Oxidative stress and inadequate redox homeostasis is crucial for tumor initiation and progression. MTH1 (NUDT1) enzyme prevents incorporation of oxidized dNTPs by sanitizing the deoxynucleoside triphosphate (dNTP) pool and is therefore vital for the survival of tumor cells. MTH1 inhibition has been found to inhibit the growth of several experimental tumors, but its role in mesothelioma progression remained elusive. Moreover, although MTH1 is nonessential to normal cells, its role in survival of host cells in tumor milieu, especially tumor endothelium, is unclear. We validated a clinically relevant MTH1 inhibitor (Karonudib) in mesothelioma treatment using human xenografts and syngeneic murine models. We show that MTH1 inhibition impedes mesothelioma progression and that inherent tumoral MTH1 levels are associated with a tumor’s response. We also identified tumor endothelial cells as selective targets of Karonudib and propose a model of intercellular signaling among tumor cells and bystander tumor endothelium. We finally determined the major biological processes associated with elevated MTH1 gene expression in human mesotheliomas.
Sophia F. Magkouta, Apostolos G. Pappas, Photene C. Vaitsi, Panagiotis C. Agioutantis, Ioannis S. Pateras, Charalampos A. Moschos, Marianthi P. Iliopoulou, Chrysavgi N. Kosti, Heleni V. Loutrari, Vassilis G. Gorgoulis, Ioannis T. Kalomenidis
Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularisation capacity was hypothesised to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared to adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. MiR expression profiling revealed miR-96 and miR-183 upregulation in adult compared to neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularisation in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183 knock-out mice had increased peri-infarct neovascularisation. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN-positive vessels were enriched in the peri-infarct area of miR-96/miR-183 knock-out mice. These findings identify miR-96 and miR-183 as regulators of neovascularisation following MI and miR-regulated genes such as anillin as potential therapeutic targets for cardiovascular disease.
Raphael F.P. Castellan, Milena Vitiello, Martina Vidmar, Steven Johnstone, Dominga Iacobazzi, David Mellis, Benjamin Cathcart, Adrian JW Thomson, Christiana Ruhrberg, Massimo Caputo, David E. Newby, Gillian A. Gray, Andrew Howard Baker, Andrea Caporali, Marco Meloni
Abnormal subretinal neovascularization is characteristic of vision-threatening retinal diseases including macular telangiectasia (MacTel) and retinal angiomatous proliferation (RAP). Subretinal neovascular tufts and photoreceptor dysfunction are observed in very low-density lipoprotein receptor mutant mice (Vldlr–/–). These changes mirror those observed in MacTel and RAP patients, but the pathogenesis is largely unknown. In this study, we show that retinal microglia are closely associated with retinal neovascular tufts in Vldlr–/– mice and retinal tissue from MacTel patients; ablation of microglia/macrophages dramatically prevents formation of retinal neovascular tufts and improves neuronal function as assessed by electroretinography. VMD2-driven retinal pigmented epithelium (RPE)-specific knockouts of VEGF greatly reduced subretinal infiltration of microglia/macrophages, subsequently reducing NV tufts. These findings highlight the contribution of microglia/macrophages to the pathogenesis of NV, provide valuable clues regarding potential causative cellular mechanisms for subretinal neovascularization in MacTel and RAP patients, and suggest that targeting microglia activation may be a therapeutic option in these diseases.
Ayumi Usui-Ouchi, Yoshihiko Usui, Toshihide Kurihara, Edith Aguilar, Michael I. Dorrell, Yoichiro Ideguchi, Susumu Sakimoto, Stephen Bravo, Martin Friedlander
Increased subchondral bone angiogenesis with blood vessels breaching the tidemark into the avascular cartilage is a diagnostic feature of human osteoarthritis. However, the mechanisms that initiate subchondral bone angiogenesis remain unclear. We show that abnormally increased platelet-derived growth factor-BB (PDGF-BB) secretion by mononuclear preosteoclasts induces subchondral bone angiogenesis, contributing to osteoarthritis development. In mice after destabilization of the medial meniscus (DMM), aberrant joint subchondral bone angiogenesis developed during an early stage of osteoarthritis, before articular cartilage damage occurred. Mononuclear preosteoclasts in subchondral bone secrete excessive amounts of PDGF-BB, which activates platelet-derived growth factor receptor β (PDGFRβ) signaling in pericytes for neo-vessel formation. Selective knockout of PDGF-BB in preosteoclasts attenuates subchondral bone angiogenesis and abrogates joint degeneration and subchondral innervation induced by DMM. Transgenic mice that express PDGF-BB in preosteoclasts recapitulate pathological subchondral bone angiogenesis and develop joint degeneration and subchondral innervation spontaneously. Our study provides the first evidence that PDGF-BB derived from preosteoclasts is a key driver of pathological subchondral bone angiogenesis during osteoarthritis development and offers a new avenue for developing early treatments for this disease.
Weiping Su, Guanqiao Liu, Xiaonan Liu, Yangying Zhou, Qi Sun, Gehua Zhen, Xiao Wang, Yihe Hu, Peisong Gao, Shadpour Demehri, Xu Cao, Mei Wan
Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow–derived CD34+ cells and vascular wall–derived endothelial colony–forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.
Sergio Li Calzi, Lynn C. Shaw, Leni Moldovan, William C. Shelley, Xiaoping Qi, Lyne Racette, Judith L. Quigley, Seth D. Fortmann, Michael E. Boulton, Mervin C. Yoder, Maria B. Grant
Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is co-opted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50-150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-cell signaling receptor EPHB2 as a promising candidate cargo to promote angiogenesis. Analysis of TCGA RNA-Seq and patient tissue microarray data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulates angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a novel model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to non-adjacent endothelial cells to induce ephrin reverse signaling.
Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver
Von Hippel–Lindau (Vhl) protein inhibits hypoxia-inducible factor (Hif), yet its deletion in murine retina does not cause the extensive angiogenesis expected with Hif induction. The mechanism is unclear. Here we show that retinoblastoma tumor suppressor (Rb1) constrains expression of Hif target genes in the Vhl-/- retina. Deleting Rb1 induced extensive retinal neovascularization and autophagic ablation of photoreceptors in the Vhl-/- retina. RNA sequencing, ChIP and reporter assays showed Rb1 recruitment to and repression of certain Hif target genes. Activating Rb1 by deleting cyclin D1 induced a partial defect in the retinal superficial vascular plexus (SVP). Unexpectedly, removing Vhl suppressed retinoblastoma formation in murine Rb1/Rbl1-deficient retina, but generated subretinal vascular growths resembling retinal angiomatous proliferation (RAP), and retinal capillary hemangioblastoma (RCH). Most stromal cells in the RAP/RCH-like lesions were Sox9+, suggesting a Müller glia origin, and expressed Lgals3, a marker of human brain hemangioblastoma. Thus, the Rb family limit Hif target gene expression in the Vhl-/- retina, and removing this inhibitory signal generates new models for RAP and RCH.
Ran Wei, Xiang Ren, Hongyu Kong, Zhongping Lv, Yongjiang Chen, Yunjing Tang, Yujiao Wang, Lirong Xiao, Sabiha Hacibekiroglu, Chen Liang, Andras Nagy, Rod Bremner, Danian Chen
Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient but not in transient hemophilia WT mice after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency, and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI’s protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These new mechanistic insights allow targeted development of new strategies to normalize vascularity and control re-bleeding in HA.
Tine Wyseure, Tingyi Yang, Jenny Y. Zhou, Esther J. Cooke, Bettina Wanko, Merissa Olmer, Ruchi Agashe, Yosuke Morodomi, Niels Behrendt, Martin Lotz, John Morser, Annette von Drygalski, Laurent O. Mosnier
We determined which metabolic pathways are activated by hypoxia-inducible factor 1–mediated (HIF-1–mediated) protection against oxygen-induced retinopathy (OIR) in newborn mice, the experimental correlate to retinopathy of prematurity, a leading cause of infant blindness. HIF-1 coordinates the change from oxidative to glycolytic metabolism and mediates flux through serine and 1-carbon metabolism (1CM) in hypoxic and cancer cells. We used untargeted metabolite profiling in vivo to demonstrate that hypoxia mimesis activates serine/1CM. Both [13C6] glucose labeling of metabolites in ex vivo retinal explants as well as in vivo [13C3] serine labeling of metabolites followed in liver lysates strongly suggest that retinal serine is primarily derived from hepatic glycolytic carbon and not from retinal glycolytic carbon in newborn pups. In HIF-1α2lox/2lox albumin-Cre–knockout mice, reduced or near-0 levels of serine/glycine further demonstrate the hepatic origin of retinal serine. Furthermore, inhibition of 1CM by methotrexate blocked HIF-mediated protection against OIR. This demonstrated that 1CM participates in protection induced by HIF-1 stabilization. The urea cycle also dominated pathway enrichment analyses of plasma samples. The dependence of retinal serine on hepatic HIF-1 and the upregulation of the urea cycle emphasize the importance of the liver to remote protection of the retina.
Charandeep Singh, George Hoppe, Vincent Tran, Leah McCollum, Youstina Bolok, Weilin Song, Amit Sharma, Henri Brunengraber, Jonathan E. Sears
While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti-VEGF therapy include the upregulation of other pro-angiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signalling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor-induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularisation in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signalling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularisation, whilst enhancing reparative angiogenesis in the ischemic retina.
Sadaf Ashraf, Samuel Bell, Caitriona O'Leary, Paul Canning, Ileana Micu, Jose A. Fernandez, Michael O'Hare, Peter Barabas, Hannah McCauley, Derek P. Brazil, Alan W. Stitt, J. Graham McGeown, Tim M. Curtis
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