Cigarette smoke stimulates clonal expansion of Jak2V617F and Tet2-/- cells.

Introduction: Somatic mutations in myeloid growth factor pathway genes, such as JAK2, and genes involved in epigenetic regulation, such as TET2, in hematopoietic stem cells (HSCs) leads to clonal hematopoiesis of indeterminate potential (CHIP) which presents a risk factor for hematologic malignancy and cardiovascular disease. Smoking behavior has been repeatedly associated with the occurrence of CHIP but whether smoking is an environmental inflammatory stressor in promoting clonal expansion has not been investigated. Methods: We performed in vivo smoke exposures in both wildtype (WT) mice and transplanted mice carrying Jak2V617F mutant and Tet2 knockout (Tet-/-) cells to determine the impact of cigarette smoke (CS) in the HSC compartment as well as favoring mutant cell expansion. Results: WT mice exposed to smoke displayed increased oxidative stress in long-term HSCs and suppression of the hematopoietic stem and progenitor compartment but smoke exposure did not translate to impaired hematopoietic reconstitution in primary bone marrow transplants. Gene expression analysis of hematopoietic cells in the bone marrow identified an imbalance between Th17 and Treg immune cells suggesting a local inflammatory environment. We also observed enhanced survival of Jak2V617F cells exposed to CS in vivo and cigarette smoke extract (CSE) in vitro. WT bone marrow hematopoietic cells from WT/Jak2V617F chimeric mice exposed to CS demonstrated an increase in neutrophil abundance and distinct overexpression of bone marrow stromal antigen 2 (Bst2) and retinoic acid early transcript 1 (Raet1) targets. Bst2 and Raet1 are indicative of increased interferon signaling and cellular stress including oxidative stress and DNA damage, respectively. In chimeric mice containing both WT and Tet2-/- cells, we observed an increased percentage of circulating mutant cells in peripheral blood post-cigarette smoke exposure when compared to pre-exposure levels while this difference was absent in air-exposed controls. Conclusion: Altogether, these findings demonstrate that CS results in an inflamed bone marrow environment that provides a selection pressure for existing CHIP mutations such as Jak2V617F and Tet2 loss-of-function.

The Microenvironment in Myeloproliferative Neoplasms.

Chronic inflammation is a hallmark of myeloproliferative neoplasms (MPNs), with elevated levels of proinflammatory cytokines being commonly found in all 3 subtypes. Systemic inflammation is responsible for the constitutional symptoms, thrombosis risk, premature atherosclerosis, and disease evolution in MPN. Although the neoplastic clone and their differentiated progeny drive the inflammatory process, they also induce ancillary cytokine secretion from nonmalignant cells. Here, the authors describe the inflammatory milieu in MPN based on soluble factors and cellular mediators. They also discuss the prognostic value of cytokine measurements in patients with MPN and potential therapeutic strategies that target the cellular players in inflammation.

E-Cigarette Exposure Decreases Bone Marrow Hematopoietic Progenitor Cells.

Electronic cigarettes (E-cigs) generate nicotine containing aerosols for inhalation and have emerged as a popular tobacco product among adolescents and young adults, yet little is known about their health effects due to their relatively recent introduction. Few studies have assessed the long-term effects of inhaling E-cigarette smoke or vapor. Here, we show that two months of E-cigarette exposure causes suppression of bone marrow hematopoietic stem and progenitor cells (HSPCs). Specifically, the common myeloid progenitors and granulocyte-macrophage progenitors were decreased in E-cig exposed animals compared to air exposed mice. Competitive reconstitution in bone marrow transplants was not affected by two months of E-cig exposure. When air and E-cig exposed mice were challenged with an inflammatory stimulus using lipopolysaccharide (LPS), competitive fitness between the two groups was not significantly different. However, mice transplanted with bone marrow from E-cigarette plus LPS exposed mice had elevated monocytes in their peripheral blood at five months post-transplant indicating a myeloid bias similar to responses of aged hematopoietic stem cells (HSC) to an acute inflammatory challenge. We also investigated whether E-cigarette exposure enhances the selective advantage of hematopoietic cells with myeloid malignancy associated mutations. E-cigarette exposure for one month slightly increased JAK2V617F mutant cells in peripheral blood but did not have an impact on TET2-/- cells. Altogether, our findings reveal that chronic E-cigarette exposure for two months alters the bone marrow HSPC populations but does not affect HSC reconstitution in primary transplants.

Impact of Host, Lifestyle and Environmental Factors in the Pathogenesis of MPN.

Philadelphia-negative myeloproliferative neoplasms (MPNs) occur when there is over-production of myeloid cells stemming from hematopoietic stem cells with constitutive activation of JAK/STAT signaling, with JAK2V617F being the most commonly occurring somatic driver mutation. Chronic inflammation is a hallmark feature of MPNs and it is now evident that inflammation is not only a symptom of MPN but can also provoke development and precipitate progression of disease. Herein we have considered major MPN driver mutation independent host, lifestyle, and environmental factors in the pathogenesis of MPN based upon epidemiological and experimental data. In addition to the traditional risk factors such as advanced age, there is evidence to indicate that inflammatory stimuli such as smoking can promote and drive MPN clone emergence and expansion. Diet induced inflammation could also play a role in MPN clonal expansion. Recognition of factors associated with MPN development support lifestyle modifications as an emerging therapeutic tool to restrain inflammation and diminish MPN progression.

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.

Diesel Exhaust Induces Mitochondrial Dysfunction, Hyperlipidemia, and Liver Steatosis.

OBJECTIVE: Air pollution is associated with increased cardiovascular morbidity and mortality, as well as dyslipidemia and metabolic syndrome. Our goal was to dissect the mechanisms involved. Approach and Results: We assessed the effects of exposure to air pollution on lipid metabolism in mice through assessment of plasma lipids and lipoproteins, oxidized fatty acids 9-HODE (9-hydroxyoctadecadienoic) and 13-HODE (13-hydroxyoctadecadienoic), lipid, and carbohydrate metabolism. Findings were corroborated, and mechanisms were further assessed in HepG2 hepatocytes in culture. ApoE knockout mice exposed to inhaled diesel exhaust (DE, 6 h/d, 5 days/wk for 16 weeks) exhibited elevated plasma cholesterol and triglyceride levels, increased hepatic triglyceride content, and higher hepatic levels of 9-HODE and 13-HODE, as compared to control mice exposed to filtered air. A direct effect of DE exposure on hepatocytes was demonstrated by treatment of HepG2 cells with a methanol extract of DE particles followed by loading with oleic acid. As observed in vivo, this led to increased triglyceride content and significant downregulation of ACAD9 mRNA expression. Treatment of HepG2 cells with DE particles and oleic acid did not alter de novo lipogenesis but inhibited total, mitochondrial, and ATP-linked oxygen consumption rate, indicative of mitochondrial dysfunction. Treatment of isolated mitochondria, prepared from mouse liver, with DE particles and oleic acid also inhibited mitochondrial complex activity and ß-oxidation. CONCLUSIONS: DE exposure leads to dyslipidemia and liver steatosis in ApoE knockout mice, likely due to mitochondrial dysfunction and decreased lipid catabolism.

Key Role of Inflammation in Myeloproliferative Neoplasms: Instigator of Disease Initiation, Progression. and Symptoms.

PURPOSE OF REVIEW: Chronic inflammation is a characteristic feature of myeloproliferative neoplasm (MPN) and impacts many aspects of the disease including initiation, progression, and symptomatology. RECENT FINDINGS: The chronic inflammatory state of MPN results from disruption of immune signaling pathways leading to overproduction of inflammatory cytokines by both the neoplastic clones and bystander immune cells. This chronic inflammation may allow for the neoplastic clone to gain a selective advantage. The symptomatic burden felt by MPN patients may be a result of the chronic inflammation associated with MPN, as several cytokines have been linked with different symptoms. Pharmacologic as well as nonpharmacologic treatments of the inflammatory component of this disease may lead to decreased symptomatic burden, prevention of disease progression, and improvement in overall disease trajectory. Inflammation plays a key role in the pathogenesis of MPN and represents an important therapeutic target.

Increased expression of transient receptor potential canonical 6 (TRPC6) in differentiating human megakaryocytes.

Members of the transient receptor potential (TRP) family of cation conducting channels are found in several tissues and cell types where they have different physiological functions. The canonical TRP channel 6 (TRPC6) is present on the platelet membrane and appears to participate in calcium influx during platelet activation. However, limited information is available on the importance of TRPC channels in megakaryocytes (MKs), the precursor cells of platelets. We determined the mRNA and protein expression of TRPC family members and investigated the role of TRPC6 for proliferation and differentiation of human MKs derived from CD34+ progenitor cells. TRPC6 transcripts were highly expressed during the differentiation of MKs and TRPC6 protein was detectable in MK cytoplasm by confocal staining. TRPC6 channel activity was modulated by pharmacological approaches using flufenamic acid (FFA) for activation and SKF96365 for inhibition. Upon FFA stimulation in MKs, an increase in intracellular calcium was observed, which was blocked by SKF96365 at 10 µM concentration. Incubation of MKs with SKF96365 resulted in a reduction in thrombopoietin-stimulated cell proliferation. Our results suggest a role of TRPC6 in calcium homeostasis during MK development, particularly for cell proliferation.

Cigarette smoking is associated with dose-dependent adverse effects on paraoxonase activity and fibrinogen in young women.

CONTEXT: Smoking is associated with increased fibrinogen and decreased paraoxonase (PON) activity, markers of inflammation and oxidative stress, in patients with coronary artery disease. OBJECTIVE: We tested the hypothesis that the adverse effect of smoking on these biomarkers of inflammation and oxidative stress would be detectable in otherwise healthy young female habitual smokers. MATERIALS AND METHODS: Thirty-eight young women participated in the study (n = 20 habitual smokers, n = 18 non-smokers). Fibrinogen, PON-1 activity and HDL oxidant index (HOI) were measured. RESULTS: Mean values of fibrinogen, PON-1 activity and log HOI were not different between the groups. Importantly, however, decreased PON-1 activity (rs = -0.51, p = 0.03) and increased fibrinogen (rs = 0.49, p = 0.04) were significantly correlated with increasing number of cigarettes smoked per day in habitual smokers. DISCUSSION AND CONCLUSION: Cigarette smoking is associated with a dose-dependent adverse effect on PON-1 activity and fibrinogen in young women, which may have implications for future cardiovascular risk.

No effect of acute exposure to coarse particulate matter air pollution in a rural location on high-density lipoprotein function.

CONTEXT: High-density lipoprotein (HDL) particles perform numerous vascular-protective functions. Animal studies demonstrate that exposure to fine or ultrafine particulate matter (PM) can promote HDL dysfunction. However, the impact of PM on humans remains unknown. OBJECTIVE: We aimed to determine the effect of exposure to coarse concentrated ambient particles (CAP) on several metrics of HDL function in healthy humans. METHODS: Thirty-two adults (25.9 ± 6.6 years) were exposed to coarse CAP [76.2 ± 51.5 µg·m(-3)] in a rural location and filtered air (FA) for 2 h in a randomized double-blind crossover study. Venous blood collected 2- and 20-h post-exposures was measured for HDL-mediated efflux of [(3)H]-cholesterol from cells and 20-h exposures for HDL anti-oxidant capacity by a fluorescent assay and paraoxonase activity. The changes [median (first, third quartiles)] between exposures among 29 subjects with available results were compared by matched Wilcoxon tests. RESULTS: HDL-mediated cholesterol efflux capacity did not differ between exposures at either time point [16.60% (15.17, 19.19) 2-h post-CAP versus 17.56% (13.43, 20.98) post-FA, p = 0.768 and 14.90% (12.47, 19.15) 20-h post-CAP versus 17.75% (13.22, 23.95) post-FA, p = 0.216]. HOI [0.26 (0.24, 0.35) versus 0.28 (0.25, 0.40), p = 0.198] and paraoxonase activity [0.54 (0.39, 0.82) versus 0.60 µmol·min(-1 )ml plasma(-1) (0.40, 0.85), p = 0.137] did not differ 20-h post-CAP versus FA, respectively. CONCLUSIONS: Brief inhalation of coarse PM from a rural location did not acutely impair several facets of HDL functionality. Whether coarse PM derived from urban sites, fine particles or longer term PM exposures can promote HDL dysfunction warrant future investigations.

EVI1 and MDS1/EVI1 expression during primary human hematopoietic progenitor cell differentiation into various myeloid lineages.

BACKGROUND AND AIM: Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in myeloid leukemia. We therefore studied its expression and function in cluster of differentiation 34-positive (CD34(+)) primary human hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) cells were differentiated into various myeloid lineages using the appropriate cytokines. EVI1 expression was measured by quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) and intranuclear fluorescence-activated cell sorting (FACS). Experimental manipulation of EVI1 levels was achieved using retroviral infection. RESULTS: EVI1 mRNA and its variant myelodysplastic syndrome 1 (MDS1)/EVI1, which gives rise to a partially antagonistic protein, were detectable in CD34(+) cells, but their levels declined rapidly during differentiation into the granulocyte, monocyte, dendritic, erythroid, and megakaryocyte lineages. Similarly, EVI1 protein levels decreased during myeloid differentiation. Attempts to experimentally express EVI1 in CD34(+) and U937 cells indicated that ectopic expression of EVI1 may cause growth arrest, apoptosis and/or senescence of human hematopoietic cells. CONCLUSION: EVI1 is expressed in human hematopoietic progenitor cells, but is down-regulated during differentiation. Ectopic expression of EVI1 may activate cellular safeguards against oncogene activation.