A fully 3D-printed versatile tumor-on-a-chip allows multi-drug screening and correlation with clinical outcomes for personalized medicine.

Optimal clinical outcomes in cancer treatments could be achieved through the development of reliable, precise ex vivo tumor models that function as drug screening platforms for patient-targeted therapies. Microfluidic tumor-on-chip technology is emerging as a preferred tool since it enables the complex set-ups and recapitulation of the physiologically relevant physical microenvironment of tumors. In order to overcome the common hindrances encountered while using this technology, a fully 3D-printed device was developed that sustains patient-derived multicellular spheroids long enough to conduct multiple drug screening tests. This tool is both cost effective and possesses four necessary characteristics of effective microfluidic devices: transparency, biocompatibility, versatility, and sample accessibility. Compelling correlations which demonstrate a clinical proof of concept were found after testing and comparing different chemotherapies on tumor spheroids, derived from ten patients, to their clinical outcomes. This platform offers a potential solution for personalized medicine by functioning as a predictive drug-performance tool.

Expert review: preeclampsia Type I and Type II.

Pregnancy involves an interplay between maternal and fetal factors affecting changes to maternal anatomy and physiology to support the developing fetus and ensure the well-being of both the mother and offspring. A century of research has provided evidence of the imperative role of the placenta in the development of preeclampsia. Recently, a growing body of evidence has supported the adaptations of the maternal cardiovascular system during normal pregnancy and its maladaptation in preeclampsia. Debate surrounds the roles of the placenta vs the maternal cardiovascular system in the pathophysiology of preeclampsia. We proposed an integrated model of the maternal cardiac-placental-fetal array and the development of preeclampsia, which reconciles the disease phenotypes and their proposed origins, whether placenta-dominant or maternal cardiovascular system-dominant. These phenotypes are sufficiently diverse to define 2 distinct types: preeclampsia Type I and Type II. Type I preeclampsia may present earlier, characterized by placental dysfunction or malperfusion, shallow trophoblast invasion, inadequate spiral artery conversion, profound syncytiotrophoblast stress, elevated soluble fms-like tyrosine kinase-1 levels, reduced placental growth factor levels, high peripheral vascular resistance, and low cardiac output. Type I is more often accompanied by fetal growth restriction, and low placental growth factor levels have a measurable impact on maternal cardiac remodeling and function. Type II preeclampsia typically occurs in the later stages of pregnancy and entails an evolving maternal cardiovascular intolerance to the demands of pregnancy, with a moderately dysfunctional placenta and inadequate blood supply. The soluble fms-like tyrosine kinase-1-placental growth factor ratio may be normal or slightly disturbed, peripheral vascular resistance is low, and cardiac output is high, but these adaptations still fail to meet demand. Emergent placental dysfunction, coupled with an increasing inability to meet demand, more often appears with fetal macrosomia, multiple pregnancies, or prolonged pregnancy. Support for the notion of 2 types of preeclampsia observable on the molecular level is provided by single-cell transcriptomic survey of gene expression patterns across different cell classes. This revealed widespread dysregulation of gene expression across all cell types, and significant imbalance in fms-like tyrosine kinase-1 (FLT1) and placental growth factor, particularly marked in the syncytium of early preeclampsia cases. Classification of preeclampsia into Type I and Type II can inform future research to develop targeted screening, prevention, and treatment approaches.

ZnT1 induces a crosstalk between T-type and L-type calcium channels through interactions with Raf-1 kinase and the calcium channel ß2 subunit.

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.

Ferroptosis, trophoblast lipotoxic damage, and adverse pregnancy outcome.

Programmed cell death is a central process in the control of tissue development, organismal physiology, and disease. Ferroptosis is a recently identified form of programmed cell death that is uniquely defined by redox-active iron-dependent hydroxy-peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids and a loss of lipid peroxidation repair capacity. This distinctive form of lipotoxic cell death has been recently implicated in multiple human diseases, spanning ischemia-reperfusion heart injury, brain damage, acute kidney injury, cancer, and asthma. Intriguingly, settings that have been associated with ferroptosis are linked to placental physiology and trophoblast injury. Such circumstances include hypoxia-reperfusion during placental development, physiological uterine contractions or pathological changes in placental bed perfusion, the abundance of trophoblastic iron, evidence for lipotoxicity during the pathophysiology of major placental disorders such as preeclampsia, fetal growth restriction, and preterm birth, and reduced glutathione peroxidation capacity and lipid peroxidation repair during placental injury. We recently interrogated placental ferroptosis in placental dysfunction in human and mouse pregnancy, dissected its relevance to placental injury, and validated the role of glutathione peroxidase-4 in guarding placental trophoblasts against ferroptotic injury. We also uncovered a role for the phospholipase PLA2G6 (PNPLA9) in attenuating trophoblast ferroptosis. Here, we summarize current data on trophoblast ferroptosis, and the role of several proteins and microRNAs as regulators of this process. Our text offers insights into new opportunities for regulating ferroptosis as a means for protecting placental trophoblasts against lipotoxic injury.

Recurrent pregnancy loss and future risk of female malignancies.

PURPOSE: To investigate whether patients with a history of recurrent pregnancy loss (RPL) have an increased risk for future female malignancies. METHODS: A retrospective population-based study compared the incidence of long-term female malignancies in a cohort of women with and without a history of RPL (2 or more consecutive pregnancy losses). Deliveries occurred between the years 1988 and 2013, with a mean follow-up duration of 12 years. Women with known malignancies before the index pregnancy were excluded from the analysis. Female malignancies were divided according to specific type including ovary, breast, uterine and uterine cervix. Kaplan-Meier survival curve was used to estimate the cumulative incidence of malignancies. Cox proportional hazards model was used to determine the adjusted hazard ratios (HR) for female malignancy after controlling for confounders. RESULTS: During the study period, 106,265 patients met the inclusion criteria; 6.6% (n = 7052) of patients had a diagnosis of RPL. During the follow-up period, patients with RPL had a significantly increased risk of being diagnosed with female malignancies as a group, while individually there was an increased risk of breast and uterine cervix cancer. Using a Kaplan-Meier survival curve, patients with a history of RPL had a significantly higher cumulative incidence of female malignancies. Using a Cox proportional hazards model, adjusted for confounders such as smoking, parity, and diabetes mellitus, a history of RPL remained independently associated with female malignancies (adjusted HR 1.4; P = 0.003). CONCLUSIONS: RPL is independently associated with long-term female malignancies. Patients with a history of RPL may benefit from counseling and screening for breast and uterine cervix cancer in particular.

The effects of pravastatin on the normal human placenta: Lessons from ex-vivo models.

INTRODUCTION: Research in animal models and preliminary clinical studies in humans support the use of pravastatin for the prevention of preeclampsia. However, its use during pregnancy is still controversial due to limited data about its effect on the human placenta and fetus. METHODS: In the present study, human placental cotyledons were perfused in the absence or presence of pravastatin in the maternal reservoir (PraM). In addition, placental explants were treated with pravastatin for 5, 24 and 72 h under normoxia and hypoxia. We monitored the secretion of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), endothelial nitric oxide synthase (eNOS) expression and activation and the fetal vasoconstriction response to angiotensin-II. RESULTS: The concentrations of PlGF, sFlt-1 and sEng were not significantly altered by pravastatin in PraM cotyledons and in placental explants compared to control. Under hypoxic conditions, pravastatin decreased sFlt-1 concentrations. eNOS expression was significantly increased in PraM cotyledons but not in pravastatin-treated placental explants cultured under normoxia or hypoxia. eNOS phosphorylation was not significantly affected by pravastatin. The feto-placental vascular tone and the fetal vasoconstriction response to angiotensin-II, did not change following exposure of the maternal circulation to pravastatin. CONCLUSION: We found that pravastatin does not alter the essential physiological functions of the placenta investigated in the study. The relevance of the study lays in the fact that it expands the current knowledge obtained thus far regarding the effect of the drug on the normal human placenta. This data is reassuring and important for clinicians that consider the treatment of high-risk patients with pravastatin, a treatment that exposes some normal pregnancies to the drug.

INO-8875, a highly selective A1 adenosine receptor agonist: evaluation of chronotropic, dromotropic, and hemodynamic effects in rats.

Selective pharmacological activation of the adenosine 1 receptor (A(1)R) is a promising new approach to achieve a potent block of atrioventricular (A-V)-nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A(1)R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V-nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 µM) progressively prolonged A-V-nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 µg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1-50 µg/kg) and CVT-510 (20 and 50 µg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V-nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V-nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A(1)R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling.

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

INO-8875, a Highly-Selective A1 Adenosine Receptor Agonist: Evaluation of Chronotropic, Dromotropic and Hemodynamic Effects in Rats.

Publication of this article is suspended until the authors can provide full identification and verification of the chemical structure of INO-8875.

Correlation between atrial ZnT-1 expression and atrial fibrillation in humans: a pilot study.

BACKGROUND: Until recently, the membrane protein ZnT-1 was studied mainly in the context of zinc homeostasis. However, new findings indicate that it acts as an inhibitor of L-type calcium channels. We recently found that acute rapid pacing of the rat atria in vivo augments the expression of ZnT-1, while knockdown of ZnT-1 in culture can oppose the inhibition of L-type calcium channels following rapid pacing. This pilot study, the first to assess cardiac ZnT-1 in humans, was designed to look for possible correlation between the atrial expression of ZnT-1 and atrial fibrillation. METHODS: Right atrial appendage tissue was collected from 39 patients (27 with sinus rhythm and 12 with atrial fibrillation; 6-permanent, 6- paroxysmal or persistent) undergoing open-heart surgery. The expression of ZnT-1 was analyzed by Western blot utilizing beta-actin as an internal loading control and a standard rat heart sample (STD) for inter-blot comparison. RESULTS: Overall atrial fibrillation patients (n = 12) had median ZnT-1/beta-actin of 1.80 STD (inter-quartile range 1.26 to 2.85) versus 0.73 STD (0.24 to 1.64) in the sinus rhythm group (P = 0.002). No association was found between ZnT-1 level and most other clinical parameters tested. Multivariate analysis determined that atrial fibrillation and increased body mass index were the only independent variables clearly associated with higher ZnT-1 levels (Standardized coefficients Beta = 0.62, 0.31; P = 0.002, P = 0.04, respectively). CONCLUSIONS: This pilot study provides evidence for increased ZnT-1 expression in the atria of patients with atrial fibrillation.

Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger.

Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.