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
Retinopathy of prematurity (ROP) is characterized by abnormal retinal neovascularization in response to vessel loss. Platelets regulate angiogenesis and may influence ROP progression. In preterm infants, we assessed ROP and correlated with longitudinal postnatal platelet counts (n = 202). Any episode of thrombocytopenia (<100 × 109/l) at ≥30 weeks postmenstrual age (at onset of ROP) was independently associated with severe ROP, requiring treatment. Infants with severe ROP also had a lower weekly median platelet count compared with infants with less severe ROP. In a mouse oxygen-induced retinopathy model of ROP, platelet counts were lower at P17 (peak neovascularization) versus controls. Platelet transfusions at P15 and P16 suppressed neovascularization, and platelet depletion increased neovascularization. Platelet transfusion decreased retinal of vascular endothelial growth factor A (VEGFA) mRNA and protein expression; platelet depletion increased retinal VEGFA mRNA and protein expression. Resting platelets with intact granules reduced neovascularization, while thrombin-activated degranulated platelets did not. These data suggest that platelet releasate has a local antiangiogenic effect on endothelial cells to exert a downstream suppression of VEGFA in neural retina. Low platelet counts during the neovascularization phase in ROP is significantly associated with the development of severe ROP in preterm infants. In a murine model of retinopathy, platelet transfusion during the period of neovascularization suppressed retinopathy.
Bertan Cakir, Raffael Liegl, Gunnel Hellgren, Pia Lundgren, Ye Sun, Susanna Klevebro, Chatarina Löfqvist, Clara Mannheimer, Steve Cho, Alexander Poblete, Rubi Duran, Boubou Hallberg, Jorge Canas, Viola Lorenz, Zhi-Jian Liu, Martha C. Sola-Visner, Lois E.H. Smith, Ann Hellström
Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is associated with aging and is a leading cause of blindness worldwide. Here, we demonstrate that leukotriene B4 (LTB4) receptor 1 (BLT1) promotes laser-induced choroidal neovascularization (CNV) in a mouse model for wet-type AMD. CNV was significantly less in BLT1-deficient (BLT1-KO) mice compared with BLT1-WT controls. Expression of several proangiogenic and profibrotic factors was lower in BLT1-KO eyes than in BLT1-WT eyes. LTB4 production in the eyes was substantially increased in the early phase after laser injury. BLT1 was highly expressed in M2 macrophages in vitro and in vivo, and ocular BLT1+ M2 macrophages were increased in the aged eyes after laser injury. Furthermore, M2 macrophages were rapidly attracted by LTB4 and subsequently produced VEGF-A– through BLT1-mediated signaling. Consequently, intravitreal injection of M2 macrophages augmented CNV formation, which was attenuated by BLT1 deficiency. Thus, laser-induced injury to the retina triggered LTB4 production and attracted M2 macrophages via BLT1, leading to development of CNV. A selective BLT1 antagonist (CP105696) and 3 LTB4 inhibitors (zileuton, MK-886, and bestatin) reduced CNV in a dose-dependent manner. CP105696 also inhibited the accumulation of BLT1+ M2 macrophages in the laser-injured eyes of aged mice. Together, these results indicate that the LTB4-BLT1 axis is a potentially novel therapeutic target for CNV of wet-type AMD.
Fumiyuki Sasaki, Tomoaki Koga, Mai Ohba, Kazuko Saeki, Toshiaki Okuno, Keijiro Ishikawa, Takahito Nakama, Shintaro Nakao, Shigeo Yoshida, Tatsuro Ishibashi, Hamid Ahmadieh, Mozhgan Rezaei Kanavi, Ali Hafezi-Moghadam, Josef M. Penninger, Koh-Hei Sonoda, Takehiko Yokomizo
Intraocular injections of VEGF-neutralizing proteins provide tremendous benefits in patients with choroidal neovascularization (NV) due to age-related macular degeneration (AMD), but during treatment some patients develop retinal atrophy. Suggesting that VEGF is a survival factor for retinal neurons, a clinical trial group attributed retinal atrophy to VEGF suppression and cautioned against frequent anti-VEGF injections. This recommendation may contribute to poor outcomes in clinical practice from insufficient treatment. Patients with type 3 choroidal NV have particularly high risk of retinal atrophy, an unexplained observation. Herein we show in mouse models that VEGF signaling does not contribute to photoreceptor survival and functioning: (a) neutralization of VEGFR2 strongly suppresses choroidal NV without compromising photoreceptor function or survival; (b) VEGF does not slow loss of photoreceptor function or death in mice with inherited retinal degeneration, and there is no exacerbation by VEGF suppression; and (c) mice with type 3 choroidal NV develop retinal atrophy due to oxidative damage with no contribution from VEGF suppression. Intraocular injections of VEGF-neutralizing proteins, a highly effective treatment in patients with neovascular AMD, should not be withheld or reduced due to concern that they may contribute to long-term visual loss from retinal atrophy.
Da Long, Yogita Kanan, Jikui Shen, Sean F. Hackett, Yuanyuan Liu, Zibran Hafiz, Mahmood Khan, Lili Lu, Peter A. Campochiaro
Angiogenesis, new blood vessel formation from preexisting vessels, is critical for solid tumor growth. As such, there have been efforts to inhibit angiogenesis as a means to obstruct tumor growth. However, antiangiogenic therapy faces major challenges to the selective targeting of tumor-associated-vessels, as current antiangiogenic targets also disrupt steady-state vessels. Here, we demonstrate that the developmentally critical transcription factor Etv2 is selectively upregulated in both human and mouse tumor-associated endothelial cells (TAECs) and is required for tumor angiogenesis. Two-photon imaging revealed that Etv2-deficient tumor-associated vasculature remained similar to that of steady-state vessels. Etv2-deficient TAECs displayed decreased Flk1 (also known as Vegfr2) expression, FLK1 activation, and proliferation. Endothelial tube formation, proliferation, and sprouting response to VEGF, but not to FGF2, was reduced in Etv2-deficient ECs. ROS activated Etv2 expression in ECs, and ROS blockade inhibited Etv2 expression in TAECs in vivo. Systemic administration of Etv2 siRNA nanoparticles potently inhibited tumor growth and angiogenesis without cardiovascular side effects. These studies highlight a link among vascular oxidative stress, Etv2 expression, and VEGF response that is critical for tumor angiogenesis. Targeting the ETV2 pathway might offer a unique opportunity for more selective antiangiogenic therapies.
Ashraf Ul Kabir, Tae-Jin Lee, Hua Pan, Jeffrey C. Berry, Karen Krchma, Jun Wu, Fang Liu, Hee-Kyoung Kang, Kristina Hinman, Lihua Yang, Samantha Hamilton, Qingyu Zhou, Deborah J. Veis, Robert P. Mecham, Samuel A. Wickline, Mark J. Miller, Kyunghee Choi
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