Experimental venous thrombus resolution is driven by IL-6 mediated monocyte actions.

Deep venous thrombosis and residual thrombus burden correlates with circulating IL-6 levels in humans. To investigate the cellular source and role of IL-6 in thrombus resolution, Wild type C57BL/6J (WT), and IL-6-/- mice underwent induction of VT via inferior vena cava (IVC) stenosis or stasis. Vein wall (VW) and thrombus were analyzed by western blot, immunohistochemistry, and flow cytometry. Adoptive transfer of WT bone marrow derived monocytes was performed into IL6-/- mice to assess for rescue. Cultured BMDMs from WT and IL-6-/- mice underwent quantitative real time PCR and immunoblotting for fibrinolytic factors and matrix metalloproteinase activity. No differences in baseline coagulation function or platelet function were found between WT and IL-6-/- mice. VW and thrombus IL-6 and IL-6 leukocyte-specific receptor CD126 were elevated in a time-dependent fashion in both VT models. Ly6Clo Mo/MØ were the predominant leukocyte source of IL-6. IL-6-/- mice demonstrated larger, non-resolving stasis thrombi with less neovascularization, despite a similar number of monocytes/macrophages (Mo/MØ). Adoptive transfer of WT BMDM into IL-6-/- mice undergoing stasis VT resulted in phenotype rescue. Human specimens of endophlebectomized tissue showed co-staining of Monocyte and IL-6 receptor. Thrombosis matrix analysis revealed significantly increased thrombus fibronectin and collagen in IL-6-/- mice. MMP9 activity in vitro depended on endogenous IL-6 expression in Mo/MØ, and IL-6-/- mice exhibited stunted matrix metalloproteinase activity. Lack of IL-6 signaling impairs thrombus resolution potentially via dysregulation of MMP-9 leading to impaired thrombus recanalization and resolution. Restoring or augmenting monocyte-mediated IL-6 signaling in IL-6 deficient or normal subjects, respectively, may represent a non-anticoagulant target to improve thrombus resolution.

Therapeutic targeting of prenatal pontine ID1 signaling in diffuse midline glioma.

BACKGROUND: Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. METHODS: Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. RESULTS: Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. CONCLUSIONS: H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

Ly6CLo Monocyte/Macrophages are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis.

Venous thrombosis (VT) resolution is a complex process, resembling sterile wound healing. Infiltrating blood-derived monocyte/macrophages (Mo/MΦs) are essential for the regulation of inflammation in tissue repair. These cells differentiate into inflammatory (CD11b+Ly6CHi) or proreparative (CD11b+Ly6CLo) subtypes. Previous studies have shown that infiltrating Mo/MΦs are important for VT resolution, but the precise roles of different Mo/MΦs subsets are not well understood. Utilizing murine models of stasis and stenosis inferior vena cava thrombosis in concert with a Mo/MΦ depletion model (CD11b-diphtheria toxin receptor [DTR]-expressing mice), we examined the effect of Mo/MΦ depletion on thrombogenesis and VT resolution. In the setting of an 80 to 90% reduction in circulating CD11b+Mo/MΦs, we demonstrated that Mo/MΦs are not essential for thrombogenesis, with no difference in thrombus size, neutrophil recruitment, or neutrophil extracellular traps found. Conversely, CD11b+Mo/MΦ are essential for VT resolution. Diphtheria toxoid (DTx)-mediated depletion after thrombus creation depleted primarily CD11b+Ly6CLo Mo/MΦs and resulted in larger thrombi. DTx-mediated depletion did not alter CD11b+Ly6CHi Mo/MΦ recruitment, suggesting a protective effect of CD11b+Ly6CLo Mo/MΦs in VT resolution. Confirmatory Mo/MΦ depletion with clodronate lysosomes showed a similar phenotype, with failure to resolve VT. Adoptive transfer of CD11b+Ly6CLo Mo/MΦs into Mo/MΦ-depleted mice reversed the phenotype, restoring normal thrombus resolution. These findings suggest that CD11b+Ly6CLo Mo/MΦs are essential for normal VT resolution, consistent with the known proreparative function of this subset, and that further study of Mo/MΦ subsets may identify targets for immunomodulation to accelerate and improve thrombosis resolution.

Acute experimental venous thrombosis impairs venous relaxation but not contraction.

OBJECTIVE: Venous thrombosis (VT) damages the vein wall, both physically by prolonged distension and from inflammation. These factors contribute to post-thrombotic syndrome (PTS). Interleukin (IL)-6 might play a role in experimental PTS and vein wall responses. Previous assessments of post-thrombotic vein wall injury used static measures such as histologic examination and immunologic assays. The purpose of the present study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute VT. METHODS: Wild-type (WT) C57BL/6 mice were used to determine the baseline vein wall passive tension on a DMT 610m myograph (DMT-USA, Inc., Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6-/- mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), with no-surgery mice used as controls. The mice were harvested at 2 days (2D) and analyzed using a myograph. The vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. The endothelial responses to VT were quantified by CD31 immunohistochemistry, Greiss assay, polymerase chain reaction, and Evans blue assay. RESULTS: Optimal passive tension was determined to be 2 mN, with an optimal concentration of Phe and Ach of 7E-3M and 1E-5M, respectively. No significant differences were found in the contractions when exposed to Phe between the WT control, WT 2D ligation, and WT 2D stenosis IVC segments and the IL-6-/- mice with and without thrombus (P > .05 for all). When treated with Ach, significantly more relaxation was found in the nonthrombosed control IVC segments than in those IVC segments that had had a 2D thrombus from either ligation- or stenosis-derived thrombotic mechanisms in both WT and IL-6-/- mice. CD31 staining showed ∼20% less luminal endothelium after stasis thrombosis (P ≤ .01) but no loss in the controls (P > .05). Evans blue staining showed a trend toward increased leakiness in post-thrombotic vein walls. No significant difference in the endothelial gene markers or nitric oxide production was found. CONCLUSIONS: Compared with the controls, acute thrombosis in the total or partial stasis models did not impair IVC contractile responses, suggesting no effect on the medial vascular smooth muscle response. The relaxation response was significantly reduced in the post-thrombotic groups, likely from direct endothelial injury. These findings suggest, at acute points, that VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and might be a mechanism that promotes PTS.

The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.

Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period.

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepRKiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IRKiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.

Genetic factors modulate the impact of pubertal androgen excess on insulin sensitivity and fertility.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. The syndrome is caused by a combination of environmental influences and genetic predisposition. Despite extensive efforts, the heritable factors contributing to PCOS development are not fully understood. The objective of this study was to test the hypothesis that genetic background contributes to the development of a PCOS-like reproductive and metabolic phenotype in mice exposed to excess DHEA during the pubertal transition. We tested whether the PCOS phenotype would be more pronounced on the diabetes-prone C57BL/6 background than the previously used strain, BALB/cByJ. In addition, we examined strain-dependent upregulation of the expression of ovarian and extra-ovarian candidate genes implicated in human PCOS, genes containing known strain variants, and genes involved with steroidogenesis or insulin sensitivity. These studies show that there are significant strain-related differences in metabolic response to excess androgen exposure during puberty. Additionally, our results suggest the C57BL/6J strain provides a more robust and uniform experimental platform for PCOS research than the BALB/cByJ strain.

Delayed puberty but normal fertility in mice with selective deletion of insulin receptors from Kiss1 cells.

Pubertal onset only occurs in a favorable, anabolic hormonal environment. The neuropeptide kisspeptin, encoded by the Kiss1 gene, modifies GnRH neuronal activity to initiate puberty and maintain fertility, but the factors that regulate Kiss1 neurons and permit pubertal maturation remain to be clarified. The anabolic factor insulin may signal nutritional status to these neurons. To determine whether insulin sensing plays an important role in Kiss1 neuron function, we generated mice lacking insulin receptors in Kiss1 neurons (IR(ΔKiss) mice). IR(ΔKiss) females showed a delay in vaginal opening and in first estrus, whereas IR(ΔKiss) males also exhibited late sexual maturation. Correspondingly, LH levels in IR(ΔKiss) mice were reduced in early puberty in both sexes. Adult reproductive capacity, body weight, fat composition, food intake, and glucose regulation were comparable between the 2 groups. These data suggest that impaired insulin sensing by Kiss1 neurons delays the initiation of puberty but does not affect adult fertility. These studies provide insight into the mechanisms regulating pubertal timing in anabolic states.

Adipocyte dysfunction in a mouse model of polycystic ovary syndrome (PCOS): evidence of adipocyte hypertrophy and tissue-specific inflammation.

Clinical research shows an association between polycystic ovary syndrome (PCOS) and chronic inflammation, a pathological state thought to contribute to insulin resistance. The underlying pathways, however, have not been defined. The purpose of this study was to characterize the inflammatory state of a novel mouse model of PCOS. Female mice lacking leptin and insulin receptors in pro-opiomelanocortin neurons (IR/LepR(POMC) mice) and littermate controls were evaluated for estrous cyclicity, ovarian and adipose tissue morphology, and body composition by QMR and CT scan. Tissue-specific macrophage infiltration and cytokine mRNA expression were measured, as well as circulating cytokine levels. Finally, glucose regulation during pregnancy was evaluated as a measure of risk for diabetes development. Forty-five percent of IR/LepR(POMC) mice showed reduced or absent ovulation. IR/LepR(POMC) mice also had increased fat mass and adipocyte hypertrophy. These traits accompanied elevations in macrophage accumulation and inflammatory cytokine production in perigonadal adipose tissue, liver, and ovary. These mice also exhibited gestational hyperglycemia as predicted. This report is the first to show the presence of inflammation in IR/LepR(POMC) mice, which develop a PCOS-like phenotype. Thus, IR/LepR(POMC) mice may serve as a new mouse model to clarify the involvement of adipose and liver tissue in the pathogenesis and etiology of PCOS, allowing more targeted research on the development of PCOS and potential therapeutic interventions.