Angiogenesis
Abstract
Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2–specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.
Authors
Jing Zhang, Yaowu Qin, Mireya Martinez, Miguel Flores-Bellver, Murilo Rodrigues, Aumreetam Dinabandhu, Xuan Cao, Monika Deshpande, Yu Qin, Silvia Aparicio-Domingo, Yuan Rui, Stephany Y. Tzeng, Shaima Salman, Jin Yuan, Adrienne W. Scott, Jordan J. Green, M. Valeria Canto-Soler, Gregg L. Semenza, Silvia Montaner, Akrit Sodhi
Abstract
Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2 and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1a in astrocytes, resulting in increased VEGF production and expression of a “hypoxic” program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1a target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.
Authors
Miguel Alejandro Lopez-Ramirez, Catherine Chinhchu Lai, Shady Ibrahim Soliman, Preston Hale, Angela Pham, Esau J. Estrada, Sara McCurdy, Romuald Girard, Riya Verma, Thomas Moore, Rhonda Lightle, Nicholas Hobson, Robert Shenkar, Orit Poulsen, Gabriel G. Haddad, Richard Daneman, Brendan Gongol, Hao Sun, Frederic Lagarrigue, Issam A. Awad, Mark H. Ginsberg
Abstract
Familial exudative vitreoretinopathy (FEVR) is a severe retinal vascular disease that causes blindness. FEVR has been linked to mutations in several genes associated with inactivation of the Norrin/β-catenin signaling pathway, but these account for only approximately 50% of cases. We report that mutations in CTNNA1 (α-catenin) cause FEVR by overactivating the β-catenin pathway and disrupting cell adherens junctions. Three heterozygous mutations in CTNNA1 (p.F72S, p.R376Cfs*27 and p.P893L) were identified by exome-sequencing. We further demon-strated that FEVR-associated mutations led to overactivation of Norrin/β-catenin signaling due to impaired protein interactions within the cadherin/catenin complex. The clinical features of FEVR were reproduced in mice lacking Ctnna1 in vascular endothelial cells (ECs) or with overactivat-ed β-catenin signaling by an EC-specific gain-of-function allele of Ctnnb1. In isolated mouse lung endothelial cells, both CTNNA1-P893L and F72S mutants failed to rescue either the dis-rupted F-ACTIN arrangement or VE-Cadherin and CTNNB1 distribution. Moreover, we discov-ered that compound heterozygous Ctnna1 F72S and a deletion allele could cause similar pheno-type. Furthermore, a LRP5 mutation, which activates Norrin/β-catenin signaling, was identified in a FEVR family and the corresponding knock-in mice exhibited partial FEVR-like phenotype. Our study demonstrates that precise regulation of β-catenin activation is critical for retinal vascu-lar development and provides new insights into the pathogenesis of FEVR.
Authors
Xianjun Zhu, Mu Yang, Peiquan Zhao, Shujin Li, Lin Zhang, Lulin Huang, Yi Huang, Ping Fei, Yeming Yang, Shanshan Zhang, Huijuan Xu, Ye Yuan, Xiang Zhang, Xiong Zhu, Shi Ma, Fang Hao, Periasamy Sundaresan, Weiquan Zhu, Zhenglin Yang
Abstract
Tumors depend on a blood supply to deliver oxygen and nutrients, making tumor vasculature an attractive anti-cancer target. However, only a fraction of cancer patients benefits from angiogenesis inhibitors. Whether anti-angiogenic therapy would be more effective if targeted to individuals with specific tumor characteristics is unknown. To better characterize the tumor vascular environment both within and between cancer types, we developed a standardized metric – the Endothelial Index (EI) – to estimate vascular density in over 10,000 human tumors, corresponding to 31 solid tumor types, from transcriptome data. We then used this index to compare hyper- and hypo-vascular tumors, enabling the classification of human tumors into six vascular microenvironment signatures (VMSs) based on the expression of a panel of 24 vascular hub genes. EI and VMS correlated with known tumor vascular features and were independently associated with prognosis in certain cancer types. Retrospective testing of clinical trial data identified VMS2 classification as a powerful biomarker for response to bevacizumab. Our studies thus provide an unbiased picture of human tumor vasculature which may enable more precise deployment of anti-angiogenesis therapy.
Authors
Benjamin M. Kahn, Alfredo Lucas, Rohan Alur, Maximilian D. Wengyn, Gregory W. Schwartz, Jinyang Li, Kathryn Sun, H. Carlo Maurer, Kenneth P. Olive, Robert B. Faryabi, Ben Stanger
Abstract
Astrocytes play multiple functions in the brain, including blood vessel (BV) homeostasis and function. However, the underlying mechanisms remain elusive. Here, we provide evidence for astrocytic neogenin (NEO1), a member of deleted in colorectal cancer (DCC) family netrin receptors, to be involved in this event. Mice with Neo1 depletion in astrocytes exhibited clustered astrocyte distribution and increased BVs in their cortex. These BVs were leaky with reduced blood flow, disrupted basement membranes (vBMs), decreased pericytes, impaired endothelial cell (EC) barrier, and elevated tip EC proliferation. Increased proliferation was also detected in cultured ECs exposed to the conditional medium (CM) of NEO1 depleted astrocytes. Further screening for angiogenetic factors in the CM identifies netrin-1 (NTN1), whose expression was decreased in NEO1 depleted cortical astrocytes. Adding NTN1 into the CM of NEO1 depleted astrocytes attenuated EC proliferation. Expressing NTN1 in NEO1 mutant cortical astrocytes ameliorated phenotypes in blood–brain barrier (BBB), EC, and astrocyte distribution. NTN1 depletion in astrocytes resulted in similar BV/BBB deficits in the cortex as those of Neo1 mutant mice. In aggregates, these results uncovered an unrecognized pathway, astrocytic NEO1 to NTN1, not only regulating astrocyte distribution, but also promoting cortical BV homeostasis and function.
Authors
Ling-ling Yao, Jin-xia Hu, Qiang Li, Daehoon Lee, Xiao Ren, Jun-shi Zhang, Dong Sun, Hong-sheng Zhang, Yong-gang Wang, Lin Mei, Wen-Cheng Xiong
Abstract
Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary non-perfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularisation. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularisation. Vessel reassembly was not limited by VEGFA neutralisation, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralisation of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue re-vascularisation by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.
Authors
Zoe L. Grant, Lachlan Whitehead, Vickie H. Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
Abstract
Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler Syndrome by inactivating Flvcr2 in mice. We show that Flvcr2 is necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood brain barrier. Endothelial cells lacking Flvcr2 have altered expression of angiogenic factors, fail to adopt tip-cell properties and display reduced sprouting leading to vascular malformations similar to those seen in humans with Fowler Syndrome. Brain hypo-vascularization is associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier is maintained in Flvcr2 mutant mice. Our new Fowler syndrome models therefore define the pathobiology of this disease, and provide new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
Authors
Nicolas Santander, Carlos Omar Lizama, Eman Meky, Gabriel L. McKinsey, Bongnam Jung, Dean Sheppard, Christer Betsholtz, Thomas D. Arnold
Abstract
Current antiangiogenic therapy is limited by its cytostatic property, scarce drug delivery to the tumor, and side toxicity. To address these limitations, we unveiled the role of ZEB1, a tumor endothelium–enriched zinc-finger transcription factor, during tumor progression. We discovered that the patients who had lung adenocarcinomas with high ZEB1 expression in tumor endothelium had increased prevalence of metastases and markedly reduced overall survival after the diagnosis of lung cancer. Endothelial ZEB1 deletion in tumor-bearing mice diminished tumor angiogenesis while eliciting persistent tumor vascular normalization by epigenetically repressing TGF-β signaling. This consequently led to improved blood and oxygen perfusion, enhanced chemotherapy delivery and immune effector cell infiltration, and reduced tumor growth and metastasis. Moreover, targeting vascular ZEB1 remarkably potentiated the anticancer activity of nontoxic low-dose cisplatin. Treatment with low-dose anti–programmed cell death protein 1 (anti–PD-1) antibody elicited tumor regression and markedly extended survival in ZEB1-deleted mice, conferring long-term protective anticancer immunity. Collectively, we demonstrated that inactivation of endothelial ZEB1 may offer alternative opportunities for cancer therapy with minimal side effects. Targeting endothelium-derived ZEB1 in combination with conventional chemotherapy or immune checkpoint blockade therapy may yield a potent and superior anticancer effect.
Authors
Rong Fu, Yi Li, Nan Jiang, Bo-Xue Ren, Chen-Zi Zang, Li-Juan Liu, Wen-Cong Lv, Hong-Mei Li, Stephen Weiss, Zheng-Yu Li, Tao Lu, Zhao-Qiu Wu
Abstract
Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell–mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.
Authors
Simone Ragusa, Borja Prat-Luri, Alejandra González-Loyola, Sina Nassiri, Mario Leonardo Squadrito, Alan Guichard, Sabrina Cavin, Nikolce Gjorevski, David Barras, Giancarlo Marra, Matthias P. Lutolf, Jean Perentes, Emily Corse, Roberta Bianchi, Laureline Wetterwald, Jaeryung Kim, Guillermo Oliver, Mauro Delorenzi, Michele De Palma, Tatiana V. Petrova
Abstract
microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor- associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophage-mediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications.
Authors
Mahnaz Sahraei, Balkrishna Chaube, Yuting Liu, Jonathan Sun, Alanna Kaplan, Nathan L. Price, Wen Ding, Stanley Oyaghire, Rolando García-Milian, Sameet Mehta, Yana K. Reshetnyak, Raman Bahal, Paolo Fiorina, Peter M. Glazer, David L. Rimm, Carlos Fernández-Hernando, Yajaira Suárez
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Copyright © 2021 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)