N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm.

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs), though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms, including platelet activation and neutrophil-extracellular trap formation; therefore, an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here, we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC), a sulfhydryl-containing compound with broad effects on glutathione replenishment, free radical scavenging, and reducing disulfide bonds, to investigate its antithrombotic effects in the context of MPN. Strikingly, NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo, we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore, NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN.

The SMAC mimetic LCL-161 selectively targets JAK2V617F mutant cells.

BACKGROUND: Evasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo. METHODS: To investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2V617F-driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2V617F-driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis. RESULTS: We found that JAK2V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2V617F-driven MPN. CONCLUSION: LCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN.

Defective negative regulation of Toll-like receptor signaling leads to excessive TNF-α in myeloproliferative neoplasm.

Patients with myeloproliferative neoplasms (MPN) have high levels of inflammatory cytokines, some of which drive many of the debilitating constitutional symptoms associated with the disease and may also promote expansion of the neoplastic clone. We report here that monocytes from patients with MPN have defective negative regulation of Toll-like receptor (TLR) signaling that leads to unrestrained production of the inflammatory cytokine tumor necrosis factor α (TNF-α) after TLR activation. Specifically, monocytes of patients with MPN are insensitive to the anti-inflammatory cytokine interleukin 10 (IL-10) that negatively regulates TLR-induced TNF-α production. This inability to respond to IL-10 is a not a direct consequence of JAK2 V617F , as the phenotype of persistent TNF-α production is a feature of JAK2 V617F and wild-type monocytes alike from JAK2 V617F -positive patients. Moreover, persistent TNF-α production was also discovered in the unaffected identical twin of a patient with MPN, suggesting it could be an intrinsic feature of those predisposed to acquire MPN. This work implicates sustained TLR signaling as not only a contributor to the chronic inflammatory state of MPN patients but also a potential predisposition to acquire MPN.

The Critical Role of Inflammation in the Pathogenesis and Progression of Myeloid Malignancies.

Hematopoietic stem cells (HSCs) maintain an organism's immune system for a lifetime, and derangements in HSC proliferation and differentiation result in hematologic malignancies. Chronic inflammation plays a contributory if not causal role in HSC dysfunction. Inflammation induces HSC exhaustion, which promotes the emergence of mutant clones that may be resistant to an inflammatory microenvironment; this likely promotes the onset of a myeloid hematologic malignancy. Inflammatory cytokines are characteristically high in patients with myeloid malignancies and are linked to disease initiation, symptom burden, disease progression, and worsened prognostic survival. This review will cover our current understanding of the role of inflammation in the initiation, progression, and complications of myeloid hematologic malignancies, drawing from clinical studies as well as murine models. We will also highlight inflammation as a therapeutic target in hematologic malignancies.

A vascularized and perfused organ-on-a-chip platform for large-scale drug screening applications.

There is a growing awareness that complex 3-dimensional (3D) organs are not well represented by monolayers of a single cell type - the standard format for many drug screens. To address this deficiency, and with the goal of improving screens so that drugs with good efficacy and low toxicity can be identified, microphysiological systems (MPS) are being developed that better capture the complexity of in vivo physiology. We have previously described an organ-on-a-chip platform that incorporates perfused microvessels, such that survival of the surrounding tissue is entirely dependent on delivery of nutrients through the vessels. Here we describe an arrayed version of the platform that incorporates multiple vascularized micro-organs (VMOs) on a 96-well plate. Each VMO is independently-addressable and flow through the micro-organ is driven by hydrostatic pressure. The platform is easy to use, requires no external pumps or valves, and is highly reproducible. As a proof-of-concept we have created arrayed vascularized micro tumors (VMTs) and used these in a blinded screen to assay a small library of compounds, including FDA-approved anti-cancer drugs, and successfully identified both anti-angiogenic and anti-tumor drugs. This 3D platform is suitable for efficacy/toxicity screening against multiple tissues in a more physiological environment than previously possible.

3D surface analysis of hippocampal microvasculature in the irradiated brain.

Cranial irradiation used to control CNS malignancies can also disrupt the vasculature and impair neurotransmission and cognition. Here we describe two distinct methodologies for quantifying early and late radiation injury in CNS microvasculature. Intravascular fluorescently labeled lectin was used to visualize microvessels in the brain of the irradiated mouse 2 days post exposure and RECA-1 immunostaining was similarly used to visualize microvessels in the brain of the irradiated rat 1-month post exposure. Confocal microscopy, image deconvolution and 3-dimensional rendering methods were used to define vascular structure in a ∼4 × 10(7) µm(3) defined region of the brain. Quantitative analysis of these 3D images revealed that irradiation caused significant short- and long-term reductions in capillary density, diameter and volume. In mice, irradiation reduced mean vessel volume from 2,250 to 1,470 µm(3) and mean vessel diameter from 5.0 to 4.5 µm, resulting in significant reductions of 34% and 10%, in the hippocampus respectively. The number of vessel branch points and area was also found to also drop significantly in mice 2 days after irradiation. For rats, immunostaining revealed a significant, three-fold drop in capillary density 1 month after exposure compared to controls. Such radiation-induced disruption of the CNS microvasculature may be contributory if not causal to any number of neurocognitive side effects that manifest in cancer patients following cranial radiotherapy. This study demonstrates the utility of two distinct methodologies for quantifying these important adverse effects of radiotherapy. Environ. Mol. Mutagen. 57:341-349, 2016. © 2016 Wiley Periodicals, Inc.

What happens to your brain on the way to Mars.

As NASA prepares for the first manned spaceflight to Mars, questions have surfaced concerning the potential for increased risks associated with exposure to the spectrum of highly energetic nuclei that comprise galactic cosmic rays. Animal models have revealed an unexpected sensitivity of mature neurons in the brain to charged particles found in space. Astronaut autonomy during long-term space travel is particularly critical as is the need to properly manage planned and unanticipated events, activities that could be compromised by accumulating particle traversals through the brain. Using mice subjected to space-relevant fluences of charged particles, we show significant cortical- and hippocampal-based performance decrements 6 weeks after acute exposure. Animals manifesting cognitive decrements exhibited marked and persistent radiation-induced reductions in dendritic complexity and spine density along medial prefrontal cortical neurons known to mediate neurotransmission specifically interrogated by our behavioral tasks. Significant increases in postsynaptic density protein 95 (PSD-95) revealed major radiation-induced alterations in synaptic integrity. Impaired behavioral performance of individual animals correlated significantly with reduced spine density and trended with increased synaptic puncta, thereby providing quantitative measures of risk for developing cognitive decrements. Our data indicate an unexpected and unique susceptibility of the central nervous system to space radiation exposure, and argue that the underlying radiation sensitivity of delicate neuronal structure may well predispose astronauts to unintended mission-critical performance decrements and/or longer-term neurocognitive sequelae.

Consequences of low dose ionizing radiation exposure on the hippocampal microenvironment.

The response of the brain to irradiation is complex, involving a multitude of stress inducible pathways that regulate neurotransmission within a dynamic microenvironment. While significant past work has detailed the consequences of CNS radiotherapy following relatively high doses (≥ 45 Gy), few studies have been conducted at much lower doses (≤ 2 Gy), where the response of the CNS (like many other tissues) may differ substantially from that expected from linear extrapolations of high dose data. Low dose exposure could elicit radioadaptive modulation of critical CNS processes such as neurogenesis, that provide cellular input into hippocampal circuits known to impact learning and memory. Here we show that mice deficient for chemokine signaling through genetic disruption of the CCR2 receptor exhibit a neuroprotective phenotype. Compared to wild type (WT) animals, CCR2 deficiency spared reductions in hippocampal neural progenitor cell survival and stabilized neurogenesis following exposure to low dose irradiation. While radiation-induced changes in microglia levels were not found in WT or CCR2 deficient animals, the number of Iba1+ cells did differ between each genotype at the higher dosing paradigms, suggesting that blockade of this signaling axis could moderate the neuroinflammatory response. Interestingly, changes in proinflammatory gene expression were limited in WT animals, while irradiation caused significant elevations in these markers that were attenuated significantly after radioadaptive dosing paradigms in CCR2 deficient mice. These data point to the importance of chemokine signaling under low dose paradigms, findings of potential significance to those exposed to ionizing radiation under a variety of occupational and/or medical scenarios.

Persistent oxidative stress in human neural stem cells exposed to low fluences of charged particles.

Exposure to the space radiation environment poses risks for a range of deleterious health effects due to the unique types of radiation encountered. Galactic cosmic rays are comprised of a spectrum of highly energetic nuclei that deposit densely ionizing tracks of damage along the particle trajectory. These tracks are distinct from those generated by the more sparsely ionizing terrestrial radiations, and define the geometric distribution of the complex cellular damage that results when charged particles traverse the tissues of the body. The exquisite radiosensitivity of multipotent neural stem and progenitor cells found within the neurogenic regions of the brain predispose the central nervous system to elevated risks for radiation induced sequelae. Here we show that human neural stem cells (hNSC) exposed to different charged particles at space relevant fluences exhibit significant and persistent oxidative stress. Radiation induced oxidative stress was found to be most dependent on total dose rather than on the linear energy transfer of the incident particle. The use of redox sensitive fluorogenic dyes possessing relative specificity for hydroxyl radicals, peroxynitrite, nitric oxide (NO) and mitochondrial superoxide confirmed that most irradiation paradigms elevated reactive oxygen and nitrogen species (ROS and RNS, respectively) in hNSC over a 1 week interval following exposure. Nitric oxide synthase (NOS) was not the major source of elevated nitric oxides, as the use of NOS inhibitors had little effect on NO dependent fluorescence. Our data provide extensive evidence for the capability of low doses of charged particles to elicit marked changes in the metabolic profile of irradiated hNSC. Radiation induced changes in redox state may render the brain more susceptible to the development of neurocognitive deficits that could affect an astronaut's ability to perform complex tasks during extended missions in deep space.

Persistent changes in neuronal structure and synaptic plasticity caused by proton irradiation.

Cranial radiotherapy is used routinely to control the growth of primary and secondary brain tumors, but often results in serious and debilitating cognitive dysfunction. In part due to the beneficial dose depth distributions that may spare normal tissue damage, the use of protons to treat CNS and other tumor types is rapidly gaining popularity. Astronauts exposed to lower doses of protons in the space radiation environment are also at risk for developing adverse CNS complications. To explore the consequences of whole body proton irradiation, mice were subjected to 0.1 and 1 Gy and analyzed for morphometric changes in hippocampal neurons 10 and 30 days following exposure. Significant dose-dependent reductions (~33 %) in dendritic complexity were found, when dendritic length, branching and area were analyzed 30 days after exposure. At equivalent doses and times, significant reductions in the number (~30 %) and density (50-75 %) of dendritic spines along hippocampal neurons of the dentate gyrus were also observed. Immature spines (filopodia, long) exhibited the greatest sensitivity (1.5- to 3-fold) to irradiation, while more mature spines (mushroom) were more resistant to changes over a 1-month post-irradiation timeframe. Irradiated granule cell neurons spanning the subfields of the dentate gyrus showed significant and dose-responsive reductions in synaptophysin expression, while the expression of postsynaptic density protein (PSD-95) was increased significantly. These findings corroborate our past work using photon irradiation, and demonstrate for the first time, dose-responsive changes in dendritic complexity, spine density and morphology and synaptic protein levels following exposure to low-dose whole body proton irradiation.

Targeted overexpression of mitochondrial catalase prevents radiation-induced cognitive dysfunction.

AIMS: Radiation-induced disruption of mitochondrial function can elevate oxidative stress and contribute to the metabolic perturbations believed to compromise the functionality of the central nervous system. To clarify the role of mitochondrial oxidative stress in mediating the adverse effects of radiation in the brain, we analyzed transgenic (mitochondrial catalase [MCAT]) mice that overexpress human catalase localized to the mitochondria. RESULTS: Compared with wild-type (WT) controls, overexpression of the MCAT transgene significantly decreased cognitive dysfunction after proton irradiation. Significant improvements in behavioral performance found on novel object recognition and object recognition in place tasks were associated with a preservation of neuronal morphology. While the architecture of hippocampal CA1 neurons was significantly compromised in irradiated WT mice, the same neurons in MCAT mice did not exhibit extensive and significant radiation-induced reductions in dendritic complexity. Irradiated neurons from MCAT mice maintained dendritic branching and length compared with WT mice. Protected neuronal morphology in irradiated MCAT mice was also associated with a stabilization of radiation-induced variations in long-term potentiation. Stabilized synaptic activity in MCAT mice coincided with an altered composition of the synaptic AMPA receptor subunits GluR1/2. INNOVATION: Our findings provide the first evidence that neurocognitive sequelae associated with radiation exposure can be reduced by overexpression of MCAT, operating through a mechanism involving the preservation of neuronal morphology. CONCLUSION: Our article documents the neuroprotective properties of reducing mitochondrial reactive oxygen species through the targeted overexpression of catalase and how this ameliorates the adverse effects of proton irradiation in the brain.

Mitochondrial-targeted human catalase affords neuroprotection from proton irradiation.

Significant past work has linked radiation exposure of the CNS to elevated levels of oxidative stress and inflammation. These secondary reactive processes are both dynamic and persistent and are believed to compromise the functionality of the CNS, in part, by disrupting endogenous neurogenesis in the hippocampus. While evidence has shown neurogenesis to be sensitive to irradiation and redox state, the mechanistic basis underlying these effects is incompletely understood. To clarify the role of reactive oxygen species (ROS) in mediating radiation-induced changes in neurogenesis we have analyzed transgenic mice that overexpress human catalase localized to the mitochondria. With this model, we investigated the consequences of low dose and clinically relevant proton irradiation on neurogenesis, and how that process is modified in response to genetic disruption of mitochondrial ROS levels. In unirradiated animals, basal neurogenesis was improved significantly by reductions in mitochondrial ROS. In animals subjected to proton exposure, hippocampal progenitor cell proliferation was attenuated significantly by overexpression of human catalase in the mitochondria. Furthermore, expression of the MCAT transgene significantly improved neurogenesis in WT animals after low-dose proton exposure (0.5 Gy), with similar trends observed at higher dose (2 Gy). Our report documents for the first time the impact of proton irradiation on hippocampal neurogenesis, and the neuroprotective properties of reducing mitochondrial ROS through the targeted overexpression of catalase.