Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies and surgical specimens of abdominal aortic aneurysms (AAA) from EH patients were used. Cell-based assays, NETs/human aortic endothelial cells co-cultures and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly-diagnosed, EH patients. Stimulation of control neutrophils with plasma from untreated EH patients generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via a reactive oxygen species (ROS)/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced significantly in EH patients starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants were accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAA. These data reveal the important pathogenic role of Ang II/ROS/NETs/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.
Akrivi Chrysanthopoulou, Eugenia Gkaliagkousi, Antonios Lazaridis, Stella Arelaki, Panagiotis Pateinakis, Maria Ntinopoulou, Alexandros Mitsios, Christina Antoniadou, Christos Argyriou, George S. Georgiadis, Vasileios Papadopoulos, Alexandra Giatromanolaki, Konstantinos Ritis, Panagiotis Skendros
Creatine transporter (CrT) upholds the brain creatine (Cr) levels, but the impacts of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments of CrT-deficiency, the second most common cause of X-linked intellectual disabilities. Herein we examined the consequences of CrT-deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplement. We found that CrT-deficient (CrT-/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT-/y mice maintained the baseline brain ATP level with a tendency towards the pAMPK/autophagy from mTOR signaling activity. Starvation elevated the signaling imbalance and reduced the brain ATP level in P3 CrT-/y mice. Similarly, CrT-/y neurons and P10 CrT-/y mice showed an imbalance between autophagy/mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/NAA (N-acetylaspartate) ratio, partially averted the signaling imbalance, and reduced the infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions of CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplement may be an effective treatment of congenital CrT-deficiency and acute brain injury.
Hong-Ru Chen, Xiaohui Zhang-Brotzge, Yury M. Morozov, Yuancheng Li, Siming Wang, Helen Zhang, Irena S. Kuan, Elizabeth M. Fugate, Hui Mao, Yu-Yo Sun, Pasko Rakic, Diana M. Lindquist, Ton DeGrauw, Chia-Yi Kuan
Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than one thousand nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here we show that histone demethylase LSD1 knockout from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in nucleus. Lsd1 knockout reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.
Yang Cao, Lingyi Tang, Kang Du, Kitt Paraiso, Qiushi Sun, Zhengxia Liu, Xiaolong Ye, Yuan Fang, Fang Yuan, Yu-Han Chen, Yumay Chen, Xiaorong Wang, Clinton Yu, Ira L. Blitz, Ping H. Wang, Lan Huang, Haibo Cheng, Xiang Lu, Ken W.Y Cho, Marcus Seldin, Zhuyuan Fang, Qin Yang
Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbate disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in COVID-19 patients and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, two signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in COVID-19 patients. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation leading to production of pathological HA fragments which are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Kimberly A. Queisser, Rebecca A. Mellema, Elizabeth A. Middleton, Irina Portier, Bhanu Kanth Manne, Frederik Denorme, Ellen J. Beswick, Matthew T. Rondina, Robert A. Campbell, Aaron C. Petrey
Macrophage activation syndrome (MAS) is a life-threatening cytokine storm complicating systemic juvenile idiopathic arthritis (SJIA) driven by IFNγ. SJIA and MAS are associated with an unexplained emerging lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFNγ pathways pathologically linking SJIA-LD and MAS. Our objective was to mechanistically define the novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFNγ, IFN-induced chemokines, and AMΦ expression of IFNγ-induced genes. Single-cell RNA-sequencing confirmed IFN-driven transcriptional changes across lung cell types with myeloid expansion and detection of MAS-specific macrophage populations. Systemic MAS resolution was associated with increased AMΦ and interstitial lymphocytic infiltration. AMΦ transcriptomic analysis confirmed IFNγ-induced proinflammatory polarization during acute MAS, which switches towards an anti-inflammatory phenotype after systemic MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and reset AMΦ polarization towards a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFNγ, both systemic feature of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFNγ-driven pulmonary inflammation replicating key features of SJIA-LD, and provides a model system for testing novel treatments directed towards SJIA-LD.
Denny K. Gao, Nathan Salomonis, Maggie Henderlight, Christopher Woods, Kairavee Thakkar, Alexei A. Grom, Sherry Thornton, Michael B. Jordan, Kathryn A. Wikenheiser-Brokamp, Grant S. Schulert
The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue (AT) is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific knockout mouse models, we show that CD36 in both adipocytes and endothelial cells mediates both LCFA deposition into and release from AT. We demonstrate the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by AT-derived LCFA. We show that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediates intercellular LCFA transport. We demonstrate that lipolysis induction in adipocytes triggers CD36 de-acylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB), and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S- acylation and its interactions with PHB and ANX2.
Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin
Immunotherapies are needed in the clinic that effectively suppress beta cell autoimmunity and reestablish long-term self-tolerance in type 1 diabetes. We previously demonstrated that nondepleting αCD4 and αCD8α antibodies establish rapid and indefinite remission in recent-onset diabetic NOD mice. Diabetes reversal by coreceptor therapy (CoRT) is induced by suppression of pathogenic effector T cells (Teff) and the selective egress of T cells from the pancreatic lymph nodes and islets that remain free of infiltration long-term. Here, we defined CoRT-induced events regulating early Teff function and pancreatic residency, and long-term tolerance. TCR-driven gene expression controlling autoreactive Teff expansion and proinflammatory activity was suppressed by CoRT, and islet T cell egress was sphingosine-1 phosphate-dependent. In both murine and human T cells, CoRT upregulated the Foxo1 transcriptional axis, which in turn was required for suppression and efficient pancreatic egress of Teff. Interestingly, long-term tolerance induced in late-preclinical NOD mice was marked by reseeding of the pancreas by a reduced CD8+ Teff pool exhibiting an exhausted phenotype. Notably, PD-1 blockade, which rescues exhausted Teff, resulted in diabetes onset in protected animals. These findings demonstrate that CoRT has distinct intrinsic effects on Teff that impact events early in induction and later in maintenance of self-tolerance.
Matthew Clark, Charles J. Kroger, Qi Ke, Rui Zhang, Karen Statum, J. Justin Milner, Aaron J. Martin, Bo Wang, Roland Tisch
Hyperstimulation of the cholecystokinin receptor (CCK1R), a Gq-protein coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand acetylcholine but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with three recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.
Jianhua Wan, Jiale Wang, Larry E. Wagner II, Oliver H. Wang, Fu Gui, Jiaxiang Chen, Xiaohui Zhu, Ashley N. Haddock, Brandy H. Edenfield, Brian Haight, Debabrata Mukhopadhyay, Ying Wang, David I. Yule, Yan Bi, Baoan Ji
Lack of sustained response to therapeutic agents in patients with KRAS mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-to-mesenchymal transition (EMT) is a biologic phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in vitro and in vivo models to identify and confirm an increased dependence of mesenchymal tumor cells on CDK4 for survival, as well as a mechanism of resistance to MEK inhibitors. High ZEB1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Co-administration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single agent treatment.
Aparna Padhye, Jessica M. Konen, B. Leticia Rodriguez, Jared J. Fradette, Joshua K. Ochieng, Lixia Diao, Jing Wang, Wei Lu, Luisa S. Solis, Harsh Batra, Maria G. Raso, Michael D. Peoples, Rosalba Minelli, Alessandro Carugo, Christopher A. Bristow, Don L. Gibbons
The metabolic environment is important for neuronal cells, such as photoreceptors. When photoreceptors undergo degeneration, as occurs during retinitis pigmentosa (RP), patients have progressive loss of vision that proceeds to full blindness. Currently, there are no available treatments for the majority of RP diseases. We performed metabolic profiling of the neural retina in a preclinical model of RP and found that tricarboxylic acid (TCA) cycle intermediates were reduced during disease. We then determined that, 1) promoting citrate production within the TCA cycle in retinal neurons during disease progression protects the photoreceptors from cell death and prolongs visual function, 2) that supplementation with single metabolites within the TCA cycle can provide this therapeutic effect in vivo over time, and, 3) that this therapeutic effect is not specific to a particular genetic mutation but has broad applicability for patients with RP and other retinal degenerative diseases. Overall, targeting TCA cycle activity in the neural retina promotes photoreceptor survival and visual function during neurodegenerative disease.
Ashley A. Rowe, Pinkal D. Patel, Ruth Gordillo, Katherine J. Wert
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