT/mG placentas showed Cre-mediated recombinase activity as demonstrated by placental enhanced green fluorescent protein (EGFP) expression. Further genetic excision analysis of the placentas revealed consistent results showing the absence of excision of the tdTomato in all of the Cyp19-Cre;ROSAmT/mG placentas lacking EGFP expression. Moreover, among the EGFP-expressing placentas, there was wide variability in recombination efficiency, even in placentas from the same litter, leading to a mosaic pattern of EGFP expression in different zones and cell types of the placentas. In addition, we observed a significantly higher percentage of Cre recombination activity in placentas with maternal Cre inheritance. Our results show frequent mosaicism, inconsistent recombination activity, and parent-of-origin effects in placentas from Cyp19-Cre;ROSAmT/mG mice, suggesting that tail-biopsy genotype results may not necessarily indicate the excision of floxed genes in Cyp19-Cre positive placentas. Thus, placenta-specific mutagenesis studies using the Cyp19-Cre model require extensive characterization and careful interpretation of the placental phenotypes for each floxed allele.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Prostaglandin-Endoperoxide Synthase 2 (PTGS2) in the Oviduct: Roles in Fertilization and Early Embryo Development.

The mammalian oviduct is a dynamic organ where important events such as final maturation of oocytes, transport of gametes, sperm capacitation, fertilization, embryo development, and transport take place. Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), is the rate-limiting enzyme in the production of prostaglandins (PGs) and plays an essential role during early pregnancy, including ovulation, fertilization, implantation, and decidualization. Even though the maternal-embryo communication originates in the oviduct, not many studies have systemically investigated PTGS2 signaling during early development. Most of the studies investigating implantation and decidualization processes in Ptgs2-/- mice employed embryo transfer into the uterus, thereby bypassing the mammalian oviduct. Consequently, an understanding of the mechanistic action as well as the regulation of PTGS2 and derived PGs in oviductal functions is far from complete. In this review, we aim to focus on the importance of PTGS2 and associated PGs signaling in the oviduct particularly in humans, farm animals, and laboratory rodents to provide a broad perspective to guide further research in this field. Specifically, we review the role of PTGS2-derived PGs in fertilization, embryo development, and transport. We focus on the actions of ovarian steroid hormones on PTGS2 regulation in the oviduct. Understanding of cellular PTGS2 function during early embryo development and transport in the oviduct will be an important step toward a better understanding of reproduction and may have potential implication in the assisted reproductive technology.

Mechanism of semen liquefaction and its potential for a novel non-hormonal contraception†.

Semen liquefaction is a proteolytic process where a gel-like ejaculated semen becomes watery due to the enzymatic activity of prostate-derived serine proteases in the female reproductive tract. The liquefaction process is crucial for the sperm to gain their motility and successful transport to the fertilization site in Fallopian tubes (or oviducts in animals). Hyperviscous semen or failure in liquefaction is one of the causes of male infertility. Therefore, the biochemical inhibition of serine proteases in the female reproductive tract after ejaculation is a prime target for novel contraceptive development. Herein, we will discuss protein components in the ejaculates responsible for semen liquefaction and any developments of contraceptive methods in the past that involve the liquefaction process.

Serine protease inhibitor disrupts sperm motility leading to reduced fertility in female mice†.

Inhibition of the sperm transport process in the female reproductive tract could lead to infertility. We previously showed that a pan-serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), blocked semen liquefaction in vivo and resulted in a drastic decrease in the number of sperm in the oviduct of female mice. In this study, we used a mouse model to test the efficacy of AEBSF as a reversible contraceptive, a sperm motility inhibitor, and a spermicide. Additionally, this study evaluated the toxicity of AEBSF on mouse vaginal tissues in vivo and human endocervical cells in vitro. We found that female mice treated with AEBSF had significantly less pups born per litter as well as fertilization rates in vivo compared to the vehicle control. We then showed that AEBSF reduced sperm motility and fertilization capability in vitro in a dose-dependent manner. Furthermore, AEBSF also exhibited spermicidal effects. Lastly, AEBSF treatment in female mice for 10 min or 3 consecutive days did not alter vaginal cell viability in vivo, similar to that of the vehicle and non-treated controls. However, AEBSF decreased cell viability of human ectocervical (ECT) cell line in vitro, suggesting that cells in the lower reproductive tract in mice and humans responded differently to AEBSF. In summary, our study showed that AEBSF can be used as a prototype compound for the further development of novel non-hormonal contraceptives for women by targeting sperm transport in the female reproductive tract.

Roles of steroid hormones in oviductal function

The oviduct (known as the fallopian tube in humans) is the site for fertilization and pre-implantation embryo development. Female steroid hormones, estrogen and progesterone, are known to modulate the morphology and function of cells in the oviduct. In this review, we focus on the actions of estrogen and progesterone on secretory, ciliated, and muscle cell functions and morphologies during fertilization, pre-implantation embryo development, and embryo transport in humans, laboratory rodents and farm animals. We review some aspects of oviductal anatomy and histology and discuss current assisted reproductive technologies (ARTs) that bypass the oviduct and their effects on embryo quality. Lastly, we review the causes of alterations in secretory, ciliated, and muscle cell functions that could result in embryo transport defects.

Steroidogenic Factor 1 (Nr5a1) is Required for Sertoli Cell Survival Post Sex Determination.

The elevated level of Steroidogenic Factor 1 (Nr5a1, Sf-1) expression in the male gonadal development pathway, post sex determination, implies a vital role in testis gonadal differentiation. In this study we generated Sertoli cell-specific Nr5a1 KO mice (SC-SF-1-/-) at E14.5, which coincides with testis development post sex determination, using the Amh-Cre mouse model. Analysis of SC-SF-1-/- (Sertoli cell specific Nr5a1 knockout) testes demonstrated apoptosis as early as E15. Further analysis revealed that SC-SF-1-/- gonads displayed lower MDM2 levels resulting in elevated TP53 levels, which we believe may lead to apoptosis of the Sertoli cell population, inferring the possibility that NR5A1 directly regulates MDM2 expression. By E15.5, the Sertoli cell and germ cell population declined in SC-SF-1-/- mice resulting in the disruption of seminiferous cords with limited cord structure remaining at E18.5. Due to the loss of Sertoli and germ cells, the testis weights of SC-SF-1-/- mice at 6-weeks were much reduced; however, SC-SF-1-/- seminal vesicles weights were comparable suggesting intact Leydig cell androgen production. We conclude that NR5A1 regulates the TP53 pathway during development, is essential for fetal Sertoli cell survival and controls the cell cycle of Sertoli cells during differentiation.

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy.

BACKGROUND: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. METHODS: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. FINDINGS: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. INTERPRETATION: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. FUND: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA).

Hypothalamic PDE3B deficiency alters body weight and glucose homeostasis in mouse

Pharmacological studies have suggested hypothalamic phosphodiesterase-3B to mediate leptin and insulin action in regulation of energy homeostasis. Whereas Pde3b-null mice show altered energy homeostasis, it is unknown whether this is due to ablation of Pde3b in the hypothalamus. Thus, to address the functional significance of hypothalamic phosphodiesterase-3B, we used Pde3bflox/flox and Nkx2.1-Cre mice to generate Pde3b Nkx2.1KD mice that showed 50% reduction of phosphodiesterase-3B in the hypothalamus. To determine the effect of partial ablation of phosphodiesterase-3B in the hypothalamus on energy and glucose homeostasis, males and females were subjected to either a low- or high-fat diet for 19­21 weeks. Only female but not male Pde3b Nkx2.1KD mice on the low-fat diet showed increased body weight from 13 weeks onward with increased food intake, decreased fat pad weights and hypoleptinemia. Glucose tolerance was improved in high-fat diet-fed male Pde3b Nkx2.1KD mice in association with decreased phosphoenolpyruvate carboxykinase-1 and glucose-6-phosphatase mRNA levels in the liver. Also, insulin sensitivity was increased in male Pde3b Nkx2.1KD mice on the low-fat diet. Changes in body weight or in glucose homeostasis were not associated with any alteration in hypothalamic proopiomelanocortin, neuropepide Y and agouti-related peptide mRNA levels. These results suggest that partial loss of phosphodiesterase-3B in the hypothalamus produces a sex-specific response in body weight and glucose homeostasis, and support a role, at least in part, for hypothalamic phosphodiesterase-3B in regulation of energy and glucose homeostasis in mice.

Preconditioning the uterine unfolded protein response maintains non-apoptotic Caspase 3-dependent quiescence during pregnancy.

The prevention of apoptotic caspase 3 activation through biological preconditioning, mediated through the modulation of the unfolded protein response has been demonstrated to ameliorate multiple pathophysiologies. The maintenance of non-apoptotic caspase 3 activity by the unfolded protein response within the pregnant uterus has previously been proven to be critical in inhibiting uterine myocyte contractility during pregnancy. Here we report that the pregnant uterus utilizes an unfolded protein response-preconditioning paradigm to conserve myometrial caspase 3 in a non-apoptotic state in order to effectively inhibit uterine contractility thereby preventing the onset of preterm labor. In the absence of appropriate endogenous preconditioning during pregnancy, uterine caspase 3 is transformed from a non-apoptotic to an apoptotic phenotype. Apoptotic caspase 3 activation results in the precocious triggering of local uterine inflammatory signaling and prostaglandin production, consequently resulting in an increased incidence of preterm birth. These findings represent a paradigm shift in our understanding of how preconditioning promotes the maintenance of uterine non-apoptotic caspase 3 action during pregnancy preventing the onset of premature uterine contraction and therefore defining the timing of the onset of labor.

Hypothalamic Phosphodiesterase 3B Pathway Mediates Anorectic and Body Weight-Reducing Effects of Insulin in Male Mice.

BACKGROUND: Insulin action in the hypothalamus plays a critical role in the regulation of energy homeostasis, yet the intracellular signaling mechanisms mediating insulin action are incompletely understood. Although phosphodiesterase 3B (PDE3B) mediates insulin action in the adipose tissue and it is highly expressed in the hypothalamic areas implicated in energy homeostasis, its role, if any, in mediating insulin action in the hypothalamus is unknown. We tested the hypothesis that insulin action in the hypothalamus is mediated by PDE3B. METHODS: Using enzymatic assay, we examined the effects of peripheral or central administration of insulin on hypothalamic PDE3B activity in adult mice. Western blotting and immunohistochemistry also examined p-Akt and p-STAT3 levels in the hypothalamus. Effects of leptin on these parameters were also compared. We injected cilostamide, a PDE3 inhibitor, prior to central injection of insulin and examined the 12- to 24-hour food intake and 24-hour body weight. Finally, we examined the effect of cilostamide on insulin-induced proopiomelanocortin (Pomc), neurotensin (Nt), neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression in the hypothalamus by qPCR. RESULTS: Peripheral or central injection of insulin significantly increased PDE3B activity in the hypothalamus in association with increased p-Akt levels but without any change in p-STAT3 levels. However, leptin-induced increase in PDE3B activity was associated with an increase in both p-Akt and p-STAT3 levels in the hypothalamus. Prior administration of cilostamide reversed the anorectic and body weight-reducing effects as well as stimulatory effect of insulin on hypothalamic Pomc mRNA levels. Insulin did not alter Nt, Npy and Agrp mRNA levels. CONCLUSION: Insulin induction of hypothalamic PDE3B activity and the reversal of the anorectic and body weight-reducing effects and stimulatory effect of insulin on hypothalamic Pomc gene expression by cilostamide suggest that activation of PDE3B is a novel mechanism of insulin signaling in the hypothalamus.

Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7-dependent.

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.

Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60-70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

Restoration of spermatogenesis and male fertility using an androgen receptor transgene.

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3' to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR-null background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.