Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor–deficient (
Bishuang Cai, Edward B. Thorp, Amanda C. Doran, Brian E. Sansbury, Mat J.A.P. Daemen, Bernhard Dorweiler, Matthew Spite, Gabrielle Fredman, Ira Tabas
Controlled angiogenesis and lymphangiogenesis are essential for tissue development, function, and repair. However, aberrant neovascularization is an essential pathogenic mechanism in many human diseases, including diseases involving tumor growth and survival. Here, we have demonstrated that mice deficient in C-type lectin family 14 member A (CLEC14A) display enhanced angiogenic sprouting and hemorrhage as well as enlarged jugular lymph sacs and lymphatic vessels. CLEC14A formed a complex with VEGFR-3 in endothelial cells (ECs), and CLEC14A KO resulted in a marked reduction in VEGFR-3 that was concomitant with increases in VEGFR-2 expression and downstream signaling. Implanted tumor growth was profoundly reduced in CLEC14A-KO mice compared with that seen in WT littermates, but tumor-bearing CLEC14A-KO mice died sooner. Tumors in CLEC14A-KO mice had increased numbers of nonfunctional blood vessels and severe hemorrhaging. Blockade of VEGFR-2 signaling suppressed these vascular abnormalities and enhanced the survival of tumor-bearing CLEC14A-KO mice. We conclude that CLEC14A acts in vascular homeostasis by fine-tuning VEGFR-2 and VEGFR-3 signaling in ECs, suggesting its relevance in the pathogenesis of angiogenesis-related human disorders.
Sungwoon Lee, Seung-Sik Rho, Hyojin Park, Jeong Ae Park, Jihye Kim, In-Kyu Lee, Gou Young Koh, Naoki Mochizuki, Young-Myeong Kim, Young-Guen Kwon
Krishnaraj S. Rathod, Vikas Kapil, Shanti Velmurugan, Rayomand S. Khambata, Umme Siddique, Saima Khan, Sven Van Eijl, Lorna C. Gee, Jascharanpreet Bansal, Kavi Pitrola, Christopher Shaw, Fulvio D’Acquisto, Romain A. Colas, Federica Marelli-Berg, Jesmond Dalli, Amrita Ahluwalia
Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.
Giuseppe Faraco, Yukio Sugiyama, Diane Lane, Lidia Garcia Bonilla, Haejoo Chang, Monica M. Santisteban, Gianfranco Racchumi, Michelle Murphy, Nico Van Rooijen, Joseph Anrather, Costantino Iadecola
Homeostatic control of tissue oxygenation is achieved largely through changes in blood flow that are regulated by the classic physiological response of hypoxic vasodilation. The role of nitric oxide (NO) in the control of blood flow is a central tenet of cardiovascular biology. However, extensive evidence now indicates that hypoxic vasodilation entails
Rongli Zhang, Douglas T. Hess, James D. Reynolds, Jonathan S. Stamler
Arterial blood pressure is controlled by vasodilatory factors such as nitric oxide (NO) that are released from the endothelium under the influence of fluid shear stress exerted by flowing blood. Flow-induced endothelial release of ATP and subsequent activation of Gq/G11–coupled purinergic P2Y2 receptors have been shown to mediate fluid shear stress–induced stimulation of NO formation. However, the mechanism by which fluid shear stress initiates these processes is unclear. Here, we have shown that the endothelial mechanosensitive cation channel PIEZO1 is required for flow-induced ATP release and subsequent P2Y2/Gq/G11–mediated activation of downstream signaling that results in phosphorylation and activation of AKT and endothelial NOS. We also demonstrated that PIEZO1-dependent ATP release is mediated in part by pannexin channels. The PIEZO1 activator Yoda1 mimicked the effect of fluid shear stress on endothelial cells and induced vasorelaxation in a PIEZO1-dependent manner. Furthermore, mice with induced endothelium-specific PIEZO1 deficiency lost the ability to induce NO formation and vasodilation in response to flow and consequently developed hypertension. Together, our data demonstrate that PIEZO1 is required for the regulation of NO formation, vascular tone, and blood pressure.
ShengPeng Wang, Ramesh Chennupati, Harmandeep Kaur, Andras Iring, Nina Wettschureck, Stefan Offermanns
Carcinoma cells can acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT). However, the significance of EMT in cancer metastasis has been controversial, and the exact fates and functions of EMT cancer cells in vivo remain inadequately understood. Here, we tracked epithelial cancer cells that underwent inducible or spontaneous EMT in various tumor transplantation models. Unlike epithelial cells, the majority of EMT cancer cells were specifically located in the perivascular space and closely associated with blood vessels. EMT markedly activated multiple pericyte markers in carcinoma cells, in particular PDGFR-β and N-cadherin, which enabled EMT cells to be chemoattracted towards and physically interact with endothelium. In tumor xenografts generated from carcinoma cells that were prone to spontaneous EMT, a substantial fraction of the pericytes associated with tumor vasculature were derived from EMT cancer cells. Depletion of such EMT cells in transplanted tumors diminished pericyte coverage, impaired vascular integrity, and attenuated tumor growth. These findings suggest that EMT confers key pericyte attributes on cancer cells. The resulting EMT cells phenotypically and functionally resemble pericytes and are indispensable for vascular stabilization and sustained tumor growth. This study thus proposes a previously unrecognized role for EMT in cancer.
Anitha K. Shenoy, Yue Jin, Huacheng Luo, Ming Tang, Christine Pampo, Rong Shao, Dietmar W. Siemann, Lizi Wu, Coy D. Heldermon, Brian K. Law, Lung-Ji Chang, Jianrong Lu
Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during
Minah Kim, Breanna Allen, Emilia A. Korhonen, Maximilian Nitschké, Hee Won Yang, Peter Baluk, Pipsa Saharinen, Kari Alitalo, Christopher Daly, Gavin Thurston, Donald M. McDonald
The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin–dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial
Emilia A. Korhonen, Anita Lampinen, Hemant Giri, Andrey Anisimov, Minah Kim, Breanna Allen, Shentong Fang, Gabriela D’Amico, Tuomas J. Sipilä, Marja Lohela, Tomas Strandin, Antti Vaheri, Seppo Ylä-Herttuala, Gou Young Koh, Donald M. McDonald, Kari Alitalo, Pipsa Saharinen
Dysregulation of vascular stiffness and cellular metabolism occurs early in pulmonary hypertension (PH). However, the mechanisms by which biophysical properties of the vascular extracellular matrix (ECM) relate to metabolic processes important in PH remain undefined. In this work, we examined cultured pulmonary vascular cells and various types of PH-diseased lung tissue and determined that ECM stiffening resulted in mechanoactivation of the transcriptional coactivators YAP and TAZ (WWTR1). YAP/TAZ activation modulated metabolic enzymes, including glutaminase (GLS1), to coordinate glutaminolysis and glycolysis. Glutaminolysis, an anaplerotic pathway, replenished aspartate for anabolic biosynthesis, which was critical for sustaining proliferation and migration within stiff ECM. In vitro, GLS1 inhibition blocked aspartate production and reprogrammed cellular proliferation pathways, while application of aspartate restored proliferation. In the monocrotaline rat model of PH, pharmacologic modulation of pulmonary vascular stiffness and YAP-dependent mechanotransduction altered glutaminolysis, pulmonary vascular proliferation, and manifestations of PH. Additionally, pharmacologic targeting of GLS1 in this model ameliorated disease progression. Notably, evaluation of simian immunodeficiency virus–infected nonhuman primates and HIV-infected subjects revealed a correlation between YAP/TAZ–GLS activation and PH. These results indicate that ECM stiffening sustains vascular cell growth and migration through YAP/TAZ-dependent glutaminolysis and anaplerosis, and thereby link mechanical stimuli to dysregulated vascular metabolism. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in PH.
Thomas Bertero, William M. Oldham, Katherine A. Cottrill, Sabrina Pisano, Rebecca R. Vanderpool, Qiujun Yu, Jingsi Zhao, Yiyin Tai, Ying Tang, Ying-Yi Zhang, Sofiya Rehman, Masataka Sugahara, Zhi Qi, John Gorcsan III, Sara O. Vargas, Rajan Saggar, Rajeev Saggar, W. Dean Wallace, David J. Ross, Kathleen J. Haley, Aaron B. Waxman, Victoria N. Parikh, Teresa De Marco, Priscilla Y. Hsue, Alison Morris, Marc A. Simon, Karen A. Norris, Cedric Gaggioli, Joseph Loscalzo, Joshua Fessel, Stephen Y. Chan
Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL–derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex–driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor–deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.
Takuro Miyazaki, Kazuo Tonami, Shoji Hata, Toshihiro Aiuchi, Koji Ohnishi, Xiao-Feng Lei, Joo-ri Kim-Kaneyama, Motohiro Takeya, Hiroyuki Itabe, Hiroyuki Sorimachi, Hiroki Kurihara, Akira Miyazaki
Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, a dual Src and BCR-ABL tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). Today, key questions remain regarding the mechanisms involved in the long-term development of dasatinib-induced PAH. Here, we demonstrated that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents. We found that dasatinib treatment attenuated hypoxic pulmonary vasoconstriction responses and increased susceptibility to experimental pulmonary hypertension (PH) in rats, but these effects were absent in rats treated with imatinib, another BCR-ABL tyrosine kinase inhibitor. Furthermore, dasatinib treatment induced pulmonary endothelial cell apoptosis in a dose-dependent manner, while imatinib did not. Dasatinib treatment mediated endothelial cell dysfunction via increased production of ROS that was independent of Src family kinases. Consistent with these findings, we observed elevations in markers of endothelial dysfunction and vascular damage in the serum of CML patients who were treated with dasatinib, compared with CML patients treated with imatinib. Taken together, our findings indicate that dasatinib causes pulmonary vascular damage, induction of ER stress, and mitochondrial ROS production, which leads to increased susceptibility to PH development.
Christophe Guignabert, Carole Phan, Andrei Seferian, Alice Huertas, Ly Tu, Raphaël Thuillet, Caroline Sattler, Morane Le Hiress, Yuichi Tamura, Etienne-Marie Jutant, Marie-Camille Chaumais, Stéphane Bouchet, Benjamin Manéglier, Mathieu Molimard, Philippe Rousselot, Olivier Sitbon, Gérald Simonneau, David Montani, Marc Humbert
The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in
Anees Fatima, Ying Wang, Yutaka Uchida, Pieter Norden, Ting Liu, Austin Culver, William H. Dietz, Ford Culver, Meredith Millay, Yoh-suke Mukouyama, Tsutomu Kume
Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.
Hung M. Bui, David Enis, Marius R. Robciuc, Harri J. Nurmi, Jennifer Cohen, Mei Chen, Yiqing Yang, Veerpal Dhillon, Kathy Johnson, Hong Zhang, Robert Kirkpatrick, Elizabeth Traxler, Andrey Anisimov, Kari Alitalo, Mark L. Kahn
The chromatin-remodeling enzyme CHD4 maintains vascular integrity at mid-gestation; however, it is unknown whether this enzyme contributes to later blood vessel or lymphatic vessel development. Here, we addressed this issue in mice harboring a deletion of
Patrick L. Crosswhite, Joanna J. Podsiadlowska, Carol D. Curtis, Siqi Gao, Lijun Xia, R. Sathish Srinivasan, Courtney T. Griffin
The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology.
Do-Geun Kim, Margaret S. Bynoe
Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in
Stoyan Ivanov, Joshua P. Scallan, Ki-Wook Kim, Kathrin Werth, Michael W. Johnson, Brian T. Saunders, Peter L. Wang, Emma L. Kuan, Adam C. Straub, Melissa Ouhachi, Erica G. Weinstein, Jesse W. Williams, Carlos Briseño, Marco Colonna, Brant E. Isakson, Emmanuel L. Gautier, Reinhold Förster, Michael J. Davis, Bernd H. Zinselmeyer, Gwendalyn J. Randolph
Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II–induced AAA and elastase perfusion–stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of
Chao Zhang, Dustin van der Voort, Hong Shi, Rongli Zhang, Yulan Qing, Shuichi Hiraoka, Minoru Takemoto, Koutaro Yokote, Joseph V. Moxon, Paul Norman, Laure Rittié, Helena Kuivaniemi, G. Brandon Atkins, Stanton L. Gerson, Guo-Ping Shi, Jonathan Golledge, Nianguo Dong, Bernard Perbal, Domenick A. Prosdocimo, Zhiyong Lin
Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2–dependent C-terminal phosphorylation of sortilin. In a murine model,
Claudia Goettsch, Joshua D. Hutcheson, Masanori Aikawa, Hiroshi Iwata, Tan Pham, Anders Nykjaer, Mads Kjolby, Maximilian Rogers, Thomas Michel, Manabu Shibasaki, Sumihiko Hagita, Rafael Kramann, Daniel J. Rader, Peter Libby, Sasha A. Singh, Elena Aikawa
The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor
Shao-Qing Kuang, Olga Medina-Martinez, Dong-chuan Guo, Limin Gong, Ellen S. Regalado, Corey L. Reynolds, Catherine Boileau, Guillaume Jondeau, Siddharth K. Prakash, Callie S. Kwartler, Lawrence Yang Zhu, Andrew M. Peters, Xue-Yan Duan, National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions (GenTAC) Investigators, National Heart, Lung, and Blood Institute (NHLBI) Grand Opportunity (GO) Exome Sequencing Project (ESP), Michael J. Bamshad, Jay Shendure, Debbie A. Nickerson, Regie L. Santos-Cortez, Xiurong Dong, Suzanne M. Leal, Mark W. Majesky, Eric C. Swindell, Milan Jamrich, Dianna M. Milewicz