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Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The [JAK2.sup.V617F] ([JAK2.sup.VF]) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. [JAK2.sup.VF] mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid [JAK2.sup.VF] expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express [JAK2.sup.VF] in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or [JAK2.sup.VF] RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in [JAK2.sup.VF] chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage [JAK2.sup.VF] expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk., Introduction Elevated hematocrit is associated with increased atherothrombotic cardiovascular disease (ACD), the number one cause of death and disability in Western societies (1, 2). Observational studies have shown that human [...]

Aims Neutrophil extracellular trap formation (NETosis) increases atherosclerotic plaque vulnerability and athero-thrombosis. However, mechanisms promoting NETosis during atherogenesis are poorly understood. We have shown that cholesterol accumulation due to myeloid cell deficiency of the cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/G1) promotes NLRP3 inflammasome activation in macrophages and neutrophils and induces prominent NETosis in atherosclerotic plaques. We investigated whether NETosis is a cell-intrinsic effect in neutrophils or is mediated indirectly by cellular crosstalk from macrophages to neutrophils involving IL-1β. Methods and results We generated mice with neutrophil or macrophage-specific Abca1/g1 deficiency (S100A8CreAbca1fl/flAbcg1fl/fl or CX3CR1CreAbca1fl/flAbcg1fl/fl mice, respectively), and transplanted their bone marrow into low-density lipoprotein receptor knockout mice. We then fed the mice a cholesterol-rich diet. Macrophage, but not neutrophil Abca1/g1 deficiency activated inflammasomes in macrophages and neutrophils, reflected by caspase-1 cleavage, and induced NETosis in plaques. NETosis was suppressed by administering an interleukin (IL)-1β neutralizing antibody. The extent of NETosis in plaques correlated strongly with the degree of neutrophil accumulation, irrespective of blood neutrophil counts, and neutrophil accumulation was decreased by IL-1β antagonism. In vitro , IL-1β or media transferred from Abca1/g1 -deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1β on NETosis was blocked by an NLRP3 inhibitor. Conclusion These studies establish a new link between inflammasome-mediated IL-1β production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1β appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Background: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency.Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr-/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2V617F).Results: Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk-/- platelets promoted increased NETosis when incubated with Lnk-/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk-/- platelets, as well as increased priming and response of Lnk-/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk-/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ controls.Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations. [ABSTRACT FROM AUTHOR]

Objective: Microvascular brain injury (mVBI) is a common pathological correlate of vascular contributions to cognitive impairment and dementia (VCID) that leads to white matter (WM) injury (WMI). VCID appears to arise from chronic recurrent white matter ischemia that triggers oxidative stress and an increase in total oligodendrocyte lineage cells. We hypothesized that mVBI involves vasodilator dysfunction of white matter penetrating arterioles and aberrant oligodendrocyte progenitor cell (OPC) responses to WMI.Methods: We analyzed cases of mVBI with low Alzheimer's disease neuropathological change in prefrontal cortex WM from rapid autopsies in a population-based cohort where VCID frequently occurs. Arteriolar vasodilator function was quantified by videomicroscopy. OPC maturation was quantified using lineage specific markers.Results: Acetylcholine-mediated arteriolar dilation in mVBI was significantly reduced in WM penetrators relative to pial arterioles. Astrogliosis-defined WMI was positively associated with increased OPCs and was negatively associated with decreased mature oligodendrocytes.Interpretation: Selectively impaired vasodilator function of WM penetrating arterioles in mVBI occurs in association with aberrant differentiation of OPCs in WMI, which supports that myelination disturbances in VCID are related to disrupted maturation of myelinating oligodendrocytes. Ann Neurol 2018;83:142-152. [ABSTRACT FROM AUTHOR]

Dyslipidemia associated with triglyceride-rich lipoproteins (TRLs) represents an important residual risk factor for cardiovascular and chronic kidney disease in patients with type 1 diabetes (T1D). Levels of growth hormone (GH) are elevated in T1D, which aggravates both hyperglycemia and dyslipidemia. The hypothalamic growth hormone- releasing hormone (GHRH) regulates the release of GH by the pituitary but also exerts separate actions on peripheral GHRH receptors, the functional role ofwhich remains elusive in T1D. In a rat model of streptozotocin (STZ)-induced T1D, GHRH receptor expression was found to be up-regulated in the distal small intestine, a tissue involved in chylomicron synthesis. Treatment of T1D rats with a GHRH antagonist,MIA-602, at a dose that did not affect plasma GH levels, significantly reduced TRL, as well as markers of renal injury, and improved endothelial-dependent vasorelaxation. Glucagon-like peptide 1 (GLP-1) reduces hyperglucagonemia and postprandial TRL, the latter in part through a decreased synthesis of apolipoprotein B-48 (ApoB-48) by intestinal cells. Although plasma GLP-1 levels were elevated in diabetic animals, this was accompanied by increased rather than reduced glucagon levels, suggesting impaired GLP-1 signaling. Treatment with MIA-602 normalized GLP-1 and glucagon to control levels in T1D rats. MIA-602 also decreased secretion of ApoB-48 from rat intestinal epithelial cells in response to oleic acid stimulation in vitro, in part through a GLP-1-dependent mechanism. Our findings support the hypothesis that antagonizing the signaling of GHRH in T1D may improve GLP-1 function in the small intestine, which, in turn, diminishes TRL and reduces renal and vascular complications. [ABSTRACT FROM AUTHOR]

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk. [ABSTRACT FROM AUTHOR]

Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1–3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide. [ABSTRACT FROM AUTHOR]