Carbohydrate-binding protein CLEC14A regulates VEGFR-2– and VEGFR-3–dependent signals during angiogenesis and lymphangiogenesis

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Abstract

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.

Authors

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

Matricellular protein SPARCL1 regulates tumor microenvironment–dependent endothelial cell heterogeneity in colorectal carcinoma

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Abstract

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Authors

Elisabeth Naschberger, Andrea Liebl, Vera S. Schellerer, Manuela Schütz, Nathalie Britzen-Laurent, Patrick Kölbel, Ute Schaal, Lisa Haep, Daniela Regensburger, Thomas Wittmann, Ludger Klein-Hitpass, Tilman T. Rau, Barbara Dietel, Valérie S. Méniel, Alan R. Clarke, Susanne Merkel, Roland S. Croner, Werner Hohenberger, Michael Stürzl

Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation

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Abstract

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 Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.

Authors

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

Tie1 controls angiopoietin function in vascular remodeling and inflammation

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Abstract

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 Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

Authors

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

Lymphatic vessels regulate immune microenvironments in human and murine melanoma

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Abstract

Lymphatic remodeling in tumor microenvironments correlates with progression and metastasis, and local lymphatic vessels play complex and poorly understood roles in tumor immunity. Tumor lymphangiogenesis is associated with increased immune suppression, yet lymphatic vessels are required for fluid drainage and immune cell trafficking to lymph nodes, where adaptive immune responses are mounted. Here, we examined the contribution of lymphatic drainage to tumor inflammation and immunity using a mouse model that lacks dermal lymphatic vessels (K14-VEGFR3-Ig mice). Melanomas implanted in these mice grew robustly, but exhibited drastically reduced cytokine expression and leukocyte infiltration compared with those implanted in control animals. In the absence of local immune suppression, transferred cytotoxic T cells more effectively controlled tumors in K14-VEGFR3-Ig mice than in control mice. Furthermore, gene expression analysis of human melanoma samples revealed that patient immune parameters are markedly stratified by levels of lymphatic markers. This work suggests that the establishment of tumor-associated inflammation and immunity critically depends on lymphatic vessel remodeling and drainage. Moreover, these results have implications for immunotherapies, the efficacies of which are regulated by the tumor immune microenvironment.

Authors

Amanda W. Lund, Marek Wagner, Manuel Fankhauser, Eli S. Steinskog, Maria A. Broggi, Stefani Spranger, Thomas F. Gajewski, Kari Alitalo, Hans P. Eikesdal, Helge Wiig, Melody A. Swartz

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

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Abstract

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Authors

Khalil Miloudi, François Binet, Ariel Wilson, Agustin Cerani, Malika Oubaha, Catherine Menard, Sullivan Henriques, Gaelle Mawambo, Agnieszka Dejda, Phuong Trang Nguyen, Flavio A. Rezende, Steve Bourgault, Timothy E. Kennedy, Przemyslaw Sapieha

Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation

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Abstract

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 FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell–specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.

Authors

Anees Fatima, Ying Wang, Yutaka Uchida, Pieter Norden, Ting Liu, Austin Culver, William H. Dietz, Ford Culver, Meredith Millay, Yoh-suke Mukouyama, Tsutomu Kume

Motif mimetic of epsin perturbs tumor growth and metastasis

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Abstract

Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium–targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin–interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium–specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.

Authors

Yunzhou Dong, Hao Wu, H.N. Ashiqur Rahman, Yanjun Liu, Satish Pasula, Kandice L. Tessneer, Xiaofeng Cai, Xiaolei Liu, Baojun Chang, John McManus, Scott Hahn, Jiali Dong, Megan L. Brophy, Lili Yu, Kai Song, Robert Silasi-Mansat, Debra Saunders, Charity Njoku, Hoogeun Song, Padmaja Mehta-D’Souza, Rheal Towner, Florea Lupu, Rodger P. McEver, Lijun Xia, Derek Boerboom, R. Sathish Srinivasan, Hong Chen

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

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