Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a pandemic. Severe disease is associated with dysfunction of multiple organs, but some infected cells do not express ACE2, the canonical entry receptor for SARS-CoV-2. Here, we report that the C-type lectin receptor L-SIGN interacted in a Ca2+-dependent manner with high-mannose–type N-glycans on the SARS-CoV-2 spike protein. We found that L-SIGN was highly expressed on human liver sinusoidal endothelial cells (LSECs) and lymph node lymphatic endothelial cells but not on blood endothelial cells. Using high-resolution confocal microscopy imaging, we detected SARS-CoV-2 viral proteins within the LSECs from liver autopsy samples from patients with COVID-19. We found that both pseudo-typed virus enveloped with SARS-CoV-2 spike protein and authentic SARS-CoV-2 virus infected L-SIGN–expressing cells relative to control cells. Moreover, blocking L-SIGN function reduced CoV-2–type infection. These results indicate that L-SIGN is a receptor for SARS-CoV-2 infection. LSECs are major sources of the clotting factors vWF and factor VIII (FVIII). LSECs from liver autopsy samples from patients with COVID-19 expressed substantially higher levels of vWF and FVIII than LSECs from uninfected liver samples. Our data demonstrate that L-SIGN is an endothelial cell receptor for SARS-CoV-2 that may contribute to COVID-19–associated coagulopathy.
Yuji Kondo, Jason L. Larabee, Liang Gao, Huiping Shi, Bojing Shao, Christopher M. Hoover, J. Michael McDaniel, Yen-Chun Ho, Robert Silasi-Mansat, Stephanie A. Archer-Hartmann, Parastoo Azadi, R. Sathish Srinivasan, Alireza R. Rezaie, Alain Borczuk, Jeffrey C. Laurence, Florea Lupu, Jasimuddin Ahamed, Rodger P. McEver, James F. Papin, Zhongxin Yu, Lijun Xia
Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.
Hui Shi, Alex A. Gandhi, Stephanie A. Smith, Qiuyu Wang, Diane Chiang, Srilakshmi Yalavarthi, Ramadan A. Ali, Chao Liu, Gautam Sule, Pei-Suen Tsou, Yu Zuo, Yogendra Kanthi, Evan A. Farkash, Jiandie D. Lin, James H. Morrissey, Jason S. Knight
Apolipoprotein B (ApoB) is the primary protein of chylomicrons, VLDLs, and LDLs and is essential for their production. Defects in ApoB synthesis and secretion result in several human diseases, including abetalipoproteinemia and familial hypobetalipoproteinemia (FHBL1). In addition, ApoB-related dyslipidemia is linked to nonalcoholic fatty liver disease (NAFLD), a silent pandemic affecting billions globally. Due to the crucial role of APOB in supplying nutrients to the developing embryo, ApoB deletion in mammals is embryonic lethal. Thus, a clear understanding of the roles of this protein during development is lacking. Here, we established zebrafish mutants for 2 apoB genes: apoBa and apoBb.1. Double-mutant embryos displayed hepatic steatosis, a common hallmark of FHBL1 and NAFLD, as well as abnormal liver laterality, decreased numbers of goblet cells in the gut, and impaired angiogenesis. We further used these mutants to identify the domains within ApoB responsible for its functions. By assessing the ability of different truncated forms of human APOB to rescue the mutant phenotypes, we demonstrate the benefits of this model for prospective therapeutic screens. Overall, these zebrafish models uncover what are likely previously undescribed functions of ApoB in organ development and morphogenesis and shed light on the mechanisms underlying hypolipidemia-related diseases.
Hanoch Templehof, Noga Moshe, Inbal Avraham-Davidi, Karina Yaniv
Glioblastoma is the most aggressive type of brain tumor with poor therapeutic response and prognosis. Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (EC). Tumor vessels in GBM are abnormal and more permeable, but the heterogeneity of BBB breakdown in different parts of the tumor vasculature and at the tumor’s invasive front is largely unknown. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.
Yuan Xie, Liqun He, Roberta Lugano, Yanyu Zhang, Haiyan Cao, Qiyuan He, Min Chao, Boxuan Liu, Qingze Cao, Jianhao Wang, Yang Jiao, Yaqin Hu, Liying Han, Yong Zhang, Hua Huang, Lene Uhrbom, Christer Betsholtz, Liang Wang, Anna Dimberg, Lei Zhang
Previously we reported heightened expression of human endogenous retroviral protein HERV-K deoxyuridine triphosphate nucleotidohydrolase (dUTPase) in circulating monocytes and pulmonary arterial (PA) adventitial macrophages of PA hypertension (PAH) patients. Furthermore, recombinant HERV-K dUTPase increased IL6 in PA endothelial cells (PAECs) and caused pulmonary hypertension in rats. Here we show that monocytes overexpressing HERV-K dUTPase as opposed to GFP, can release HERV-K dUTPase in extracellular vesicles (EVs) that cause pulmonary hypertension in mice in association with endothelial mesenchymal transition (EndMT) related to induction of SNAIL/SLUG, and proinflammatory molecules IL6 as well as VCAM1. In PAECs, HERV-K dUTPase requires TLR4-myeloid differentiation primary response (MYD)-88 to increase IL6 and SNAIL/SLUG, and HERV-K dUTPase interaction with melanoma cell adhesion molecule (MCAM) is necessary to upregulate VCAM1. TLR4 engagement induces p-p38 activation of NF-κB in addition to pJNK-pSMAD3 required for SNAIL, and pSTAT1 for IL6. HERV-K dUTPase interaction with MCAM also induces p-p38 activation of NF-κB in addition to pERK1/2-activating transcription factor (ATF)-2 to increase VCAM1. Thus in PAH, monocytes or macrophages can release HERV-K dUTPase in EVs, and HERV-K dUTPase can engage dual receptors and signaling pathways to subvert PAEC transcriptional machinery to induce EndMT and associated pro-inflammatory molecules.
Shoichiro Otsuki, Toshie Saito, Shalina Taylor, Dan Li, Jan-Renier Moonen, David P. Marciano, Rebecca L. Harper, Aiqin Cao, Lingli Wang, Maria E. Ariza, Marlene Rabinovitch
AAA is a disease with high morbidity and mortality, especially when ruptured. The rational of this study was to evaluate the repurposing of lenvatinib, a multi tyrosine kinase inhibitor, in limiting experimental abdominal aortic aneurysm (AAA) growth targeting vascular smooth muscle cells (VSMC) and angiogenesis. We applied systemic and local lenvatinib treatment to elastase-induced murine aortic aneurysms, and RNA profiling identified myosin heavy chain 11 (Myh11) as the most deregulated transcript. Daily oral treatment significantly reduced aneurysm formation in two independent mouse models. In addition, a large animal aneurysm model in hypercholesterolemic low-density lipoprotein receptor knockout (LDLR-/-) Yucatan minipigs was applied to endovascularly deliver Lenvatinib via drug-eluting balloons (DEB). Here, a single local endovascular delivery blocked AAA progression successfully compared to a DEB-delivered control treatment. Reduced VSMC proliferation and a restored contractile phenotype were observed in animal tissues (murine and porcine), as well as AAA patient-derived cells. Apart from increasing MYH11 levels, lenvatinib reduced downstream ERK signaling. Hence, Lenvatinib is a promising new therapy to limit aortic aneurysm expansion upon local endovascular delivery. The tyrosine kinase inhibitor was able to positively affect pathways of key relevance to human AAA disease, even in a potentially new local delivery using DEBs.
Albert Busch, Jessica Pauli, Greg Winski, Sonja Bleichert, Ekaterina Chernogubova, Susanne Metschl, Hanna Winter, Matthias Trenner, Armin Wiegering, Christoph Otto, Johannes Fischer, Judith Reiser, Julia Werner, Joy Roy, Christine Brostjan, Christoph Knappich, Hans-Henning Eckstein, Valentina Paloschi, Lars Maegdefessel
Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatic vessels in bone, gradually lose bone, and can have life-threatening complications such as chylothorax. The etiology of GSD is poorly understood and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a GSD patient and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.
Nassim Homayun Sepehr, Anna L. McCarter, Raphaël Helaers, Christine Galant, Laurence M. Boon, Pascal Brouillard, Miikka Vikkula, Michael T. Dellinger
The stimulator of interferon genes (STING) protein senses cyclic di-nucleotides released in response to double stranded DNA, and functions as an adaptor molecule for type I interferon (IFN-I) signaling by activating IFN-I stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared to control EC, whereas T cells adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell expressed molecules ICAM-1 and VCAM-1 compared to WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC and genetic loss or pharmacologic antagonism of IFN-type I interferon receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC STING during T cell TEM that is dependent on the ISG CXCL10 and on IFN-I-IFNAR signaling.
Marina Anastasiou, Gail A. Newton, Kuljeet Kaur, Francisco J. Carrillo-Salinas, Sasha A. Smolgovsky, Abraham L. Bayer, Vladimir Ilyukha, Shruti Sharma, Alexander Poltorak, Francis W. Luscinskas, Pilar Alcaide
SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3.
Nina Martino, Ramon Bossardi Ramos, Shuhan Lu, Kara Leyden, Lindsay Tomaszek, Sudeshna Sadhu, Gabrielle Fredman, Ariel Jaitovich, Peter A. Vincent, Alejandro P. Adam
Excessive proliferation of vascular smooth muscle cells (SMCs) remains a significant cause of in-stent restenosis. Integrins, which are heterodimeric transmembrane receptors, play a crucial role in SMC biology by binding to the extracellular matrix protein with the actin cytoskeleton within the SMC. Integrin α9 plays an important role in cell motility and autoimmune diseases; however, its role in SMC biology and remodeling remains unclear. Herein, we demonstrate that stimulated human coronary SMCs upregulate α9 expression. Targeting α9 in stimulated human coronary SMCs, using anti–integrin α9 antibody, suppresses synthetic phenotype and inhibits SMC proliferation and migration. To provide definitive evidence, we generated an SMC-specific α9-deficient mouse strain. Genetic ablation of α9 in SMCs suppressed synthetic phenotype and reduced proliferation and migration in vitro. Mechanistically, suppressed synthetic phenotype and reduced proliferation were associated with decreased focal adhesion kinase/steroid receptor coactivator signaling and downstream targets, including phosphorylated ERK, p38 MAPK, glycogen synthase kinase 3β, and nuclear β-catenin, with reduced transcriptional activation of β-catenin target genes. Following vascular injury, SMC-specific α9-deficient mice or wild-type mice treated with murine anti–integrin α9 antibody exhibited reduced injury-induced neointimal hyperplasia at day 28 by limiting SMC migration and proliferation. Our findings suggest that integrin α9 regulates SMC biology, suggesting its potential therapeutic application in vascular remodeling.
Manish Jain, Rishabh Dev, Prakash Doddapattar, Shigeyuki Kon, Nirav Dhanesha, Anil K. Chauhan
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