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

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

KIAA1549-BRAF Expression Establishes A Permissive Tumor Microenvironment Through Nfκb-Mediated CCL2 Production

KIAA1549-BRAF is the most frequently identified genetic mutation in sporadic pilocytic astrocytoma (PA), creating a fusion BRAF (f-BRAF) protein with increased BRAF activity. Fusion-BRAF-expressing neural stem cells (NSCs) exhibit increased cell growth and can generate glioma-like lesions following injection into the cerebella of naïve mice. Increased Iba1+ monocyte (microglia) infiltration is associated with murine f-BRAF-expressing NSC-induced glioma-like lesion formation, suggesting that f-BRAF-expressing NSCs attract microglia to establish a microenvironment supportive of tumorigenesis. Herein, we identify Ccl2 as the chemokine produced by f-BRAF-expressing NSCs, which is critical for creating a permissive stroma for gliomagenesis. In addition, f-BRAF regulation of Ccl2 production operates in an ERK- and NFκB-dependent manner in cerebellar NSCs. Finally, Ccr2-mediated microglia recruitment is required for glioma-like lesion formation in vivo, as tumor do not form in Ccr2-deficient mice following f-BRAF-expressing NSC injection. Collectively, these results demonstrate that f-BRAF expression creates a supportive tumor microenvironment through NFκB-mediated Ccl2 production and microglia recruitment

The Effects of Dexamethasone on Mitogen Activated Protein Kinase-14 Signaling in Diffuse Intrinsic Pontine Glioma (DIPG) Cells

Objective: Determine the effects of dexamethasone (DEX) on diffuse intrinsic pontine glioma (DIPG) cell behavior. Background: Patients with DIPG routinely receive DEX to treat vasogenic edema, but the direct effects on tumor growth and sensitivity to other therapies are unknown. Previous studies on glioblastoma cell lines have suggested that DEX interferes with nuclear translocation of p38α mitogen activated protein kinase-14 (MAPK-14), which may be required for cytokine production, cell mobility, and invasion, in addition to a metabolic shift from glycolysis to the pentose phosphate pathway. We tested the hypothesis that DEX prevents nuclear translocation of MAPK-14 in DIPG, resulting in metabolic reprogramming and decreased cell survival, migration, and colony formation. Design/Methods: We used immunocytochemistry to measure the nuclear translocation of total and phosphorylated MAPK-14 in cultured SU-DIPG-IV cells following treatment with DEX or vehicle. We also treated cells with DEX, with or without the clinically relevant chemotherapies panobinostat and imatinib on logarithmic dose curves, and measured proliferation rate, viability and apoptosis with combined assays of trypan blue exclusion and caspase 3/7 activation. Results: Treatment with DEX reduced nuclear localization of phosphorylated MAPK-14 in DIPG cells, but did not affect basal or chemotherapy-inhibited rates of cell proliferation, viability or apoptosis. Conclusions: DEX, a commonly prescribed medication for DIPG patients, decreased nuclear localization of phosphorylated MAPK-14 but had no apparent effects on basal or chemotherapy-inhibited rates of cell proliferation, viability or apoptosis. We are currently exploring the effects of DEX and MAPK-14 signaling on cell metabolism, migration, and colony formation

The Effects of Dexamethasone on Mitogen Activated Protein Kinase-14 Signaling in Diffuse Intrinsic Pontine Glioma (DIPG) Cells

Objective: Determine the effects of dexamethasone (DEX) on diffuse intrinsic pontine glioma (DIPG) cell behavior. Background: Patients with DIPG routinely receive DEX to treat vasogenic edema, but the direct effects on tumor growth and sensitivity to other therapies are unknown. Previous studies on glioblastoma cell lines have suggested that DEX interferes with nuclear translocation of p38α mitogen activated protein kinase-14 (MAPK-14), which may be required for cytokine production, cell mobility, and invasion, in addition to a metabolic shift from glycolysis to the pentose phosphate pathway. We tested the hypothesis that DEX prevents nuclear translocation of MAPK-14 in DIPG, resulting in metabolic reprogramming and decreased cell survival, migration, and colony formation. Design/Methods: We used immunocytochemistry to measure the nuclear translocation of total and phosphorylated MAPK-14 in cultured SU-DIPG-IV cells following treatment with DEX or vehicle. We also treated cells with DEX, with or without the clinically relevant chemotherapies panobinostat and imatinib on logarithmic dose curves, and measured proliferation rate, viability and apoptosis with combined assays of trypan blue exclusion and caspase 3/7 activation. Results: Treatment with DEX reduced nuclear localization of phosphorylated MAPK-14 in DIPG cells, but did not affect basal or chemotherapy-inhibited rates of cell proliferation, viability or apoptosis. Conclusions: DEX, a commonly prescribed medication for DIPG patients, decreased nuclear localization of phosphorylated MAPK-14 but had no apparent effects on basal or chemotherapy-inhibited rates of cell proliferation, viability or apoptosis. We are currently exploring the effects of DEX and MAPK-14 signaling on cell metabolism, migration, and colony formation

KIAA1549-BRAF Expression Establishes a Permissive Tumor Microenvironment Through NFκB-Mediated CCL2 Production

KIAA1549-BRAF is the most frequently identified genetic mutation in sporadic pilocytic astrocytoma (PA), creating a fusion BRAF (f-BRAF) protein with increased BRAF activity. Fusion-BRAF-expressing neural stem cells (NSCs) exhibit increased cell growth and can generate glioma-like lesions following injection into the cerebella of naïve mice. Increased Iba1+ monocyte (microglia) infiltration is associated with murine f-BRAF-expressing NSC-induced glioma-like lesion formation, suggesting that f-BRAF-expressing NSCs attract microglia to establish a microenvironment supportive of tumorigenesis. Herein, we identify Ccl2 as the chemokine produced by f-BRAF-expressing NSCs, which is critical for creating a permissive stroma for gliomagenesis. In addition, f-BRAF regulation of Ccl2 production operates in an ERK- and NFκB-dependent manner in cerebellar NSCs. Finally, Ccr2-mediated microglia recruitment is required for glioma-like lesion formation in vivo, as tumor do not form in Ccr2-deficient mice following f-BRAF-expressing NSC injection. Collectively, these results demonstrate that f-BRAF expression creates a supportive tumor microenvironment through NFκB-mediated Ccl2 production and microglia recruitment

Erratum to “KIAA1549-BRAF Expression Establishes a Permissive Tumor Microenvironment Through NFκB-Mediated CCL2 Production

The order of authors in the above-referenced article should have read as follows: Ran Chen *, Chanel Keoni †, Christopher A. Waker ‡, Robert M. Lober †, ‡, §, Yi-Hsien Chen ¶, David H. Gutmann *. The publisher would like to apologize for any inconvenience caused