Current State of Preeclampsia Mouse Models: Approaches, Relevance, and Standardization

Preeclampsia (PE) is a multisystemic, pregnancy-specific disorder and a leading cause of maternal and fetal death. PE is also associated with an increased risk for chronic morbidities later in life for mother and offspring. Abnormal placentation or placental function has been well-established as central to the genesis of PE; yet much remains to be determined about the factors involved in the development of this condition. Despite decades of investigation and many clinical trials, the only definitive treatment is parturition. To better understand the condition and identify potential targets preclinically, many approaches to simulate PE in mice have been developed and include mixed mouse strain crosses, genetic overexpression and knockout, exogenous agent administration, surgical manipulation, systemic adenoviral infection, and trophoblast-specific gene transfer. These models have been useful to investigate how biological perturbations identified in human PE are involved in the generation of PE-like symptoms and have improved the understanding of the molecular mechanisms underpinning the human condition. However, these approaches were characterized by a wide variety of physiological endpoints, which can make it difficult to compare effects across models and many of these approaches have aspects that lack physiological relevance to this human disorder and may interfere with therapeutic development. This report provides a comprehensive review of mouse models that exhibit PE-like symptoms and a proposed standardization of physiological characteristics for analysis in murine models of PE

Sequential and Batch Processing Methods of the EBP Learning Algorithm

Placental abnormalities can cause Pregnancy-Associated Disorders, including preeclampsia, intrauterine growth restriction, and placental insufficiency, resulting in complications for both the mother and fetus. Trophoblast cells within the labyrinthine layer of the placenta facilitate the exchange of nutrients, gases, and waste between mother and fetus; therefore, the development of this cell layer is critical for fetal development. As trophoblast cells differentiate, it is assumed their metabolism changes with their energy requirements. We hypothesize that proper regulation of trophoblast metabolism is a key component of normal placental development; therefore, we examined the role of AMP-activated kinase (AMPK, PRKAA1/2), a sensor of cellular energy status. Our previous studies have shown that AMPK knockdown alters both trophoblast differentiation and nutrient transport. In this study, AMPKα1/2 shRNA was used to investigate the metabolic effects of AMPK knockdown on SM10 placental labyrinthine progenitor cells before and after differentiation. Extracellular flux analysis confirmed that AMPK knockdown was sufficient to reduce trophoblast glycolysis, mitochondrial respiration, and ATP coupling efficiency. A reduction in AMPK in differentiated trophoblasts also resulted in increased mitochondrial volume. These data indicate that a reduction in AMPK disrupts cellular metabolism in both progenitors and differentiated placental trophoblasts. This disruption correlates to abortive trophoblast differentiation that may contribute to the development of Pregnancy-Associated Disorders

Mouse models of preeclampsia with preexisting comorbidities

Preeclampsia is a pregnancy-specific condition and a leading cause of maternal and fetal morbidity and mortality. It is thought to occur due to abnormal placental development or dysfunction, because the only known cure is delivery of the placenta. Several clinical risk factors are associated with an increased incidence of preeclampsia including chronic hypertension, diabetes, autoimmune conditions, kidney disease, and obesity. How these comorbidities intersect with preeclamptic etiology, however, is not well understood. This may be due to the limited number of animal models as well as the paucity of studies investigating the impact of these comorbidities. This review examines the current mouse models of chronic hypertension, pregestational diabetes, and obesity that subsequently develop preeclampsia-like symptoms and discusses how closely these models recapitulate the human condition. Finally, we propose an avenue to expand the development of mouse models of preeclampsia superimposed on chronic comorbidities to provide a strong foundation needed for preclinical testing

Metabolic Characterization of MPNST Cell Lines

Malignant transformation is the process by which cells develop cancer properties. While many causes for malignant transformation are known (i.e. common genetic mutations and/or exposure to toxins or viruses), the basic requirements that allow a cell to stay alive with altered nutrient and energy requirements are just now being studied. In some tumor types malignant cells undergo changes that result in metabolic differences compared to normal cells. These can include defects in mitophagy resulting in an accumulation of dysfunctional mitochondria and/or a metabolic switch resulting in increased glycolysis, termed the Warburg effect. Increased tumor growth and metastasis have also been associated with mitochondrial DNA mutations in some tumor types. In this study, we characterized the mitochondrial function of malignant peripheral nerve sheath tumor (MPNST) cell lines commonly used to study malignant transformation in Neurofibromatosis Type I. We identified metabolic differences between NF1-wildtype (STS26T) and NF1-deficient (ST88-14, 90-8, and S462) MPNST cell lines by measuring extracellular acidification and oxygen consumption, mitochondrial respiration protein expression, and ROS levels. Similar to findings from other malignant tumors, all MPNST cell lines were more glycolytic compared to non-tumorigenic normal human Schwann cells and surprisingly NF1-deficiency correlated with lower glycolytic and mitochondrial respiratory rate compared to wildtype MPNST. Mitochondrial respiratory rates and respiratory protein expression were significantly lower in the NF1-deficient MPNST cell lines when compared to NF1-wildtype MPNST cells. These findings demonstrate that neurofibromin affects glycolysis and mitochondrial respiration in malignant cells

DIPG-30. Differential Hypoxic Response inHuman Dipg Cell Lines

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) are rare and aggressive childhood tumors, with an abysmal prognosis and limited experimental models available for study. A recent report showed that DIPG are hypoperfused compared to surrounding brain tissue, suggesting that the tumor cells are exposed to a hypoxic microenvironment. This stimulus may induce widespread transcriptional changes through activation of Hypoxia-inducible Factors (HIF), which have been associated with invasion, metastasis, angiogenesis, and resistance to radiation therapy and chemotherapy. Therefore, in vitro models using DIPG cells cultured in conditions of normal oxygen tension may not be representative of in vivo tumors because of artificial differences in HIF activation. METHODS: To test cellular responses to hypoxia, human DIPG cell lines with H3.1 K27M and H3.3 K27M mutations were treated with the hypoxia-mimetic compound, cobalt (II) chloride, for 24 hours. Simulated hypoxic responses were assayed using Western blot analysis, proliferation assays, and extracellular flux analysis. RESULTS: The H3.3 K27M cell line increased expression of HIF1a with cobalt (II) chloride treatment and correlated with an increase in glycolytic rates and increased expression of proteins involved in glucose transport and glycolysis. The H3.1 K27M cell line increased expression of HIF2a and not HIF1a after cobalt (II) chloride treatment, correlating with an increase in some glycolysis-associated proteins but no change in glycolytic rate. CONCLUSION: These data suggest that the effects of a hypoxic microenvironment should be considered in experimental models using cultured DIPG cell lines to understand differences in cell behavior, patient prognosis, and predicted treatment responses. Activation of specific HIF isoforms may underlie differences in hypoxic responses among cell line

DIPG-30. Differential Hypoxic Response inHuman Dipg Cell Lines

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) are rare and aggressive childhood tumors, with an abysmal prognosis and limited experimental models available for study. A recent report showed that DIPG are hypoperfused compared to surrounding brain tissue, suggesting that the tumor cells are exposed to a hypoxic microenvironment. This stimulus may induce widespread transcriptional changes through activation of Hypoxia-inducible Factors (HIF), which have been associated with invasion, metastasis, angiogenesis, and resistance to radiation therapy and chemotherapy. Therefore, in vitro models using DIPG cells cultured in conditions of normal oxygen tension may not be representative of in vivo tumors because of artificial differences in HIF activation. METHODS: To test cellular responses to hypoxia, human DIPG cell lines with H3.1 K27M and H3.3 K27M mutations were treated with the hypoxia-mimetic compound, cobalt (II) chloride, for 24 hours. Simulated hypoxic responses were assayed using Western blot analysis, proliferation assays, and extracellular flux analysis. RESULTS: The H3.3 K27M cell line increased expression of HIF1a with cobalt (II) chloride treatment and correlated with an increase in glycolytic rates and increased expression of proteins involved in glucose transport and glycolysis. The H3.1 K27M cell line increased expression of HIF2a and not HIF1a after cobalt (II) chloride treatment, correlating with an increase in some glycolysis-associated proteins but no change in glycolytic rate. CONCLUSION: These data suggest that the effects of a hypoxic microenvironment should be considered in experimental models using cultured DIPG cell lines to understand differences in cell behavior, patient prognosis, and predicted treatment responses. Activation of specific HIF isoforms may underlie differences in hypoxic responses among cell line

Current State of Preeclampsia Mouse Models: Approaches, Relevance, and Standardization

Preeclampsia (PE) is a multisystemic, pregnancy-specific disorder and a leading cause of maternal and fetal death. PE is also associated with an increased risk for chronic morbidities later in life for mother and offspring. Abnormal placentation or placental function has been well-established as central to the genesis of PE; yet much remains to be determined about the factors involved in the development of this condition. Despite decades of investigation and many clinical trials, the only definitive treatment is parturition. To better understand the condition and identify potential targets preclinically, many approaches to simulate PE in mice have been developed and include mixed mouse strain crosses, genetic overexpression and knockout, exogenous agent administration, surgical manipulation, systemic adenoviral infection, and trophoblast-specific gene transfer. These models have been useful to investigate how biological perturbations identified in human PE are involved in the generation of PE-like symptoms and have improved the understanding of the molecular mechanisms underpinning the human condition. However, these approaches were characterized by a wide variety of physiological endpoints, which can make it difficult to compare effects across models and many of these approaches have aspects that lack physiological relevance to this human disorder and may interfere with therapeutic development. This report provides a comprehensive review of mouse models that exhibit PE-like symptoms and a proposed standardization of physiological characteristics for analysis in murine models of PE

DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways

DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways

DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways