Therapeutic lymphangiogenesis ameliorates established acute lung allograft rejection

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Abstract

Lung transplantation is the only viable option for patients suffering from otherwise incurable end-stage pulmonary diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Despite aggressive immunosuppression, acute rejection of the lung allograft occurs in over half of transplant recipients, and the factors that promote lung acceptance are poorly understood. The contribution of lymphatic vessels to transplant pathophysiology remains controversial, and data that directly address the exact roles of lymphatic vessels in lung allograft function and survival are limited. Here, we have shown that there is a marked decline in the density of lymphatic vessels, accompanied by accumulation of low-MW hyaluronan (HA) in mouse orthotopic allografts undergoing rejection. We found that stimulation of lymphangiogenesis with VEGF-C156S, a mutant form of VEGF-C with selective VEGFR-3 binding, alleviates an established rejection response and improves clearance of HA from the lung allograft. Longitudinal analysis of transbronchial biopsies from human lung transplant recipients demonstrated an association between resolution of acute lung rejection and decreased HA in the graft tissue. Taken together, these results indicate that lymphatic vessel formation after lung transplantation mediates HA drainage and suggest that treatments to stimulate lymphangiogenesis have promise for improving graft outcomes.

Authors

Ye Cui, Kaifeng Liu, Maria E. Monzon-Medina, Robert F. Padera, Hao Wang, Gautam George, Demet Toprak, Elie Abdelnour, Emmanuel D’Agostino, Hilary J. Goldberg, Mark A. Perrella, Rosanna Malbran Forteza, Ivan O. Rosas, Gary Visner, Souheil El-Chemaly

High salt reduces the activation of IL-4– and IL-13–stimulated macrophages

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Abstract

A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt–induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis.

Authors

Katrina J. Binger, Matthias Gebhardt, Matthias Heinig, Carola Rintisch, Agnes Schroeder, Wolfgang Neuhofer, Karl Hilgers, Arndt Manzel, Christian Schwartz, Markus Kleinewietfeld, Jakob Voelkl, Valentin Schatz, Ralf A. Linker, Florian Lang, David Voehringer, Mark D. Wright, Norbert Hubner, Ralf Dechend, Jonathan Jantsch, Jens Titze, Dominik N. Müller

Intravital imaging of intestinal lacteals unveils lipid drainage through contractility

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Abstract

Lacteals are lymphatic vessels located at the center of each intestinal villus and provide essential transport routes for lipids and other lipophilic molecules. However, it is unclear how absorbed molecules are transported through the lacteal. Here, we used reporter mice that express GFP under the control of the lymphatic-specific promoter Prox1 and a custom-built confocal microscope and performed intravital real-time visualization of the absorption and transport dynamics of fluorescence-tagged fatty acids (FAs) and various exogenous molecules in the intestinal villi in vivo. These analyses clearly revealed transepithelial absorption of these molecules via enterocytes, diffusive distribution over the lamina propria, and subsequent transport through lacteals. Moreover, we observed active contraction of lacteals, which seemed to be directly involved in dietary lipid drainage. Our analysis revealed that the smooth muscles that surround each lacteal are responsible for contractile dynamics and that lacteal contraction is ultimately controlled by the autonomic nervous system. These results indicate that the lacteal is a unique organ-specific lymphatic system and does not merely serve as a passive conduit but as an active pump that transports lipids. Collectively, using this efficient imaging method, we uncovered drainage of absorbed molecules in small intestinal villus lacteals and the involvement of lacteal contractibility.

Authors

Kibaek Choe, Jeon Yeob Jang, Intae Park, Yeseul Kim, Soyeon Ahn, Dae-Young Park, Young-Kwon Hong, Kari Alitalo, Gou Young Koh, Pilhan Kim

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature

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Abstract

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

Authors

Amélie Sabine, Esther Bovay, Cansaran Saygili Demir, Wataru Kimura, Muriel Jaquet, Yan Agalarov, Nadine Zangger, Joshua P. Scallan, Werner Graber, Elgin Gulpinar, Brenda R. Kwak, Taija Mäkinen, Inés Martinez-Corral, Sagrario Ortega, Mauro Delorenzi, Friedemann Kiefer, Michael J. Davis, Valentin Djonov, Naoyuki Miura, Tatiana V. Petrova

Lymph flow regulates collecting lymphatic vessel maturation in vivo

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Abstract

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin–like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.

Authors

Daniel T. Sweet, Juan M. Jiménez, Jeremy Chang, Paul R. Hess, Patricia Mericko-Ishizuka, Jianxin Fu, Lijun Xia, Peter F. Davies, Mark L. Kahn

GATA2 is required for lymphatic vessel valve development and maintenance

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Abstract

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema.

Authors

Jan Kazenwadel, Kelly L. Betterman, Chan-Eng Chong, Philippa H. Stokes, Young K. Lee, Genevieve A. Secker, Yan Agalarov, Cansaran Saygili Demir, David M. Lawrence, Drew L. Sutton, Sebastien P. Tabruyn, Naoyuki Miura, Marjo Salminen, Tatiana V. Petrova, Jacqueline M. Matthews, Christopher N. Hahn, Hamish S. Scott, Natasha L. Harvey

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III

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Abstract

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12^–/– mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

Authors

Jenny Björkqvist, Steven de Maat, Urs Lewandrowski, Antonio Di Gennaro, Chris Oschatz, Kai Schönig, Markus M. Nöthen, Christian Drouet, Hal Braley, Marc W. Nolte, Albert Sickmann, Con Panousis, Coen Maas, Thomas Renné

P2Y₂ and G_q/G₁₁ control blood pressure by mediating endothelial mechanotransduction

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Abstract

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress–induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y₂ and the G proteins G_q/G₁₁ mediate fluid shear stress–induced endothelial responses, including [Ca²⁺]_i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y₂ receptor. Mice with induced endothelium-specific P2Y₂ or G_q/G₁₁ deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y₂ and G_q/G₁₁ as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y₂ and G_q/G₁₁ are required for basal endothelial NO formation, vascular tone, and blood pressure.

Authors

ShengPeng Wang, András Iring, Boris Strilic, Julián Albarrán Juárez, Harmandeep Kaur, Kerstin Troidl, Sarah Tonack, Joachim C. Burbiel, Christa E. Müller, Ingrid Fleming, Jon O. Lundberg, Nina Wettschureck, Stefan Offermanns

Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis

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Abstract

The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E–deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39^+/–Apoe^–/– mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe^–/– mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress.

Authors

Yogendra Kanthi, Matthew C. Hyman, Hui Liao, Amy E. Baek, Scott H. Visovatti, Nadia R. Sutton, Sascha N. Goonewardena, Mithun K. Neral, Hanjoong Jo, David J. Pinsky

Neural crest–derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation

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Abstract

In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest–derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.

Authors

Alice Plein, Amélie Calmont, Alessandro Fantin, Laura Denti, Naomi A. Anderson, Peter J. Scambler, Christiana Ruhrberg