Atherosclerosis is a chronic inflammatory disease, and developing therapies to promote its regression is an important clinical goal. We previously established that atherosclerosis regression is characterized by an overall decrease in plaque macrophages and enrichment in markers of alternatively activated M2 macrophages. We have now investigated the origin and functional requirement for M2 macrophages in regression in normolipidemic mice that received transplants of atherosclerotic aortic segments. We compared plaque regression in WT normolipidemic recipients and those deficient in chemokine receptors necessary to recruit inflammatory Ly6Chi (Ccr2–/– or Cx3cr1–/–) or patrolling Ly6Clo (Ccr5–/–) monocytes. Atherosclerotic plaques transplanted into WT or Ccr5–/– recipients showed reduced macrophage content and increased M2 markers consistent with plaque regression, whereas plaques transplanted into Ccr2–/– or Cx3cr1–/– recipients lacked this regression signature. The requirement of recipient Ly6Chi monocyte recruitment was confirmed in cell trafficking studies. Fate-mapping and single-cell RNA sequencing studies also showed that M2-like macrophages were derived from newly recruited monocytes. Furthermore, we used recipient mice deficient in STAT6 to demonstrate a requirement for this critical component of M2 polarization in atherosclerosis regression. Collectively, these results suggest that continued recruitment of Ly6Chi inflammatory monocytes and their STAT6-dependent polarization to the M2 state are required for resolution of atherosclerotic inflammation and plaque regression.
Karishma Rahman, Yuliya Vengrenyuk, Stephen A. Ramsey, Noemi Rotllan Vila, Natasha M. Girgis, Jianhua Liu, Viktoria Gusarova, Jesper Gromada, Ada Weinstock, Kathryn J. Moore, P’ng Loke, Edward A. Fisher
The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.
Lingjuan He, Xiuzhen Huang, Onur Kanisicak, Yi Li, Yue Wang, Yan Li, Wenjuan Pu, Qiaozhen Liu, Hui Zhang, Xueying Tian, Huan Zhao, Xiuxiu Liu, Shaohua Zhang, Yu Nie, Shengshou Hu, Xiang Miao, Qing-Dong Wang, Fengchao Wang, Ting Chen, Qingbo Xu, Kathy O. Lui, Jeffery D. Molkentin, Bin Zhou
Capillary malformation–arteriovenous malformation (CM-AVM) is a blood and lymphatic vessel (LV) disorder that is caused by inherited inactivating mutations of the
Philip E. Lapinski, Beth A. Lubeck, Di Chen, Abbas Doosti, Scott D. Zawieja, Michael J. Davis, Philip D. King
Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.
Michael J. Kraakman, Man K.S. Lee, Annas Al-Sharea, Dragana Dragoljevic, Tessa J. Barrett, Emilie Montenont, Debapriya Basu, Sarah Heywood, Helene L. Kammoun, Michelle Flynn, Alexandra Whillas, Nordin M.J. Hanssen, Mark A. Febbraio, Erik Westein, Edward A. Fisher, Jaye Chin-Dusting, Mark E. Cooper, Jeffrey S. Berger, Ira J. Goldberg, Prabhakara R. Nagareddy, Andrew J. Murphy
Sarah R. Langley, Karin Willeit, Athanasios Didangelos, Ljubica Perisic Matic, Philipp Skroblin, Javier Barallobre-Barreiro, Mariette Lengquist, Gregor Rungger, Alexander Kapustin, Ludmilla Kedenko, Chris Molenaar, Ruifang Lu, Temo Barwari, Gonca Suna, Xiaoke Yin, Bernhard Iglseder, Bernhard Paulweber, Peter Willeit, Joseph Shalhoub, Gerard Pasterkamp, Alun H. Davies, Claudia Monaco, Ulf Hedin, Catherine M. Shanahan, Johann Willeit, Stefan Kiechl, Manuel Mayr
The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow–induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of
Dongwon Choi, Eunkyung Park, Eunson Jung, Young Jin Seong, Jaehyuk Yoo, Esak Lee, Mingu Hong, Sunju Lee, Hiroaki Ishida, James Burford, Janos Peti-Peterdi, Ralf H. Adams, Sonal Srikanth, Yousang Gwack, Christopher S. Chen, Hans J. Vogel, Chester J. Koh, Alex K. Wong, Young-Kwon Hong
Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of
Cyrill Géraud, Philipp-Sebastian Koch, Johanna Zierow, Kay Klapproth, Katrin Busch, Victor Olsavszky, Thomas Leibing, Alexandra Demory, Friederike Ulbrich, Miriam Diett, Sandhya Singh, Carsten Sticht, Katja Breitkopf-Heinlein, Karsten Richter, Sanna-Maria Karppinen, Taina Pihlajaniemi, Bernd Arnold, Hans-Reimer Rodewald, Hellmut G. Augustin, Kai Schledzewski, Sergij Goerdt
We recently demonstrated that selective expression of the Rho GTPase-activating protein ARHGAP42 in smooth muscle cells (SMCs) controls blood pressure by inhibiting RhoA-dependent contractility, providing a mechanism for the blood pressure–associated locus within the
Xue Bai, Kevin D. Mangum, Rachel A. Dee, George A. Stouffer, Craig R. Lee, Akinyemi Oni-Orisan, Cam Patterson, Jonathan C. Schisler, Anthony J. Viera, Joan M. Taylor, Christopher P. Mack
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