Vascular biology
Abstract
Autophagy has long been associated with longevity and it is well established that autophagy reverts and prevents vascular deterioration associated with aging and cardiovascular diseases. Currently, our understanding of how autophagy benefits the vasculature is centered on the premise that reduced autophagy leads to the accumulation of cellular debris resulting in inflammation and oxidative stress, which are then reversed by reconstitution or upregulation of autophagic activity. Evolutionarily, autophagy also functions to mobilize endogenous nutrients in response to starvation. Therefore, we hypothesized that the biosynthesis of the most physiologically abundant ketone body, β-hydroxybutyrate (βHB), would be autophagy dependent, and exert vasodilatory effects via its canonical receptor, Gpr109a. We have revealed for the first time that the biosynthesis of βHB can be impaired by preventing autophagy. Subsequently, βHB caused potent vasodilation via potassium-channels, but not Gpr109a. Finally, we observed that chronic consumption of a high salt diet negatively regulates both βHB biosynthesis and hepatic autophagy, and that reconstitution of βHB bioavailability prevents high salt diet-induced endothelial dysfunction. In summary, this work offers an alternative mechanism to the anti-inflammatory and anti-oxidative stress hypothesis of autophagy-dependent vasculoprotection. Furthermore, it reveals a direct mechanism, by which ketogenic interventions (e.g., intermittent fasting) improve vascular health.
Authors
Cameron G. McCarthy, Saroj Chakraborty, Gagandeep Singh, Beng San Yeoh, Zachary J. Schreckenberger, Avinash Singh, Blair Mell, Nicole R. Bearss, Tao Yang, Xi Cheng, Matam Vijay-Kumar, Camilla F. Wenceslau, Bina Joe
Abstract
The mutant nuclear lamin protein (progerin) produced in Hutchinson-Gilford progeria syndrome (HGPS) results in loss of arterial smooth muscle cells (SMCs), but the mechanism has been unclear. We found that progerin induces repetitive nuclear membrane (NM) ruptures, DNA damage, and cell death in cultured SMCs. Reducing lamin B1 expression and exposing cells to mechanical stress — to mirror conditions in the aorta — triggered more frequent NM ruptures. Increasing lamin B1 protein levels had the opposite effect, reducing NM ruptures and improving cell survival. Remarkably, raising lamin B1 levels increased nuclear compliance in cells and was able to offset the increased nuclear stiffness caused by progerin. In mice, lamin B1 expression in aortic SMCs is normally very low, and in mice with a targeted HGPS mutation (LmnaG609G), levels of lamin B1 decrease further with age while progerin levels increase. Those observations suggest that NM ruptures might occur in aortic SMCs in vivo. Indeed, studies in LmnaG609G mice identified NM ruptures in aortic SMCs, along with ultrastructural abnormalities in the cell nucleus that preceded SMC loss. Our studies identify NM ruptures in SMCs as likely causes of vascular pathology in HGPS.
Authors
Paul H. Kim, Natalie Y. Chen, Patrick J. Heizer, Yiping Tu, Thomas A. Weston, Jared L.-C. Fong, Navjot Kaur Gill, Amy C. Rowat, Stephen G. Young, Loren G. Fong
Abstract
The prevalence of hypertension is increasing globally, while strategies for prevention and treatment of hypertension remain limited. FG-4592 (Roxadustat) is a novel, orally active small-molecule HIF stabilizer, and is being used clinically to treat CKD anemia. In the present study, we evaluate the effects of FG-4592 on hypertension. In an Ang II hypertension model, FG-4592 abolished hypertensive responses, prevented vascular thickening, cardiac hypertrophy, and kidney injury, downregulated AGTR1 expression, and enhanced AGTR2, eNOS, and HIF1α protein levels in the aortas of mice. Additionally, the levels of thiobarbituric acid reactive substances (TBARs) in blood and urine were diminished by FG-4592 treatment. In vascular smooth muscle cells, FG-4592 treatment reduced AGTR1 and increased AGTR2 levels, while preventing Ang II-induced oxidative stress. In vascular endothelial cells, FG-4592 upregulated total and phosphorylated eNOS. Moreover, FG-4592 treatment was hypotensive in L-NAME-induced hypertension. In summary, FG-4592 treatment remarkably ameliorated hypertension and organ injury, possibly through stabilizing HIF1α and subsequently targeting eNOS, AGTR1, AGTR2, and oxidative stress. Therefore, in addition to its role in treating CKD anemia, FG-4592 could be explored as a treatment for hypertension associated with high RAS activity or eNOS defects.
Authors
Jing Yu, Shuqin Wang, Wei Shi, Wei Zhou, Yujia Niu, Songming Huang, Yue Zhang, Aihua Zhang, Zhanjun Jia
Abstract
Neutrophils provide a critical line of defense in immune responses to various pathogens, but also inflict self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients suffering from COVID-19. Here, we utilize state-of-the art mass spectrometry-based proteomics, transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic interleukin-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces SARS-CoV-2 spike protein-induced, hACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8-axis as promising therapeutic target in severe SARS-CoV-2 infection.
Authors
Rainer Kaiser, Alexander Leunig, Kami Pekayvaz, Oliver Popp, Markus Joppich, Vivien Polewka, Raphael Escaig, Afra Anjum, Marie-Louise Hoffknecht, Christoph Gold, Sophia Brambs, Anouk Engel, Sven Stockhausen, Viktoria Knottenberg, Anna Titova, Mohamed Haji, Clemens Scherer, Maximilian Muenchhoff, Johannes C. Hellmuth, Kathrin Saar, Benjamin Schubert, Anne Hilgendorff, Christian Schulz, Stefan Kääb, Ralf Zimmer, Norbert Hübner, Steffen Massberg, Philipp Mertins, Leo Nicolai, Konstantin Stark
Abstract
Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Murine and human data suggest that the NLRP3-IL-1β pathway is the main driver of KD pathophysiology. NLRP3 can be activated during defective autophagy/mitophagy. We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, to examine the role of autophagy/mitophagy on cardiovascular lesion development. LCWE-injected mice had impaired autophagy/mitophagy and increased levels of ROS in cardiovascular lesions, together with increased systemic 8-OHdG release. Enhanced autophagic flux significantly reduced cardiovascular lesions in LCWE-injected mice, whereas autophagy blockade increased inflammation. Vascular smooth muscle cell specific deletion of Atg16l1 and global Parkin-/- significantly increased disease formation, supporting the importance of autophagy/mitophagy in this model. Ogg1-/- mice had significantly increased lesions with increased NLRP3 activity, whereas treatment with MitoQ, reduced vascular tissue inflammation, ROS production and systemic 8-OHdG release. Treatment with MN58b or Metformin (increasing AMPK and reducing ROS), resulted in decreased disease formation. Our results demonstrate that impaired autophagy/mitophagy and ROS-dependent damage exacerbate the development of murine KD vasculitis. This pathway can be efficiently targeted to reduce disease severity. These findings enhance our understanding of KD pathogenesis and identify novel therapeutic avenues for KD treatment.
Authors
Stefanie Marek-Iannucci, A. Beyza Ozdemir, Debbie Moreira, Angela C. Gomez, Malcolm Lane, Rebecca A Porritt, Youngho Lee, Kenichi Shimada, Masanori Abe, Aleksandr Stotland, David Zemmour, Sarah Parker, Elsa Sanchez-Lopez, Jennifer Van Eyk, Roberta A. Gottlieb, Michael Fishbein, Michael Karin, Timothy R Crother, Magali Noval Rivas, Moshe Arditi
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
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
Abstract
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.
Authors
Hanoch Templehof, Noga Moshe, Inbal Avraham-Davidi, Karina Yaniv
Abstract
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.
Authors
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
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