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
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.
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
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.
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
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
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.
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
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.
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
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.
Daniel T. Sweet, Juan M. Jiménez, Jeremy Chang, Paul R. Hess, Patricia Mericko-Ishizuka, Jianxin Fu, Lijun Xia, Peter F. Davies, Mark L. Kahn
RASA1 (also known as p120 RasGAP) is a Ras GTPase–activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation–arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.
Philip E. Lapinski, Sunkuk Kwon, Beth A. Lubeck, John E. Wilkinson, R. Sathish Srinivasan, Eva Sevick-Muraca, Philip D. King
Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5β1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.
Boyeon Lee, Douglas Clarke, Abraham Al Ahmad, Michael Kahle, Christi Parham, Lisa Auckland, Courtney Shaw, Mehmet Fidanboylu, Anthony Wayne Orr, Omolara Ogunshola, Andrzej Fertala, Sarah A. Thomas, Gregory J. Bix
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