2-Methoxyestradiol in Pulmonary Arterial Hypertension: A New Disease Modifier

Pulmonary arterial hypertension (PAH), a debilitating and incurable disease, predominantly develops in women. Estradiol metabolism leads to the production of numerous metabolites with different levels of estrogenic activity and very often opposing biological effects. Dysregulated estradiol metabolism was recently linked to the penetrance, progression, and prognosis of the disease. Ongoing clinical trials are examining the effects of estradiol synthesis/signaling inhibition in patients with PAH. In this chapter, the effects of sex, sex hormones, and estradiol metabolism on the healthy pulmonary circulation and vascular pathobiology are discussed in the light of estradiol metabolism as potential pharmacological target in PAH. The effects of estrogens and their metabolites on vascular pathobiology and disease progression, their involvement in PAH-associated diseases, and the pros and cons for interventions at different levels of estradiol metabolism are discussed. Finally, we propose that 2-methoxyestradiol (2ME), a major non-estrogenic metabolite of estradiol, mediates at least in part the beneficial effects of estradiol and that 2ME exhibits opposing effects to estradiol on several processes relevant to the underlying pathophysiology of PAH, including angiogenesis, metabolic reprograming, inflammation, and immunity. Based on cellular and in vivo effects, 2ME should be viewed as a disease modifier in women with PAH

Sex hormones and hypertension

Gender has an important influence on blood pressure, with premenopausal women having a lower arterial blood pressure than age-matched men. Compared with premenopausal women, postmenopausal women have higher blood pressures, suggesting that ovarian hormones may modulate blood pressure. However, whether sex hormones are responsible for the observed gender-associated differences in arterial blood pressure and whether ovarian hormones account for differences in blood pressure in premenopausal versus postmenopausal women remains unclear. In this review, we provide a discussion of the potential blood pressure regulating effects of female and male sex hormones, as well as the cellular, biochemical and molecular mechanisms by which sex hormones may modify the effects of hypertension on the cardiovascular syste

Vascular consequences of menopause and hormone therapy: Importance of timing of treatment and type of estrogen

Premenopausal women have a lower risk for cardiovascular events, and mortality due to coronary vascular disease (CVD) in premenopausal women is rare. These facts suggest that endogenous estrogens, such as estradiol, protect the cardiovascular system, and several observational studies and a few small clinical studies conducted in healthy and younger postmenopausal women support this hypothesis. In contrast, two large randomized clinical trials (RCTs), using conjugated equine estrogens and conducted in older women with established CVD or without overt CVD, failed to demonstrate protection against CVD by exogenous estrogens. These divergent findings have resulted in confusion with regard to the association between estrogen deficiency and CVD in postmenopausal women. In order to reconcile these contradictory findings, it is necessary to examine the pathophysiology associated with age-dependent changes within the vessel wall and to compare the pharmacology of different types of estrogens. Understanding age-dependent changes in vascular pathology and the pharmacology of different estrogens may facilitate the development of therapeutic strategies for hormone replacement therapy (HRT) that would be effective in delaying vascular remodeling leading to CVD following menopause. In this review we provide an overview of the impact of menopause and estrogen deficiency on vascular remodeling and emphasize the importance of timing and type of estrogen to achieve maximum benefits with regard to reducing the risk of CV

Long-term caffeine consumption exacerbates renal failure in obese, diabetic, ZSF1 (fa-facp) rats

Long-term caffeine consumption exacerbates renal failure in obese, diabetic, ZSF1 (fa-facp) rats.BackgroundOur preliminary studies indicate that chronic caffeine consumption has adverse renal effects in nephropathy associated with high blood pressure and insulin resistance. The purpose of this study was to investigate the effects of early (beginning at 8 weeks of age) and long-term (30 weeks) caffeine treatment (0.1% solution) on renal function and structure in obese, diabetic ZSF1 rats.MethodsMetabolic and renal function measurements were performed at six-week intervals and in a subset of animals (N = 6 per group) heart rate (HR) and mean arterial blood pressure (MABP) were monitored by a radiotelemetric technique. At the end of the study acute, measurements of renal hemodynamics and excretory function were conducted in anesthetized animals.ResultsCaffeine produced a very mild increase (4 to 5%) of MABP and HR, but greatly augmented proteinuria (P < 0.001), reduced creatinine clearance (P < 0.05) and had a mixed effect on metabolic status in obese ZSF1 rats. Caffeine significantly reduced body weight, glycosuria, fasting glucose and insulin levels and improved glucose tolerance, had no effect on elevated plasma triglycerides levels and significantly increased plasma cholesterol level (P < 0.001). Acute measurements of renal function revealed increased renal vascular resistance (95.1 ± 11 vs. 50.7 ± 2.4 mm Hg/mL/min/g kidney, P < 0.01) and decreased inulin clearance (0.37 ± 0.11 vs. 0.97 ± 0.13 mL/min/g kidney, P < 0.002) in caffeine-treated versus control animals, respectively. Caffeine potentiated the development of more severe tubulointerstitial changes (P < 0.05) and increased focal glomerulosclerosis (14.7 ± 1.7 vs. 6.5 ± 0.9%, caffeine vs. control, P < 0.002).ConclusionThe present study provides the first evidence that caffeine (despite improving insulin sensitivity) exacerbates renal failure in obese, diabetic ZSF1 rats. Further mechanistic studies of adverse renal effects of caffeine in chronic renal failure associated with metabolic syndrome are warranted

The β-blocker Nebivolol Is a GRK/β-arrestin Biased Agonist

Nebivolol, a third generation β-adrenoceptor (β-AR) antagonist (β-blocker), causes vasodilation by inducing nitric oxide (NO) production. The mechanism via which nebivolol induces NO production remains unknown, resulting in the genesis of much of the controversy regarding the pharmacological action of nebivolol. Carvedilol is another β-blocker that induces NO production. A prominent pharmacological mechanism of carvedilol is biased agonism that is independent of Gαs and involves G protein-coupled receptor kinase (GRK)/β-arrestin signaling with downstream activation of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). Due to the pharmacological similarities between nebivolol and carvedilol, we hypothesized that nebivolol is also a GRK/β-arrestin biased agonist. We tested this hypothesis utilizing mouse embryonic fibroblasts (MEFs) that solely express β2-ARs, and HL-1 cardiac myocytes that express β1- and β2-ARs and no detectable β3-ARs. We confirmed previous reports that nebivolol does not significantly alter cAMP levels and thus is not a classical agonist. Moreover, in both cell types, nebivolol induced rapid internalization of β-ARs indicating that nebivolol is also not a classical β-blocker. Furthermore, nebivolol treatment resulted in a time-dependent phosphorylation of ERK that was indistinguishable from carvedilol and similar in duration, but not amplitude, to isoproterenol. Nebivolol-mediated phosphorylation of ERK was sensitive to propranolol (non-selective β-AR-blocker), AG1478 (EGFR inhibitor), indicating that the signaling emanates from β-ARs and involves the EGFR. Furthermore, in MEFs, nebivolol-mediated phosphorylation of ERK was sensitive to pharmacological inhibition of GRK2 as well as siRNA knockdown of β-arrestin 1/2. Additionally, nebivolol induced redistribution of β-arrestin 2 from a diffuse staining pattern into more intense punctate spots. We conclude that nebivolol is a β2-AR, and likely β1-AR, GRK/β-arrestin biased agonist, which suggests that some of the unique clinically beneficial effects of nebivolol may be due to biased agonism at β1- and/or β2-ARs. © 2013 Erickson et al

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved. This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens

Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction

Detection of PHLPP1α/β in Human and Mouse Brain by Different Anti-PHLPP1 Antibodies

Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1α (~140-150 kDa) and PHLPP1β (~180-190 kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1α/β by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1β in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1α (~145-150 kDa). The other four antibodies detect a non-specific signal at ~150 kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1α by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ~150 kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis

Multi-Terrain Impact Testing and Simulation of a Composite Energy Absorbing Fuselage Section

Comparisons of the impact performance of a 5-ft diameter crashworthy composite fuselage section were investigated for hard surface, soft soil, and water impacts. The fuselage concept, which was originally designed for impacts onto a hard surface only, consisted of a stiff upper cabin, load bearing floor, and an energy absorbing subfloor. Vertical drop tests were performed at 25-ft/s onto concrete, soft-soil, and water at NASA Langley Research Center. Comparisons of the peak acceleration values, pulse durations, and onset rates were evaluated for each test at specific locations on the fuselage. In addition to comparisons of the experimental results, dynamic finite element models were developed to simulate each impact condition. Once validated, these models can be used to evaluate the dynamic behavior of subfloor components for improved crash protection for hard surface, soft soil, and water impacts

Novel Guidewire Design and Coating for Continuous Delivery of Adenosine During Interventional Procedures.

Background: The no-reflow phenomenon compromises percutaneous coronary intervention outcomes. There is an unmet need for a device that prevents no-reflow phenomenon. Our goal was to develop a guidewire platform comprising a nondisruptive hydrophilic coating that allows continuous delivery of adenosine throughout a percutaneous coronary intervention. Methods and Results: We developed a guidewire with spaced coils to increase surface area for drug loading. Guidewires were plasma treated to attach hydroxyl groups to metal surfaces, and a methoxy-polyethylene glycol-silanol primer layer was covalently linked to hydroxyl groups. Using polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl acetate, a drug layer containing jet-milled adenosine was hydrogen-bonded to the polyethylene glycol-silanol layer and coated with an outer diffusive barrier layer. Coatings were processed with a freeze/thaw curing method. In vitro release studies were conducted followed by in vivo evaluation in pigs. Coating quality, performance, and stability with sterilization were also evaluated. Antiplatelet properties of the guidewire were also determined. Elution studies with adenosine-containing guidewires showed curvilinear and complete release of adenosine over 60 minutes. Porcine studies demonstrated that upon insertion into a coronary artery, adenosine-releasing guidewires induced immediate and robust increases (2.6-fold) in coronary blood flow velocity, which were sustained for ≈30 minutes without systemic hemodynamic effects or arrhythmias. Adenosine-loaded wires prevented and reversed coronary vasoconstriction induced by acetylcholine. The wires significantly inhibited platelet aggregation by \u3e80% in vitro. Guidewires passed bench testing for lubricity, adherence, integrity, and tracking. Conclusions: Our novel drug-releasing guidewire platform represents a unique approach to prevent/treat no-reflow phenomenon during percutaneous coronary intervention