Angiogenesis
Abstract
Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1β promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1β–induced angiogenesis and cell inflammation. IL-1β induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti–Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1β– or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1–deficient (MCP-1–/–) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1β–induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1β–induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1β (LLC/IL-1β) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1–/– mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1β. Thus, macrophage involvement might be a prerequisite for IL-1β–induced neovascularization and tumor progression.
Authors
Shintaro Nakao, Takashi Kuwano, Chikako Tsutsumi-Miyahara, Shu-ichi Ueda, Yusuke N. Kimura, Shinjiro Hamano, Koh-hei Sonoda, Yasuo Saijo, Toshihiro Nukiwa, Robert M. Strieter, Tatsuro Ishibashi, Michihiko Kuwano, Mayumi Ono
Abstract
The molecular and cellular pathways that support the maintenance and stability of tumor neovessels are not well defined. The efficacy of microtubule-disrupting agents, such as combretastatin A4 phosphate (CA4P), in inducing rapid regression of specific subsets of tumor neovessels has opened up new avenues of research to identify factors that support tumor neoangiogenesis. Herein, we show that CA4P selectively targeted endothelial cells, but not smooth muscle cells, and induced regression of unstable nascent tumor neovessels by rapidly disrupting the molecular engagement of the endothelial cell–specific junctional molecule vascular endothelial-cadherin (VE-cadherin) in vitro and in vivo in mice. CA4P increases endothelial cell permeability, while inhibiting endothelial cell migration and capillary tube formation predominantly through disruption of VE-cadherin/β-catenin/Akt signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis. Remarkably, stabilization of VE-cadherin signaling in endothelial cells with adenovirus E4 gene or ensheathment with smooth muscle cells confers resistance to CA4P. CA4P synergizes with low and nontoxic doses of neutralizing mAbs to VE-cadherin by blocking assembly of neovessels, thereby inhibiting tumor growth. These data suggest that the microtubule-targeting agent CA4P selectively induces regression of unstable tumor neovessels, in part through disruption of VE-cadherin signaling. Combined treatment with anti–VE-cadherin agents in conjunction with microtubule-disrupting agents provides a novel synergistic strategy to selectively disrupt assembly and induce regression of nascent tumor neovessels, with minimal toxicity and without affecting normal stabilized vasculature.
Authors
Loïc Vincent, Pouneh Kermani, Lauren M. Young, Joseph Cheng, Fan Zhang, Koji Shido, George Lam, Heidi Bompais-Vincent, Zhenping Zhu, Daniel J. Hicklin, Peter Bohlen, David J. Chaplin, Chad May, Shahin Rafii
Abstract
Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (Aβ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Aβ on cultured human RPE cells as well as ocular findings in neprilysin gene–disrupted mice, which leads to an increased deposition Aβ. The results showed that Aβ treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Aβ-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Aβ accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Aβ reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD.
Authors
Takeshi Yoshida, Kyoko Ohno-Matsui, Shizuko Ichinose, Tetsuji Sato, Nobuhisa Iwata, Takaomi C. Saido, Toshio Hisatomi, Manabu Mochizuki, Ikuo Morita
Abstract
Human noncollagenous domain 1 of the α1 chain of type IV collagen [α1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human α1(IV)NC1 binds to α1β1 integrin, competes with type IV collagen binding to α1β1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, α1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, α1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in α1 integrin receptor knockout ECs. Consistent with this, α1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in α1 integrin receptor knockout mice. This suggests a highly specific, α1β1 integrin–dependent antiangiogenic activity of α1(IV)NC1. In addition, α1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1α and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human α1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that α1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.
Authors
Akulapalli Sudhakar, Pia Nyberg, Venkateshwar G. Keshamouni, Arjuna P. Mannam, Jian Li, Hikaru Sugimoto, Dominic Cosgrove, Raghu Kalluri
Abstract
Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling–related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.
Authors
Michael Potente, Carmen Urbich, Ken-ichiro Sasaki, Wolf K. Hofmann, Christopher Heeschen, Alexandra Aicher, Ramya Kollipara, Ronald A. DePinho, Andreas M. Zeiher, Stefanie Dimmeler
Abstract
Although increased external load initially induces cardiac hypertrophy with preserved contractility, sustained overload eventually leads to heart failure through poorly understood mechanisms. Here we describe a conditional transgenic system in mice characterized by the sequential development of adaptive cardiac hypertrophy with preserved contractility in the acute phase and dilated cardiomyopathy in the chronic phase following the induction of an activated Akt1 gene in the heart. Coronary angiogenesis was enhanced during the acute phase of adaptive cardiac growth but reduced as hearts underwent pathological remodeling. Enhanced angiogenesis in the acute phase was associated with mammalian target of rapamycin–dependent induction of myocardial VEGF and angiopoietin-2 expression. Inhibition of angiogenesis by a decoy VEGF receptor in the acute phase led to decreased capillary density, contractile dysfunction, and impaired cardiac growth. Thus, both heart size and cardiac function are angiogenesis dependent, and disruption of coordinated tissue growth and angiogenesis in the heart contributes to the progression from adaptive cardiac hypertrophy to heart failure.
Authors
Ichiro Shiojima, Kaori Sato, Yasuhiro Izumiya, Stephan Schiekofer, Masahiro Ito, Ronglih Liao, Wilson S. Colucci, Kenneth Walsh
Abstract
NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, α–smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell–derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial–mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell–derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.
Authors
Satoshi Kashiwagi, Yotaro Izumi, Takeshi Gohongi, Zoe N. Demou, Lei Xu, Paul L. Huang, Donald G. Buerk, Lance L. Munn, Rakesh K. Jain, Dai Fukumura
Abstract
Neovascularization depends on vascular cell proliferation and on the stabilization of vessels by association of vascular smooth muscle–like pericytes with ECs. Here we show that integrin α4β1 (VLA-4) and VCAM-1 promote close intercellular adhesion between ECs and pericytes and that this interaction is required for blood vessel formation. Integrin α4β1 is expressed by proliferating but not quiescent ECs, while its ligand VCAM-1 is expressed by proliferating but not quiescent mural cells. Antagonists of this integrin-ligand pair block the adhesion of mural cells to proliferating endothelia in vitro and in vivo, thereby inducing apoptosis of ECs and pericytes and inhibiting neovascularization. These studies indicate that integrin α4β1 and VCAM-1 facilitate a critical cell-cell adhesion event required for survival of endothelial and mural cells during vascularization.
Authors
Barbara Garmy-Susini, Hui Jin, Yuhong Zhu, Rou-Jia Sung, Rosa Hwang, Judy Varner
Abstract
The neurotrophin brain-derived neurotrophic factor (BDNF) is required for the maintenance of cardiac vessel wall stability during embryonic development through direct angiogenic actions on endothelial cells expressing the tropomysin receptor kinase B (TrkB). However, the role of BDNF and a related neurotrophin ligand, neurotrophin-4 (NT-4), in the regulation of revascularization of the adult tissues is unknown. To study the potential angiogenic capacity of BDNF in mediating the neovascularization of ischemic and non-ischemic adult mouse tissues, we utilized a hindlimb ischemia and a subcutaneous Matrigel model. Recruitment of endothelial cells and promotion of channel formation within the Matrigel plug by BDNF and NT-4 was comparable to that induced by VEGF-A. The introduction of BDNF into non-ischemic ears or ischemic limbs induced neoangiogenesis, with a 2-fold increase in the capillary density. Remarkably, treatment with BDNF progressively increased blood flow in the ischemic limb over 21 days, similar to treatment with VEGF-A. The mechanism by which BDNF enhances capillary formation is mediated in part through local activation of the TrkB receptor and also by recruitment of Sca-1+CD11b+ pro-angiogenic hematopoietic cells. BDNF induces a potent direct chemokinetic action on subsets of marrow-derived Sca-1+ hematopoietic cells co-expressing TrkB. These studies suggest that local regional delivery of BDNF may provide a novel mechanism for inducing neoangiogenesis through both direct actions on local TrkB-expressing endothelial cells in skeletal muscle and recruitment of specific subsets of TrkB+ bone marrow–derived hematopoietic cells to provide peri-endothelial support for the newly formed vessels.
Authors
Pouneh Kermani, Dahlia Rafii, David K. Jin, Paul Whitlock, Wendy Schaffer, Anne Chiang, Loic Vincent, Matthias Friedrich, Koji Shido, Neil R. Hackett, Ronald G. Crystal, Shahin Rafii, Barbara L. Hempstead
Abstract
Edema occurs in asthma and other inflammatory diseases when the rate of plasma leakage from blood vessels exceeds the drainage through lymphatic vessels and other routes. It is unclear to what extent lymphatic vessels grow to compensate for increased leakage during inflammation and what drives the lymphangiogenesis that does occur. We addressed these issues in mouse models of (a) chronic respiratory tract infection with Mycoplasma pulmonis and (b) adenoviral transduction of airway epithelium with VEGF family growth factors. Blood vessel remodeling and lymphangiogenesis were both robust in infected airways. Inhibition of VEGFR-3 signaling completely prevented the growth of lymphatic vessels but not blood vessels. Lack of lymphatic growth exaggerated mucosal edema and reduced the hypertrophy of draining lymph nodes. Airway dendritic cells, macrophages, neutrophils, and epithelial cells expressed the VEGFR-3 ligands VEGF-C or VEGF-D. Adenoviral delivery of either VEGF-C or VEGF-D evoked lymphangiogenesis without angiogenesis, whereas adenoviral VEGF had the opposite effect. After antibiotic treatment of the infection, inflammation and remodeling of blood vessels quickly subsided, but lymphatic vessels persisted. Together, these findings suggest that when lymphangiogenesis is impaired, airway inflammation may lead to bronchial lymphedema and exaggerated airflow obstruction. Correction of defective lymphangiogenesis may benefit the treatment of asthma and other inflammatory airway diseases.
Authors
Peter Baluk, Tuomas Tammela, Erin Ator, Natalya Lyubynska, Marc G. Achen, Daniel J. Hicklin, Michael Jeltsch, Tatiana V. Petrova, Bronislaw Pytowski, Steven A. Stacker, Seppo Ylä-Herttuala, David G. Jackson, Kari Alitalo, Donald M. McDonald
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ISSN: 0021-9738 (print), 1558-8238 (online)