Endothelial cells (ECs) are constitutively an anticoagulant surface but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases”, such as TMEM16F. TMEM16F-dependent PS externalization is well-characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
Dysfunction of vascular endothelial cells (ECs) facilitates imbalanced immune responses and tissue hyperinflammation. However, the heterogeneous functions of skin ECs and their underlying mechanism in dermatoses remain to be solved. Here, focusing on the pathogenic role of skin ECs in psoriasis, we characterized the molecular and functional heterogeneity of skin ECs from healthy individuals and psoriasis patients at the single-cell level. We found that endothelial glycocalyx destruction, a major feature of EC dysfunction in psoriasis, was a driving force during the process of T cell extravasation. Interestingly, we identified a skin EC subset, IGFBP7high ECs, in psoriasis. This subset actively responded to psoriatic-related cytokine signaling, secreted IGFBP7, damaged the endothelial glycocalyx, exposed the adhesion molecules underneath, and prepared the endothelium for immune cell adhesion and transmigration, thus aggravating skin inflammation. More importantly, we provided evidence in a psoriasis-like mouse model that anti-IGFBP7 treatment showed promising therapeutic effects for restoring the endothelial glycocalyx and alleviating skin inflammation. Taken together, our results depicted the distinct functions of EC clusters in healthy and psoriatic skin, identified IGFBP7high ECs as an active subset modulating vascular function and cutaneous inflammation, and indicated that targeting IGFBP7 is a potential therapeutic strategy in psoriasis.
Qingyang Li, Shuai Shao, Zhenlai Zhu, Jiaoling Chen, Junfeng Hao, Yaxing Bai, Bing Li, Erle Dang, Gang Wang
Christian Lacks Lino Cardenas, Lauren C. Briere, David A. Sweetser, Mark E. Lindsay, Patricia L. Musolino
Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long non-coding RNAs are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the LncRNA that Enhances Endothelial Nitric oxide synthase Expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression is decreased in diabetic conditions in cultured endothelial cells (EC), mouse hindlimb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduces their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in leene demonstrate impaired angiogenesis and perfusion following hindlimb ischemia. Importantly, overexpression of human LEENE rescues the impaired ischemic response in leene knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promotes transcription of pro-angiogenic genes in ECs, such as KDR and eNOS, potentially by interacting with LEO1, a key component of RNA Polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.
Xiaofang Tang, Yingjun Luo, Dongqiang Yuan, Riccardo Calandrelli, Naseeb Kaur Malhi, Kiran Sriram, Yifei Miao, Chih Hong Lou, Walter Tsark, Alonso Tapia, Aleysha T. Chen, Guangyu Zhang, Daniel Roeth, Markus Kalkum, Zhao V. Wang, Shu Chien, Rama Natarajan, John P. Cooke, Sheng Zhong, Zhen Bouman Chen
Pain signals are relayed to the brain via a nociceptive system, and in rare situations, this nociceptive system contains genetic variants that can limit pain response. Here we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and further if we can target this region by a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N knock-in mouse using CRISPR/Cas9, we discovered the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral response to chemical noxious stimuli and less hypersensitivity to nerve injury-induced pain, while leaving the response to noxious heat intact. Furthermore, blocking this K710 region in wild-type rodents by a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and rescued pain hypersensitivity induced by nerve injury back to baseline. These findings identify K710 TRPV1 as a discrete site crucial for the control of nociception and provides new insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.
Shufang He, Vanessa O. Zambelli, Pritam Sinharoy, Laura Brabenec, Yang Bian, Freeborn Rwere, Rafaela C.R. Hell, Beatriz Stein Neto, Barbara Hung, Xuan Yu, Meng Zhao, Zhaofei Luo, Chao Wu, Lijun Xu, Katrin J. Svensson, Stacy L. McAllister, Creed M. Stary, Nana-Maria Wagner, Ye Zhang, Eric R. Gross
Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. BAF60c, a unique subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, is critical for cardiac and skeletal myogenesis; yet, little is known about its function in the vasculature and, specifically, in AAA pathogenesis. Here, we found that BAF60c was downregulated in human and mouse AAA tissues, with primary staining to vascular smooth muscle cells (VSMC), confirmed by single-cell RNA-sequencing. In vivo studies revealed that VSMC-specific knockout of Baf60c significantly aggravated both AngII- and elastase-induced AAA formation in mice, with a significant increase in elastin degradation, inflammatory cell infiltration, VSMC phenotypic switch, and apoptosis. In vitro studies showed that BAF60c knockdown in VSMC resulted in the loss of contractile phenotype, increased VSMC inflammation, and apoptosis. Mechanistically, we demonstrated that BAF60c preserved VSMC contractile phenotype by strengthening serum response factor (SRF) association with its co-activator P300 and the SWI/SNF complex, suppressed VSMC inflammation by promoting a repressive chromatin state of the NFκB-target genes, as well as prevented VSMC apoptosis through transcriptional activation of KLF5-dependent BCL2 expression. Together, our identification of the essential role of BAF60c in preserving VSMC homeostasis expands its therapeutic potential in preventing and treating AAA.
Guizhen Zhao, Yang Zhao, Haocheng Lu, Ziyi Chang, Hongyu Liu, Huilun Wang, Wenying Liang, Yuhao Liu, Tianqing Zhu, Oren Rom, Yanhong Guo, Lin Chang, Bo Yang, Minerva T. Garcia-Barrio, Jiandie D. Lin, Y. Eugene Chen, Jifeng Zhang
Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor-3 (VEGFR3), encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here we used gene deletion, blocking antibodies, transgene induction and gene transfer to study how Ang2, its Tie2 receptor and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) is involved in full Akt activation downstream of phosphoinositide-3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of Ang2 blocking antibody decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of PI3K catalytic p110α subunit or with small molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2-Tie-PI3K signaling.
Emilia A. Korhonen, Aino Murtomäki, Sawan Kumar Jha, Andrey Anisimov, Anne Pink, Yan Zhang, Simon Stritt, Inam Liaqat, Lukas Stanczuk, Laura Alderfer, Zhiliang Sun, Emmi Kapiainen, Abhishek Singh, Ibrahim Sultan, Anni Lantta, Veli-Matti Leppänen, Lauri Eklund, Yulong He, Hellmut G. Augustin, Kari Vaahtomeri, Pipsa Saharinen, Taija Mäkinen, Kari Alitalo
Cardiovascular disease is the major cause of morbidity and mortality in breast cancer survivors. Chemotherapy contributes to this risk. We aimed to define the mechanisms of long-term vascular dysfunction caused by neoadjuvant chemotherapy (NACT) and identify novel therapeutic targets. We studied arteries from postmenopausal women who had undergone breast cancer treatment using docetaxel, doxorubicin and cyclophosphamide (NACT), and women with no history of such treatment matched for key clinical parameters. Mechanisms were explored in wild-type and Nox4-/- mice and human microvascular endothelial cells. Endothelium-dependent vasodilatation is severely impaired in patients after NACT, while endothelium-independent responses remain normal. This was mimicked by 24-hour exposure of arteries to NACT agents ex-vivo. When applied individually, only docetaxel impaired endothelial function in human vessels. Mechanistic studies showed that NACT increased inhibitory eNOS phosphorylation of threonine 495 in a ROCK-dependent manner and augmented vascular superoxide and hydrogen peroxide production and NADPH oxidase activity. Docetaxel increased expression of NADPH oxidase NOX4 in endothelial and smooth muscle cells and NOX2 in the endothelium. NOX4 increase in human arteries may be mediated epigenetically by diminished DNA methylation of the NOX4 promoter. Docetaxel induced endothelial dysfunction and hypertension in mice. These were prevented in Nox4-/- and by pharmacological inhibition of Nox4 or Rock. Commonly used chemotherapeutic agents, and in particular, docetaxel, alter vascular function by promoting inhibitory phosphorylation of eNOS and enhancing ROS production by NADPH oxidases.
Piotr Szczepaniak, Mateusz Siedlinski, Diana Hodorowicz-Zaniewska, Ryszard Nosalski, Tomasz P. Mikolajczyk, Aneta M. Dobosz, Anna Dikalova, Sergey Dikalov, Joanna Streb, Katarzyna Gara, Pawel Basta, Jaroslaw Krolczyk, Joanna Sulicka-Grodzicka, Ewelina Jozefczuk, Anna Dziewulska, Blessy Saju, Iwona Laksa, Wei Chen, John Dormer, Maciej Tomaszewski, Pasquale Maffia, Marta Czesnikiewicz-Guzik, Filippo Crea, Agnieszka Dobrzyn, Javid Moslehi, Tomasz Grodzicki, David G. Harrison, Tomasz J. Guzik
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 (CH). Jak2VF mice with elevated white blood cells, platelets and red blood cells (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 CH, but had no impact in controls. Erythroid lineage Jak2VF expression leads to qualitative and quantitative defects in RBCs that exacerbate atherosclerosis. Phagocytosis of RBCs by plaque macrophages promotes ferroptosis, suggesting a new therapeutic target to reduce RBC-mediated cardiovascular risk.
Wenli Liu, Nataliya K. Östberg, Mustafa Yalcinkaya, Huijuan Dou, Kaori Endo-Umeda, Yang Tang, Xintong Hou, Tong Xiao, Trevor Filder, Sandra Abramowicz, Yong-Guang Yang, Oliver Soehnlein, Alan R. Tall, Nan Wang
Pericytes (PC) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated if PC can be reprogrammed to aid neovascularization. Primary PC from human and mouse hearts acquired cytoskeleton proteins typical of vascular smooth muscle cells (VSMC) upon exclusion of EGF/bFGF, which signal through ERK1/2, or exposure to the MEK-inhibitor PD0325901. Differentiated PC became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA-Sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of two angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid-binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PC during in vivo studies. In mice, implantation of Matrigel plugs containing human PC+PD0325901 promoted the formation of α-SMApos neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRβposAQP1posCRABP2pos PC, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PC have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.
Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu