Angiogeneses
Abstract
Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long non-coding RNAs are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the LncRNA that Enhances Endothelial Nitric oxide synthase Expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression is decreased in diabetic conditions in cultured endothelial cells (EC), mouse hindlimb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduces their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in leene demonstrate impaired angiogenesis and perfusion following hindlimb ischemia. Importantly, overexpression of human LEENE rescues the impaired ischemic response in leene knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promotes transcription of pro-angiogenic genes in ECs, such as KDR and eNOS, potentially by interacting with LEO1, a key component of RNA Polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.
Authors
Xiaofang Tang, Yingjun Luo, Dongqiang Yuan, Riccardo Calandrelli, Naseeb Kaur Malhi, Kiran Sriram, Yifei Miao, Chih Hong Lou, Walter Tsark, Alonso Tapia, Aleysha T. Chen, Guangyu Zhang, Daniel Roeth, Markus Kalkum, Zhao V. Wang, Shu Chien, Rama Natarajan, John P. Cooke, Sheng Zhong, Zhen Bouman Chen
Abstract
Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5′-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcγ receptor–directed (FcγR-directed), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site–specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti–PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system–modulating strategy for cancer therapy.
Authors
Haohai Zhang, Lili Feng, Paola de Andrade Mello, Changchuin Mao, Richard Near, Eva Csizmadia, Leo Li-Ying Chan, Keiichi Enjyoji, Wenda Gao, Haitao Zhao, Simon C. Robson
Abstract
Pericytes (PC) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated if PC can be reprogrammed to aid neovascularization. Primary PC from human and mouse hearts acquired cytoskeleton proteins typical of vascular smooth muscle cells (VSMC) upon exclusion of EGF/bFGF, which signal through ERK1/2, or exposure to the MEK-inhibitor PD0325901. Differentiated PC became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA-Sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of two angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid-binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PC during in vivo studies. In mice, implantation of Matrigel plugs containing human PC+PD0325901 promoted the formation of α-SMApos neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRβposAQP1posCRABP2pos PC, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PC have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.
Authors
Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu
Abstract
The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 and histone acetylation were downregulated in NGBR deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of both HBO1 and acetylated H4K5/K12 on the promotor of CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.
Authors
Zhi Fang, Xiaoran Sun, Xiang Wang, Ji Ma, Thomas Palaia, Ujala Rana, Benjamin Miao, Louis Ragolia, Wenquan Hu, Qing Robert Miao
Abstract
Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans, and that its expression correlates negatively with long-term survival. Endothelial-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed “oxygen-glucose uncoupling”, which suppresses tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the anti-tumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms non-canonical inter-endothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrate the existence of tumor-specific inter-endothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.
Authors
Tomofumi Ando, Ikue Tai-Nagara, Yuki Sugiura, Dai Kusumoto, Koji Okabayashi, Yasuaki Kido, Kohji Sato, Hideyuki Saya, Sutip Navankasattusas, Dean Y. Li, Makoto Suematsu, Yuko Kitagawa, Elena Seiradake, Satoru Yamagishi, Yoshiaki Kubota
Non-β-blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma
Abstract
Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of β-blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSC) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial differentiation. As our previous work implicated the transcription factor SRY(Sex Determining Region Y)-Box Transcription Factor-18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with a similar efficacy as the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical and quantitative molecular imaging assays we show that R(+) propranolol directly interferes with SOX18 target gene trans-activation, disrupts SOX18-chromatin binding dynamics and reduced SOX18 dimer formation. We suggest the R(+) enantiomers of widely used β-blockers could be repurposed to increase efficiency of current IH treatment and lower adverse associated side effects.
Authors
Caroline T. Seebauer, Matthew S. Graus, Lan Huang, Alex J. McCann, Jill Wylie-Sears, Frank R. Fontaine, Tara Karnezis, David Zurakowski, Steven J. Staffa, Frédéric A. Meunier, John B. Mulliken, Joyce Bischoff, Mathias Francois
Abstract
Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.
Authors
Leeanna El-Houjeiri, Marco Biondini, Mathieu Paquette, Helen Kuasne, Alain Pacis, Morag Park, Peter M. Siegel, Arnim Pause
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
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Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)