As blood transitions from steady laminar flow (S-flow) in healthy arteries to disturbed flow (D-flow) in aneurysmal arteries, platelets are subjected to external forces. Biomechanical platelet activation is incompletely understood and is a potential mechanism behind antiplatelet medication resistance. While it was demonstrated that anti-platelet drugs suppress growth of abdominal aortic aneurysms (AAA) in patients, we revealed a certain degree of platelet reactivity persisted in spite of aspirin therapy urging us to consider additional anti-platelet therapeutic targets. Transcriptomic profiling of platelets from patients with AAA revealed upregulation of a signal transduction pathway common to olfactory receptors (ORs), and this was explored as a mediator of AAA progression. Healthy platelets subjected to D-flow ex vivo, platelets from patients with AAA, and platelets in murine models of AAA demonstrated increased membrane olfactory receptor 2L13 (OR2L13) expression. A drug screen identified a molecule activating platelet OR2L13 which limited both biochemical and biomechanical platelet activation as well as AAA growth. This observation was further supported by selective deletion of the OR2L13 ortholog in a murine model of AAA that accelerated aortic aneurysm growth and rupture. These studies reveal that ORs regulate platelet activation in AAA and aneurysmal progression through platelet-derived mediators of aortic remodeling.
Craig N. Morrell, Doran Mix, Anu Aggarwal, Rohan Bhandari, Matthew Godwin, A. Phillip Owens III, Sean P. Lyden, Adam Doyle, Krystin Krauel, Matthew T. Rondina, Amy Mohan, Charles J. Lowenstein, Sharon Shim, Shaun Stauffer, Vara Prasad Josyula, Sara K. Ture, David I. Yule, Larry E. Wagner III, John M. Ashton, Ayman Elbadawi, Scott J. Cameron
Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activates chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplifies the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slows the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction and pericyte death, reduces neutrophil stalling and improves cerebrovascular reperfusion. Genetic analysis implicates altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function, and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer’s disease and vascular dementia.
Nils Korte, Zeki Ilkan, Claire L. Pearson, Thomas Pfeiffer, Prabhav Singhal, Jason R. Rock, Huma Sethi, Dipender Gill, David Attwell, Paolo Tammaro
The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 and histone acetylation were downregulated in NGBR deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of both HBO1 and acetylated H4K5/K12 on the promotor of CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.
Zhi Fang, Xiaoran Sun, Xiang Wang, Ji Ma, Thomas Palaia, Ujala Rana, Benjamin Miao, Louis Ragolia, Wenquan Hu, Qing Robert Miao
Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis post MI. Here we report that defective efferocytosis by macrophages after experimental MI leads to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic-induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA sequencing and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophages-produced VEGFC was directly effective at suppressing pro-inflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.
Kristofor E. Glinton, Wanshu Ma, Connor W. Lantz, Lubov S. Grigoryeva, Matthew DeBerge, Xiaolei Liu, Maria Febbraio, Mark Kahn, Guillermo Oliver, Edward B. Thorp
Genetic variants at the SORT1 locus in humans causing increased SORT1 expression in liver are significantly associated with reduced plasma levels of LDL cholesterol and apolipoprotein B (apoB). However, the role of hepatic sortilin remains controversial, as genetic deletion of sortilin in mice has yielded variable and conflicting effects on apoB secretion. Sort1 knockout mice on a chow diet and several Sort1-deficient hepatocyte lines displayed no difference in apoB secretion. When these models were challenged with high fat or ER stress, the loss of Sort1 expression resulted in a significant increase in apoB-100 secretion. Sort1 overexpression studies yielded reciprocal results. Importantly, diabetic carriers of SORT1 variant have larger decreases in plasma apoB, TG, and VLDL and LDL particle number as compared to non-diabetics with the same variants. We conclude that under basal non-stressed conditions, loss of sortilin has little effect on hepatocyte apoB secretion, but that in the setting of lipid-loading or ER stress, sortilin deficiency leads to increased apoB secretion. These results are consistent with the directionality of effect in human genetics studies and suggest that under stress conditions, hepatic sortilin directs apoB toward lysosomal degradation rather than secretion, potentially serving as a quality control step in the apoB secretion pathway in hepatocytes.
Donna M. Conlon, Carolin V. Schneider, Yi-An Ko, Amrith Rodrigues, Kathy Guo, Nicholas J. Hand, Daniel J. Rader
Sortilin has been positively correlated with vascular disorders in humans. No study has yet evaluated the possible direct effect of sortilin on vascular function. We used pharmacological and genetic approaches coupled with study of murine and human samples to unravel the mechanisms recruited by sortilin in the vascular system. Sortilin induced endothelial dysfunction of mesenteric arteries through NADPH oxidase 2 (NOX2) isoform activation, dysfunction that was prevented by knockdown of acid sphingomyelinase (ASMase) or sphingosine kinase 1. In vivo, recombinant sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of sphingosine-1-phosphate receptor 3 (S1P3) and gp91phox/NOX2 resulted in preservation of endothelial function and blood pressure homeostasis after 14 days of systemic sortilin administration. Translating these research findings into the clinical setting, we detected elevated sortilin levels in hypertensive patients with endothelial dysfunction. Furthermore, in a population-based cohort of 270 subjects, we showed increased plasma ASMase activity and increased plasma levels of sortilin, S1P, and soluble NOX2-derived peptide (sNOX2-dp) in hypertensive subjects, and the increase was more pronounced in hypertensive subjects with uncontrolled blood pressure. Our studies reveal what we believe is a previously unrecognized role of sortilin in the impairment of vascular function and in blood pressure homeostasis and suggest the potential of sortilin and its mediators as biomarkers for the prediction of vascular dysfunction and high blood pressure.
Paola Di Pietro, Albino Carrizzo, Eduardo Sommella, Marco Oliveti, Licia Iacoviello, Augusto Di Castelnuovo, Fausto Acernese, Antonio Damato, Massimiliano De Lucia, Fabrizio Merciai, Paola Iesu, Eleonora Venturini, Raffaele Izzo, Valentina Trimarco, Michele Ciccarelli, Giuseppe Giugliano, Roberto Carnevale, Vittoria Cammisotto, Serena Migliarino, Nicola Virtuoso, Andrea Strianese, Viviana Izzo, Pietro Campiglia, Elena Ciaglia, Bodo Levkau, Annibale A. Puca, Carmine Vecchione
Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans, and that its expression correlates negatively with long-term survival. Endothelial-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed “oxygen-glucose uncoupling”, which suppresses tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the anti-tumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms non-canonical inter-endothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrate the existence of tumor-specific inter-endothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.
Tomofumi Ando, Ikue Tai-Nagara, Yuki Sugiura, Dai Kusumoto, Koji Okabayashi, Yasuaki Kido, Kohji Sato, Hideyuki Saya, Sutip Navankasattusas, Dean Y. Li, Makoto Suematsu, Yuko Kitagawa, Elena Seiradake, Satoru Yamagishi, Yoshiaki Kubota
Piezo1 forms mechanically-activated non-selective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial apoptosis, with no effect on thrombospondin-1 (TSP1), a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without effect on Tsp1. In the endothelial cells, Piezo1 was required for normal expression of endothelial nitric oxide synthase (eNOS). The data suggest an endothelial-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.
Fiona Bartoli, Marjolaine Debant, Eulashini Chuntharpursat-Bon, Elizabeth L. Evans, Katie E. Musialowski, Gregory Parsonage, Lara C. Morley, T. Simon Futers, Piruthivi Sukumar, T. Scott Bowen, Mark T. Kearney, Laeticia Lichtenstein, Lee D. Roberts, David J. Beech
Obstructive arterial diseases including supravalvular aortic stenosis (SVAS), atherosclerosis and restenosis share two important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic or deletion mutations in the elastin gene ELN, and SVAS patients and elastin mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking as underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models as well as aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the Notch pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln(-/-) mutants. Eln(-/-) mice expressed higher levels of Notch ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln(-/-) mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.
Jui M. Dave, Raja Chakraborty, Aglaia Ntokou, Junichi Saito, Fatima Z. Saddouk, Zhonghui Feng, Ashish Misra, George Tellides, Robert K. Riemer, Zsolt Urban, Caroline Kinnear, James Ellis, Seema Mital, Robert Mecham, Kathleen A. Martin, Daniel M. Greif
Circular RNAs (circRNAs) have been recently recognized as playing a role in the pathogenesis of vascular remodeling–related diseases by modulating the functions of miRNAs. However, the interplay between circRNAs and proteins during vascular remodeling remains poorly understood. Here, we investigated a previously identified circRNA, circEsyt2, whose expression is known to be upregulated during vascular remodeling. Loss- and gain-of‑function mutation analyses in vascular smooth muscle cells (VSMCs) revealed that circEsyt2 enhanced cell proliferation and migration and inhibited apoptosis and differentiation. Furthermore, the silencing of circEsyt2 in vivo reduced neointima formation, while circEsyt2 overexpression enhanced neointimal hyperplasia in the injured carotid artery, confirming its role in vascular remodeling. Using unbiased protein–RNA screening and molecular validation, circEsyt2 was found to directly interact with polyC-binding protein 1 (PCBP1), an RNA splicing factor, and regulate PCBP1 intracellular localization. Additionally, circEsyt2 silencing substantially enhanced p53β splicing via the PCBP1–U2AF65 interaction, leading to the altered expression of p53 target genes (cyclin D1, p21, PUMA, and NOXA) and the decreased proliferation of VSMCs. Thus, we identified a potentially novel circRNA that regulated vascular remodeling, via altered RNA splicing, in atherosclerotic mouse models.
Xue Gong, Miao Tian, Nian Cao, Peili Yang, Zaicheng Xu, Shuo Zheng, Qiao Liao, Caiyu Chen, Cindy Zeng, Pedro A. Jose, Da-Zhi Wang, Zhao Jian, Yingbin Xiao, Ding-Sheng Jiang, Xiang Wei, Bing Zhang, Yibin Wang, Ken Chen, Gengze Wu, Chunyu Zeng