Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

The Association between Prenatal Nicotine Exposure and Offspring's Hearing Impairment.

OBJECTIVE: The objective of this study is to evaluate whether there is an association between in-utero exposure to nicotine and subsequent hearing dysfunction. PATIENTS AND METHODS: Secondary analysis of a multicenter randomized trial to prevent congenital cytomegalovirus (CMV) infection among gravidas with primary CMV infection was conducted. Monthly intravenous immunoglobulin hyperimmune globulin therapy did not influence the rate of congenital CMV. Dyads with missing urine, fetal or neonatal demise, infants diagnosed with a major congenital anomaly, congenital CMV infection, or with evidence of middle ear dysfunction were excluded. The primary outcome was neonatal hearing impairment in one or more ears defined as abnormal distortion product otoacoustic emissions (DPOAEs; 1 to 8 kHz) that were measured within 42 days of birth. DPOAEs were interpreted using optimized frequency-specific level criteria. Cotinine was measured via enzyme-linked immunosorbent assay kits in maternal urine collected at enrollment and in the third trimester (mean gestational age 16.0 and 36.7 weeks, respectively). Blinded personnel ran samples in duplicates. Maternal urine cotinine >5 ng/mL at either time point was defined as in-utero exposure to nicotine. Multivariable logistic regression included variables associated with the primary outcome and with the exposure (p < 0.05) in univariate analysis. RESULTS: Of 399 enrolled patients in the original trial, 150 were included in this analysis, of whom 46 (31%) were exposed to nicotine. The primary outcome occurred in 18 (12%) newborns and was higher in nicotine-exposed infants compared with those nonexposed (15.2 vs. 10.6%, odds ratio [OR] 1.52, 95% confidence interval [CI] 0.55-4.20), but the difference was not significantly different (adjusted odds ratio [aOR] = 1.0, 95% CI 0.30-3.31). This association was similar when exposure was stratified as heavy (>100 ng/mL, aOR 0.72, 95% CI 0.15-3.51) or mild (5-100 ng/mL, aOR 1.28, 95% CI 0.33-4.95). There was no association between nicotine exposure and frequency-specific DPOAE amplitude. CONCLUSION: In a cohort of parturients with primary CMV infection, nicotine exposure was not associated with offspring hearing dysfunction assessed with DPOAEs. KEY POINTS: · Nicotine exposure was quantified from maternal urine.. · Nicotine exposure was identified in 30% of the cohort.. · Exposure was not associated with offspring hearing dysfunction..

Glycemic Control and Aspirin Resistance in Patients Taking Low-Dose Aspirin for Pre-eclampsia Prevention.

OBJECTIVES: To assess the association between aspirin and glycemic control in diabetic, pregnant patients, and the risk for aspirin resistance in those with poor glycemic control across gestation taking low-dose aspirin (LDA) for pre-eclampsia (PEC) prevention. STUDY DESIGN: We performed a secondary analysis of samples collected during the Maternal-Fetal Medicine Units trial of LDA for PEC prevention. A subset of insulin-controlled diabetic patient samples on placebo or 60 mg aspirin daily were evaluated. Glycosylated hemoglobin was measured at randomization, mid-second trimester, and third trimester time points. Thromboxane B2 (TXB2) measurements were previously assessed as part of the original study. Primary outcome was the effect of LDA on glycosylated hemoglobin levels compared with placebo across gestation. RESULTS: Levels of glycosylated hemoglobin increased across gestation in the placebo group (2,067.7 [interquartile range, IQR: 1,624.6-2,713.5 µg/mL] vs. 2,461.9 [1,767.0-3,209.9 µg/mL] vs. 3,244.3 [2,691.5-4,187.0 µg/mL]; p < 0.01) compared with no difference in levels of glycosylated hemoglobin across gestation in the LDA group (2,186.4 [IQR: 1,462.3-3,097.7 µg/mL] vs. 2,337.1 [1,327.7-5,932.6 µg/mL] vs. 2,532.9 [1,804.9-5,511.8 µg/mL]; p = 0.78). Higher levels of glycosylated hemoglobin were associated with increased TXB2 levels prior to randomization (r = 0.67, p < 0.05). Incomplete TXB2 was higher in pregnancies with increasing levels of glycosylated hemoglobin compared with those with decreasing levels of glycosylated hemoglobin across gestation (69.2 vs. 18.1%, p = 0.02). CONCLUSION: LDA exposure may be beneficial to glycemic control in this patient population. Additionally, poor glycemic control is associated with a higher level of TXB2 in diabetic pregnant patients on LDA. Higher doses of aspirin may be required in these patients to prevent development of PEC. KEY POINTS: · Low-dose aspirin may improve glycemic control.. · Poor glycemic control increases risk for aspirin resistance.. · Higher doses of aspirin may be required for pre-eclampsia prevention..

Low-dose aspirin increases 15-epi-lipoxins A4 in pregnancies at high-risk for developing preeclampsia.

BACKGROUND: LDA triggers biosynthesis of endogenous anti-inflammatory molecules, aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4), which may counteract inflammatory process of preeclampsia (PE), and play role in LDA's mechanism of action in PE prevention in high-risk patients. OBJECTIVE: Investigate the effects of daily LDA on levels of 15-epi-LXA4 in pregnancies at high-risk for developing PE. MATERIALS AND METHODS: Secondary analysis of multi-centered randomized controlled trial investigating effects of daily LDA (60 mg) in high-risk pregnancies. Maternal samples were drawn at three points: before LDA initiation (13-26 weeks' gestation), 24-28 weeks' gestation (at least two weeks after LDA) and 34-36 weeks' gestation. 15-epi-LXA4 levels were measured by ELISA. RESULTS: Analysis included 82 patients: 63 receiving daily LDA and 29 receiving daily placebo starting between 13 and 25 weeks gestation. Prior to randomization, baseline 15-epi-LXA4 levels were similar between both groups (75.9 pg/mL [IQR; 63.8-114.0] vs 136.2 pg/mL [52.4-476.2]; p = 0.10). Patients receiving daily LDA were noted to have significantly increased levels of 15-epi-LXA4 after LDA administration (136.2 pg/mL [IQR; 52.4-476.2] vs 1758.2 pg/mL [905.4-6638.5]; p < 0.001). They also had higher 15-epi-LXA4 levels compared to those receiving placebo at 24-28 weeks' (50.3 [38.1-94.2] vs 1758.2 [905.4-6638.5]; p < 0.001 and 34-38 weeks' gestation (57.9 [41.9-76.7] vs 2310.3 pg/mL [656.9-10609.4]; p < 0.001). After LDA administration in the second trimester, patients who developed PE had decrease in 15-epi-LXA4 levels compared to those without PE (942 pg/mL [348.3-1810.3] vs 1758.2 pg/mL [905.4-6638.5]; p = 0.129). CONCLUSION: Daily LDA administration increases 15-epi-LXA4 levels in high-risk pregnancies for PE. In LDA group, pregnancies complicated by PE have lower levels of 15-epi-LXA4 compared to pregnancies without PE.

Progesterone Receptor Signaling Selectively Modulates Cytokine-Induced Global Gene Expression in Human Cervical Stromal Cells.

Preterm birth (PTB) is the leading cause of morbidity and mortality in infants <1 year of age. Intrauterine inflammation is a hallmark of preterm and term parturition; however, this alone cannot fully explain the pathobiology of PTB. For example, the cervix undergoes a prolonged series of biochemical and biomechanical events, including extracellular matrix (ECM) remodeling and mechanochemical changes, culminating in ripening. Vaginal progesterone (P4) prophylaxis demonstrates great promise in preventing PTB in women with a short cervix (<25 mm). We used a primary culture model of human cervical stromal fibroblasts to investigate gene expression signatures in cells treated with interleukin-1ß (IL-1ß) in the presence or absence of P4 following 17ß-estradiol (17ß-E2) priming for 7-10 days. Microarrays were used to measure global gene expression in cells treated with cytokine or P4 alone or in combination, followed by validation of select transcripts by semiquantitative polymerase chain reactions (qRT-PCR). Primary/precursor (MIR) and mature microRNAs (miR) were quantified by microarray and NanoString® platforms, respectively, and validated by qRT-PCR. Differential gene expression was computed after data normalization followed by pathway analysis using Kyoto Encyclopedia Genes and Genomes (KEGG), Panther, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) upstream regulator algorithm tools. Treatment of fibroblasts with IL-1ß alone resulted in the differential expression of 1432 transcripts (protein coding and non-coding), while P4 alone led to the expression of only 43 transcripts compared to untreated controls. Cytokines, chemokines, and their cognate receptors and prostaglandin endoperoxide synthase-2 (PTGS-2) were among the most highly upregulated transcripts following either IL-1ß or IL-1ß + P4. Other prominent differentially expressed transcripts were those encoding ECM proteins, ECM-degrading enzymes, and enzymes involved in glycosaminoglycan (GAG) biosynthesis. We also detected differential expression of bradykinin receptor-1 and -2 transcripts, suggesting (prominent in tissue injury/remodeling) a role for the kallikrein-kinin system in cervical responses to cytokine and/or P4 challenge. Collectively, this global gene expression study provides a rich database to interrogate stromal fibroblasts in the setting of a proinflammatory and endocrine milieu that is relevant to cervical remodeling/ripening during preparation for parturition.

Novel Interventions for the Prevention of Preeclampsia.

PURPOSE OF REVIEW: To review the rationale and biological plausibility and discuss the current research on novel interventions for the prevention of preeclampsia. RECENT FINDINGS: Preeclampsia affects up to 8% of pregnancies worldwide and remains a major cause of maternal and neonatal morbidity and mortality. Multiple medications have been investigated or repurposed as potential effective interventions for preeclampsia prevention. Aspirin is currently the only drug for which there is some evidence of benefit for preeclampsia prevention, and its use is recommended by professional societies for pregnancies at risk. Statins have shown promise for prevention of preeclampsia in animal models and human pilot studies, without any trend or concerns for safety signals or teratogenicity. The use of metformin has also gained popularity in experimental studies, but observations from randomized clinical trials were not consistent on its utility as a possible intervention for preeclampsia prevention. While initial studies evaluating esomeprazole were promising, randomized trials failed to show benefit. Contemporary research shows exciting new opportunities for prophylactic treatment for preeclampsia, to prevent this debilitating and life-threatening disease.

Cell Lines as Biological Models: Practical Steps for More Reliable Research.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

Myoferlin regulates epithelial cancer cell plasticity and migration through autocrine TGF-ß1 signaling.

Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231MYOFKD) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231MYOFKD cells were treated with TGF-ß1, a potent stimulus of EMT. After 48 hr with TGF-ß1, MDA-231MYOFKD cells underwent an EMT. TGF-ß1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-ß1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-ß1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-ß1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-ß1 and observed a decrease in TGF-ß1 protein secretion and mRNA transcription. To determine if TGF-ß1 was required to maintain the mesenchymal phenotype, TGF-ß receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-ß signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1ß, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1ß or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1ß induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1ß inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Agonist-Dependent Downregulation of Progesterone Receptors in Human Cervical Stromal Fibroblasts.

Progesterone (P(4)) maintains uterine quiescence during the majority of pregnancy, whereas diminished progesterone receptor (PR) expression and/or activity (ie, functional P(4) withdrawal) promotes parturition. To investigate the regulation of PR expression in cervical stroma, fibroblasts from premenopausal hysterectomy specimens were prepared. Greater than 99% of the cultures were vimentin positive (mesenchymal cell marker) with only occasional cytokeratin-8 positivity (epithelial cell marker) and no evidence of CD31-positive (endothelial cell marker) cells. Cells were immunolabeled with antibodies directed against PRs (PR-A and PR-B), estrogen receptor α (ER-α), and glucocorticoid receptor-α/ß (GR-α/ß). All cells were uniformly immunopositive for ER-α and GR-α/ß but did not express PRs. Incubation of cells with 10(-8) mol/L 17ß-estradiol induced a time-dependent increase in PR-A and PR-B messenger RNAs (mRNAs) by quantitative real-time polymerase chain reactions and proteins by immunoblotting and immunofluorescence. Incubation of cervical fibroblasts with PR ligands (medroxyprogesterone acetate or Org-2058) downregulated PR-A and PR-B levels. Coincubation of cells with PR ligands plus RU-486, a PR antagonist, partially abrogated agonist-induced receptor downregulation. Dexamethasone, a pure glucocorticoid, had no inhibitory effect on PR expression. These results indicate that progestins and estrogens regulate PR expression in cervical fibroblasts. We postulate that hormonal regulation of PR expression in the cervical stroma may contribute to functional P(4) withdrawal in preparation for parturition.

Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition.

BACKGROUND: Inflammation is a proximate mediator of preterm birth and fetal injury. During inflammation several microRNAs (22 nucleotide noncoding ribonucleic acid (RNA) molecules) are up-regulated in response to cytokines such as interleukin-1ß. MicroRNAs, in most cases, fine-tune gene expression, including both up-regulation and down-regulation of their target genes. However, the role of pro- and antiinflammatory microRNAs in this process is poorly understood. OBJECTIVE: The principal goal of the work was to examine the inflammatory genomic profile of human decidual cells challenged with a proinflammatory cytokine known to be present in the setting of preterm parturition. We determined the coding (messenger RNA) and noncoding (microRNA) sequences to construct a network of interacting genes during inflammation using an in vitro model of decidual stromal cells. STUDY DESIGN: The effects of interleukin-1ß exposure on mature microRNA expression were tested in human decidual cell cultures using the multiplexed NanoString platform, whereas the global inflammatory transcriptional response was measured using oligonucleotide microarrays. Differential expression of select transcripts was confirmed by quantitative real time-polymerase chain reaction. Bioinformatics tools were used to infer transcription factor activation and regulatory interactions. RESULTS: Interleukin-1ß elicited up- and down-regulation of 350 and 78 nonredundant transcripts (false discovery rate < 0.1), respectively, including induction of numerous cytokines, chemokines, and other inflammatory mediators. Whereas this transcriptional response included marked changes in several microRNA gene loci, the pool of fully processed, mature microRNA was comparatively stable following a cytokine challenge. Of a total of 6 mature microRNAs identified as being differentially expressed by NanoString profiling, 2 (miR-146a and miR-155) were validated by quantitative real time-polymerase chain reaction. Using complementary bioinformatics approaches, activation of several inflammatory transcription factors could be inferred downstream of interleukin-1ß based on the overall transcriptional response. Further analysis revealed that miR-146a and miR-155 both target genes involved in inflammatory signaling, including Toll-like receptor and mitogen-activated protein kinase pathways. CONCLUSION: Stimulation of decidual cells with interleukin-1ß alters the expression of microRNAs that function to temper proinflammatory signaling. In this setting, some microRNAs may be involved in tissue-level inflammation during the bulk of gestation and assist in pregnancy maintenance.

Loss of myoferlin redirects breast cancer cell motility towards collective migration.

Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231(MYOF-KD)) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA-231(LTVC)) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD) cells migrated directionally and collectively, while MDA-231(LTVC) cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231(MYOF-KD) cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA-231(MYOF-KD) cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA-231(MYOF-KD) tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC)-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research.

Finite element analysis of traction force microscopy: influence of cell mechanics, adhesion, and morphology.

The interactions between adherent cells and their extracellular matrix (ECM) have been shown to play an important role in many biological processes, such as wound healing, morphogenesis, differentiation, and cell migration. Cells attach to the ECM at focal adhesion sites and transmit contractile forces to the substrate via cytoskeletal actin stress fibers. This contraction results in traction stresses within the substrate/ECM. Traction force microscopy (TFM) is an experimental technique used to quantify the contractile forces generated by adherent cells. In TFM, cells are seeded on a flexible substrate and displacements of the substrate caused by cell contraction are tracked and converted to a traction stress field. The magnitude of these traction stresses are normally used as a surrogate measure of internal cell contractile force or contractility. We hypothesize that in addition to contractile force, other biomechanical properties including cell stiffness, adhesion energy density, and cell morphology may affect the traction stresses measured by TFM. In this study, we developed finite element models of the 2D and 3D TFM techniques to investigate how changes in several biomechanical properties alter the traction stresses measured by TFM. We independently varied cell stiffness, cell-ECM adhesion energy density, cell aspect ratio, and contractility and performed a sensitivity analysis to determine which parameters significantly contribute to the measured maximum traction stress and net contractile moment. Results suggest that changes in cell stiffness and adhesion energy density can significantly alter measured tractions, independent of contractility. Based on a sensitivity analysis, we developed a correction factor to account for changes in cell stiffness and adhesion and successfully applied this correction factor algorithm to experimental TFM measurements in invasive and noninvasive cancer cells. Therefore, application of these types of corrections to TFM measurements can yield more accurate estimates of cell contractility.

Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion.

Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF) transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.

Mechanistic modeling of the effects of myoferlin on tumor cell invasion.

Myoferlin (MYOF) is a member of the evolutionarily conserved ferlin family of proteins, noted for their role in a variety of membrane processes, including endocytosis, repair, and vesicular transport. Notably, ferlins are implicated in Caenorhabditis elegans sperm motility (Fer-1), mammalian skeletal muscle development and repair (MYOF and dysferlin), and presynaptic transmission in the auditory system (otoferlin). In this paper, we demonstrate that MYOF plays a previously unrecognized role in cancer cell invasion, using a combination of mathematical modeling and in vitro experiments. Using a real-time impedance-based invasion assay (xCELLigence), we have shown that lentiviral-based knockdown of MYOF significantly reduced invasion of MDA-MB-231 breast cancer cells in Matrigel bioassays. Based on these experimental data, we developed a partial differential equation model of MYOF effects on cancer cell invasion, which we used to generate mechanistic hypotheses. The mathematical model predictions revealed that matrix metalloproteinases (MMPs) may play a key role in modulating this invasive property, which was supported by experimental data using quantitative RT-PCR screens. These results suggest that MYOF may be a promising target for biomarkers or drug target for metastatic cancer diagnosis and therapy, perhaps mediated through MMPs.

Inflammatory gene regulatory networks in amnion cells following cytokine stimulation: translational systems approach to modeling human parturition.

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1ß, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.