Causal Players Research Articles

Neuroinflammatory reactive astrocyte formation correlates with adverse outcomes in perinatal white matter injury.

Perinatal white matter injury (WMI) is the leading cause of long-term neurological morbidity in infants born preterm. Neuroinflammation during a critical window of early brain development plays a key role in WMI disease pathogenesis. The mechanisms linking inflammation with the long-term myelination failure that characterizes WMI, however, remain unknown. Here, we investigate the role of astrocyte reactivity in WMI. In an experimental mouse model of WMI, we demonstrate that WMI disease outcomes are improved in mutant mice lacking secretion of inflammatory molecules TNF-α, IL-1α, and C1q known, in addition to other roles, to induce the formation of a neuroinflammatory reactive astrocyte substate. We show that astrocytes express molecular signatures of the neuroinflammatory reactive astrocyte substate in both our WMI mouse model and human tissue affected by WMI, and that this gene expression pattern is dampened in injured mutant mice. Our data provide evidence that a neuroinflammatory reactive astrocyte substate correlates with adverse WMI disease outcomes, thus highlighting the need for further investigation of these cells as potential causal players in WMI pathology.

Placental mTOR Signaling and Sexual Dimorphism in Metabolic Health across the Lifespan of Offspring

Robust evidence of fetal programming of adult disease has surfaced in the last several decades. Human and preclinical investigations of intrauterine insults report perturbations in placental nutrient sensing by the mechanistic target of rapamycin (mTOR). This review focuses on pregnancy complications associated with placental mTOR regulation, such as fetal growth restriction (FGR), fetal overgrowth, gestational diabetes mellitus (GDM), polycystic ovarian syndrome (PCOS), maternal nutrient restriction (MNR), preeclampsia (PE), maternal smoking, and related effects on offspring birthweight. The link between mTOR-associated birthweight outcomes and offspring metabolic health trajectory with a focus on sexual dimorphism are discussed. Both human physiology and animal models are summarized to facilitate in depth understanding. GDM, PCOS and fetal overgrowth are associated with increased placental mTOR, whereas FGR, MNR and maternal smoking are linked to decreased placental mTOR activity. Generally, birth weight is reduced in complications with decreased mTOR (i.e., FGR, MNR, maternal smoking) and higher with increased mTOR (GDM, PCOS). Offspring display obesity or a higher body mass index in childhood and adulthood, impaired glucose and insulin tolerance in adulthood, and deficiencies in pancreatic beta-cell mass and function compared to offspring from uncomplicated pregnancies. Defining causal players in the fetal programming of offspring metabolic health across the lifespan will aid in stopping the vicious cycle of obesity and type II diabetes.

Peptidyl Inhibition of Nox2 Enhances Stress Response and Mitigates Muscle Fiber Atrophy with Simulated Microgravity

Skeletal muscle is a dynamic and pliable tissue, which contains up to 75% of all proteins in the body. The regulation of proteins synthesis and degradation determines the muscle responsive adaptations to alterations in mechanical loading, nutrition availability, exercise intensity, etc. The pathway by which skeletal muscle cells are equipped with the ability of converting changes in mechanical force to biochemical signals is entitled “mechanotransduction”. The translocation of mu-splice variant of neuronal Nitric Oxide Synthase (nNOSµ) from sarcolemma to sarcoplasm is ascertained a causal player in mechanotransduction in unloaded skeletal muscle. In addition, Reactive Oxygen Species (ROS) are demonstrated to be causal in the translocation of nNOSμ, suppression of anabolic signaling, and muscle fiber atrophy. Mitochondria is known to be one of the sources of ROS production during unloading. Moreover, the sarcolemmal NADPH oxidase-2 (Nox2), a mechano-sensitive source of ROS, is also revealed to be increased in unloaded skeletal muscles. The upregulated Nox2 would further contribute directly to nNOSμ translocation, perturbation in heat shock proteins (e.g., HSP70) and antioxidant enzyme (e.g., MnSOD), and muscle atrophy during unloading. Upstream mechanisms and downstream effects involved in the regulation of Nox2 activity during exposure to weightlessness are partially understood due to their complex nature. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor reported to protect cells in response to stressors. We postulate that peptidyl inhibition of Nox2 would alleviate the disruption of HSP70, MnSOD, and Nrf2, and the translocation of sarcolemmal nNOS during unloading and, therefore, muscle fiber atrophy. 4-month-old F344 rats were partitioned into 3 groups: ambulatory control (CON), hindlimb unloaded (HU), and hindlimb unloaded + 5 mg/kg/day peptidyl Nox2 inhibitor gp91dstat (HUG). At the end of the study (7 days), soleus muscles were removed and frozen in isopentane chilled in liquid N2. Samples were then fractionized for further analysis by western blot, immunofluorescence, etc. Our data revealed that exposure to mechanical unloading significantly decreases the protein level of HSP70, MnSOD, and NrF2. Although, no significant protective effect of gp91dstat treatment was observed in MnSOD protein abundance, Nrf2 and HSP70 levels were notably protected during unloading when Nox2 was inhibited, suggestive of the preservation of antioxidant protection. In addition, the nNOS sarcolemmal activity, which was reduced in the hindlimb unloading study group, was elevated with the use of peptidyl Nox2 inhibitor. In conclusion, this study provides direct evidence that upregulated Nox2 can act as a causal role in skeletal muscle mechanotransduction and cellular remodeling during mechanical unloading.

Growth differentiation factor 15 in adverse cardiac remodelling: from biomarker to causal player.

Heart failure is a growing health issue as a negative consequence of improved survival upon myocardial infarction, unhealthy lifestyle, and the ageing of our population. The large and complex pathology underlying heart failure makes diagnosis and especially treatment very difficult. There is an urgent demand for discriminative biomarkers to aid disease management of heart failure. Studying cellular pathways and pathophysiological mechanisms contributing to disease initiation and progression is crucial for understanding the disease process and will aid to identification of novel biomarkers and potential therapeutic targets. Growth differentiation factor 15 (GDF15) is a proven valuable biomarker for different pathologies, including cancer, type 2 diabetes, and cardiovascular diseases. Although the prognostic value of GDF15 in heart failure is robust, the biological function of GDF15 in adverse cardiac remodelling is not fully understood. GDF15 is a distant member of the transforming growth factor‐β family and involved in various biological processes including inflammation, cell cycle, and apoptosis. However, more research is suggesting a role in fibrosis, hypertrophy, and endothelial dysfunction. As GDF15 is a pleiotropic protein, elucidating the exact role of GDF15 in complex disease processes has proven to be a challenge. In this review, we provide an overview of the role GDF15 plays in various intracellular and extracellular processes underlying heart failure, and we touch upon crucial points that need consideration before GDF15 can be integrated as a biomarker in standard care or when considering GDF15 for therapeutic intervention.

Klinefelter syndrome comorbidities linked to increased X chromosome gene dosage and altered protein interactome activity.

Klinefelter syndrome (KS) (47,XXY) is the most common male sex chromosome aneuploidy. Diagnosis and clinical supervision remain a challenge due to varying phenotypic presentation and insufficient characterization of the syndrome. Here we combine health data-driven epidemiology and molecular level systems biology to improve the understanding of KS and the molecular interplay influencing its comorbidities. In total, 78 overrepresented KS comorbidities were identified using in- and out-patient registry data from the entire Danish population covering 6.8 million individuals. The comorbidities extracted included both clinically well-known (e.g. infertility and osteoporosis) and still less established KS comorbidities (e.g. pituitary gland hypofunction and dental caries). Several systems biology approaches were applied to identify key molecular players underlying KS comorbidities: Identification of co-expressed modules as well as central hubs and gene dosage perturbed protein complexes in a KS comorbidity network build from known disease proteins and their protein–protein interactions. The systems biology approaches together pointed to novel aspects of KS disease phenotypes including perturbed Jak-STAT pathway, dysregulated genes important for disturbed immune system (IL4), energy balance (POMC and LEP) and erythropoietin signalling in KS. We present an extended epidemiological study that links KS comorbidities to the molecular level and identify potential causal players in the disease biology underlying the identified comorbidities.

ANGPTL2 - A New Causal Player in Accelerating Heart Disease Development in the Aging.

In parallel with the increase in the number of elderly people worldwide, the number of patients with heart disease is also rapidly increasing. Of the heart diseases, cardiovascular disease (CVD) and heart failure (HF) are strongly associated with adverse health outcomes that decrease productivity in later years. Recently, ANGPTL2, a secreted glycoprotein and member of the angiopoietin-like protein family, has received attention as a causal player in the development of CVD and HF. Prolonged ANGPTL2 autocrine/paracrine signaling in vascular tissue leads to chronic inflammation and pathologic tissue remodeling, accelerating CVD development. Excess ANGPTL2 autocrine/paracrine signaling induced in the pathologically stressed heart accelerates cardiac dysfunction by decreasing myocardial energy metabolism. Conversely, ANGPTL2 inactivation in vascular tissue and the heart delays development or progression of CVD and HF, respectively. Moreover, there is increased evidence for an association between elevated circulating ANGPTL2 levels and CVD and HF. Interestingly, ANGPTL2 expression is also associated with cellular senescence, which may promote premature aging and development of aging-associated diseases, including CVD and HF. Overall, ANGPTL2 autocrine/paracrine signaling is a new factor in accelerating heart disease development in the aging. Here, we focus on current topics relevant to ANGPTL2 function in heart disease.

MicroRNA in late lung development and bronchopulmonary dysplasia: the need to demonstrate causality.

MicroRNA are emerging as powerful regulators of cell differentiation and tissue and organ development. Several microRNA have been described to play a role in branching morphogenesis, a key step in early lung development. However, considerably less attention has been paid to microRNA as regulators of the process of secondary septation, which drives lung alveolarization during late lung development. Secondary septation is severely perturbed in bronchopulmonary dysplasia (BPD), a common complication of preterm birth characterized by blunted alveolarization. A number of studies to date have reported microRNA microarray screens in animal models of BPD; however, only two studies have attempted to demonstrate causality. Although the expression of miR-150 was altered in experimental BPD, a miR-150−/− knockout mouse did not exhibit appreciable protection in a BPD animal model. Similarly, while the expression of miR-489 in the lung was reduced in clinical and experimental BPD, antagomiR and over-expression approaches could not validate a role for miR-489 in the impaired alveolarization associated with experimental BPD. This mini-review aims to highlight microRNA that have been revealed by multiple microarray studies to be potential causal players in normal and pathological alveolarization. Additionally, the challenges faced in attempting to demonstrate a causal role for microRNA in lung alveolarization are discussed. These include the tremendous variability in the animal models employed, and the limitations and advantages offered by the available tools, including antagomiRs and approaches for the validation of a specific microRNA-mRNA interaction during lung alveolarization.

Abstract A66: The CD73+/CD24- subpopulation of ovarian cancer cells is enriched in cancer stem cells.

Abstract Recent data suggest that drug resistance and/or disease recurrence in ovarian cancer (OC) are driven by a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of OC. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional traits. Specifically, we are pursuing these objectives through the accomplishment of the following milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow has been applied to surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. OCSC have been derived from primary cultures exploiting their ability to resist anoikis and form monoclonal spheroids when cultured under nonadherent conditions. The transcriptome of OC-derived spheroids was compared with that of their normal counterparts, yielding a set of differentially expressed genes. Among these, for the initial characterization we have selected two cell surface markers CD73, upregulated in spheroids, and CD24, downregulated in spheroids. The CD73+/CD24- subpopulation was enriched in sphere-forming cells and, most importantly, exhibited higher tumorigenic capacity when xenografted in immunocompromised mice. Thus, by utilizing clinically relevant samples we have characterized the transcriptome of OCSC and of their normal counterparts. Furthermore, we found that the CD73+/CD24- phenotype in ovarian cancer is associated to CSC-like traits. On one hand, our approach revealed novel OCSC biomarkers that can be exploited for imaging and purification purposes. On the other hand, we have identified potential therapeutic targets, such as CD73, that might set the stage for innovative treatments aimed at the eradication of OC through the elimination of OCSC. Citation Format: Michela Lupia, Giovanni Bertalot, Pier Paolo Di Fiore, Nicoletta Colombo, Fabrizio Bianchi, Ugo Cavallaro. The CD73+/CD24- subpopulation of ovarian cancer cells is enriched in cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A66.

Androgen receptor profiling predicts prostate cancer outcome.

Prostate cancer is the second most prevalent malignancy in men. Biomarkers for outcome prediction are urgently needed, so that high-risk patients could be monitored more closely postoperatively. To identify prognostic markers and to determine causal players in prostate cancer progression, we assessed changes in chromatin state during tumor development and progression. Based on this, we assessed genomewide androgen receptor/chromatin binding and identified a distinct androgen receptor/chromatin binding profile between primary prostate cancers and tumors with an acquired resistance to therapy. These differential androgen receptor/chromatin interactions dictated expression of a distinct gene signature with strong prognostic potential. Further refinement of the signature provided us with a concise list of nine genes that hallmark prostate cancer outcome in multiple independent validation series. In this report, we identified a novel gene expression signature for prostate cancer outcome through generation of multilevel genomic data on chromatin accessibility and transcriptional regulation and integration with publically available transcriptomic and clinical datastreams. By combining existing technologies, we propose a novel pipeline for biomarker discovery that is easily implementable in other fields of oncology.

Abstract 1414: Molecular and functional characterization of ovarian cancer stem cells

Abstract Epithelial Ovarian Cancer (OC) is one of the leading causes of death for women, and no significant therapeutic progress has been made in the last decades. Recent data suggest that one of the mechanisms accounting for resistant and/or relapsing disease is a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of the disease. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional profile. Specifically, we are pursuing the following objectives through the accomplishment of these milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow is being applied to a series of fresh surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. Thus, we obtained a collection of primary cells that recapitulate many traits of the original tissue both in vitro and in vivo. The gene expression profiles of sphere-forming SC has been compared to that of adherent, parental cells, aimed at identifying stemness-associated genes. This screening has been extended to OC, OSE, and FTE, resulting in a set of genes that are differentially expressed in OCSC as compared to their normal counterparts. A subset of such genes has been selected for the subsequent validation, both as OCSC biomarkers that could have clinical applications and as drivers in the biological and pathogenic function of OCSC. Our study might set the stage for innovative therapeutic approaches aimed at the selective elimination of OCSC, thus preventing tumor recurrence and chemoresistance, namely the two main causes of ovarian cancer lethality. Citation Format: Michela Lupia, Giovanni Bertalot, Nicoletta Colombo, Stefano Confalonieri, Pier Paolo Di Fiore, Ugo Cavallaro. Molecular and functional characterization of ovarian cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2015-1414

External Actors and Regime Change

This article explores how the study of post-communism has transformed comparative politics by adding a substantial role for external actors to existing theories of democratization. The big, overarching finding is dramatic: external actors can, under certain conditions, tip the balance in favor of democracy by offering strong rewards to elites, conditional on complying with tough requirements. External actors can also influence the performance of the state—and how the state treats its citizens. The main causal player is the European Union. This simple finding is consequential for all three major strands of democratization theory. The article goes on to explore how the leverage of the European Union has shaped the trajectories of political change in the new and credible future EU members in East Central and South Eastern Europe. The great variation among existing and candidate members can be largely explained by different domestic conditions, even though there are many areas where the EU’s use of its leverage could have been improved.

Identification of MAGEA antigens as causal players in the development of tamoxifen-resistant breast cancer

The antiestrogen tamoxifen is a well-tolerated, effective treatment for estrogen receptor-α-positive (ER+) breast cancer, but development of resistance eventually limits its use. Here we show that expression of MAGEA2, and related members of this cancer-testis antigen family, is upregulated in tamoxifen-resistant tumor cells. Expression of MAGEA2 in tumor lines grown in vitro or as xenografts led to continued proliferation in the presence of tamoxifen. At the molecular level, we demonstrate that MAGEA2 protein localizes to the nucleus and forms complexes with p53 and ERα, resulting in repression of the p53 pathway but increased ER-dependent signaling. In a series of ER+, tamoxifen-treated breast cancer patients, we show a highly significant (P=0.006) association between MAGEA (melanoma-associated antigen) expression and reduced overall survival, confirming the clinical significance of our observations.

Neuroscience as a Human Science: Integrating Phenomenology and Empiricism in the Study of Action and Consciousness

This paper considers where contemporary neuroscience leaves us in terms of how human consciousness fits into the material world, and whether consciousness is reducible to merely mechanical physical systems, or on the contrary whether consciousness is a self-organizing system that can in a sense use the brain for its own purposes. The paper discusses how phenomenology can be integrated with new findings about “neural plasticity” to yield new approaches to the mind–body problem and the place of consciousness as a causal player in the physical world. By phenomenology, I mean simply any attempt to have introspective or reflective access to the meaning of our own conscious states, and to carefully take account of the notorious pitfalls of subjective introspection (often subsumed within the concept of “folk psychology” in the empirically oriented cognitive theory literature).

Genetic analysis of albuminuria in aging mice and concordance with loci for human diabetic nephropathy found in a genome-wide association scan

Aging in the kidney can cause albuminuria, and discovering molecular mechanisms responsible for this might offer a new perspective on the etiology of this abnormality. Haplotype association mapping in the mouse is a novel approach which uses the haplotypes of the relatively closely related mouse inbred strains and the phenotypic variation among these strains to find associations between haplotypes and phenotype. The albumin-to-creatinine ratios, a measure of urinary albumin excretion, were determined in 30 inbred mouse strains at 12, 18, and 24 months of age. Mapping was performed for males and females separately at all three time points using a high density set of 63,222 single-nucleotide polymorphisms to determine genetic loci involved in albuminuria. One significant and eight suggestive loci were found, some of which map to previously identified loci for traits associated with kidney damage in the mouse, but with a much higher resolution thus narrowing their chromosomal location. These nine loci were then compared with genome-wide association scans for diabetic nephropathy (DN) in human type I diabetes. Our study found that two of the nine mouse loci for age-related albuminuria were significantly associated with DN and consistent across male and female strata. This suggests common underlying genes predispose to kidney disease in mice and humans.

Building a new bridge between metabolism, free radicals and longevity

Building a new bridge between metabolism, free radicals and longevity

C-reactive protein and cardiovascular disease: Weighing the evidence

C-reactive protein (CRP) has been widely promoted as a strong, independent predictor of cardiovascular events and metabolic syndrome, both in general populations and in patients with clinical cardiovascular disease, and as a causal player in atherothrombosis. However, recent evidence shows that the association of CRP with cardiovascular events is weaker than previously thought, that it may be largely attributed to confounding by established causal risk factors, and that CRP is, therefore, probably not a clinically useful risk predictor. The lack of association of noncoding CRP gene polymorphisms (which determine different baseline CRP values) with coronary events or metabolic syndrome does not support a causal role for CRP, and most of the putatively proatherothrombotic in vitro effects claimed for CRP were caused by contaminants in commercial CRP preparations and not by CRP. Future clinical trials of specific CRP inhibitors now in development could directly test the contribution of CRP to pathogenesis of cardiovascular disease.