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
Myocardial infarctions (MIs) cause the loss of myocytes due to lack of sufficient oxygenation and latent revascularization. Although the administration of histone deacetylase (HDAC) inhibitors reduces the size of infarctions and improves cardiac physiology in small-animal models of MI injury, the cellular targets of the HDACs, which the drugs inhibit, are largely unspecified. Here, we show that WNT-inducible secreted protein-1 (Wisp-1), a matricellular protein that promotes angiogenesis in cancers as well as cell survival in isolated cardiac myocytes and neurons, is a target of HDACs. Further, Wisp-1 transcription is regulated by HDACs and can be modified by the HDAC inhibitor, suberanilohydroxamic acid (SAHA/vorinostat), after MI injury. We observe that, at 7 days after MI, Wisp-1 is elevated 3-fold greater in the border zone of infarction in mice that experience an MI injury and are injected daily with SAHA, relative to MI alone. Additionally, human coronary artery endothelial cells (HCAECs) produce WISP-1 and are responsive to autocrine WISP-1–mediated signaling, which functionally promotes their proangiogenic behavior. Altering endogenous expression of WISP-1 in HCAECs directly impacts their network density in vitro. Therapeutic interventions after a heart attack define the extent of infarct injury, cell survival, and overall prognosis. Our studies shown here identify a potentially novel cardiac angiokine, Wisp-1, that may contribute to beneficial post-MI treatment modalities.
Lillianne H. Wright, Daniel J. Herr, Symone S. Brown, Harinath Kasiganesan, Donald R. Menick
Intestinal epithelial cells condition tolerogenic properties in DCs. Aqueous-deficient dry eye is associated with goblet cell (GC) loss and increased IFN-γ expression in the conjunctiva. We hypothesized that loss of GCs reduces tolerance-inducing properties of antigen presenting cells (APCs) in the conjunctiva and draining nodes. Mice lacking the SAM pointed domain containing ETS transcription factor (Spdef) that is required for GC differentiation had an increased frequency of macrophages in the conjunctiva and CD11b+CD11c+ DCs in the conjunctiva and draining nodes, and these cells had greater IL-12 expression than WT mice. Conditioned media from cultured WT conjunctival GCs suppressed LPS-induced IL-12 production by conjunctival APCs. OVA antigen–specific OTII CD4+ T cells primed by Spdef-KO draining lymph node APCs showed greater proliferation, lower frequency of Foxp3+, increased frequency of IFN-γ+ and IL-17+ cells, and greater IFN-γ production than those primed by WT APCs. The immune tolerance to OVA antigen topically applied to the conjunctiva measured by cutaneous delayed type hypersensitivity (DTH) reaction, OVA-specific T cell proliferation, Foxp3 induction, and IFN-γ production observed in WT mice was lost in the Spdef-KO mice. We concluded that conjunctival GCs condition tolerogenic properties in APCs that suppress IL-12 production and Th1 polarization.
Byung Yi Ko, Yangyan Xiao, Flavia L. Barbosa, Cintia S. de Paiva, Stephen C. Pflugfelder
Oxygen-induced retinopathy (OIR) is a widely used model to study ischemia-driven neovascularization (NV) in the retina and to serve in proof-of-concept studies in evaluating antiangiogenic drugs for ocular, as well as nonocular, diseases. The primary parameters that are analyzed in this mouse model include the percentage of retina with vaso-obliteration (VO) and NV areas. However, quantification of these two key variables comes with a great challenge due to the requirement of human experts to read the images. Human readers are costly, time-consuming, and subject to bias. Using recent advances in machine learning and computer vision, we trained deep learning neural networks using over a thousand segmentations to fully automate segmentation in OIR images. While determining the percentage area of VO, our algorithm achieved a similar range of correlation coefficients to that of expert inter-human correlation coefficients. In addition, our algorithm achieved a higher range of correlation coefficients compared with inter-expert correlation coefficients for quantification of the percentage area of neovascular tufts. In summary, we have created an open-source, fully automated pipeline for the quantification of key values of OIR images using deep learning neural networks.
Sa Xiao, Felicitas Bucher, Yue Wu, Ariel Rokem, Cecilia S. Lee, Kyle V. Marra, Regis Fallon, Sophia Diaz-Aguilar, Edith Aguilar, Martin Friedlander, Aaron Y. Lee
Occurrence of transient ischemic attacks (TIA) and cerebral strokes is a recognized risk associated with cocaine abuse. Here, we use a rodent model along with optical imaging to study cocaine-induced TIA and the associated dynamic changes in cerebral blood flow velocity (CBFv) and cerebrovasculature. We show that chronic cocaine exposure in mice resulted in marked cortical hypoperfusion, in significant arterial and venous vasoconstriction, and in a sensitized vascular response to an acute cocaine injection. Starting after 10 days of exposure, an acute cocaine challenge to these mice resulted in a TIA, which presented as hemiparalysis and was associated with an abrupt exacerbation of CBFv. The severity of the TIA correlated with the decreases in cortical CBFv such that the greater the decreases in flow, the longer the TIA duration. The severity of TIA peaked around 17–22 days of cocaine exposure and decreased thereafter in parallel to a reorganization of CBFv from superficial to deep cortical layers, along with an increase in vessel density into these layers. Here, we document for the first time to our knowledge evidence of a TIA in an animal model of chronic cocaine exposure that was associated with profound decreases in CBFv, and we revealed that while the severity of the TIA initially increased with repeated exposures, it subsequently improved in parallel to an increase in the vessel density. This suggests that strategies to accelerate cerebrovascular recovery might be therapeutically beneficial in cocaine abusers.
Jiang You, Nora D. Volkow, Kicheon Park, Quijia Zhang, Kevin Clare, Congwu Du, Yingtian Pan
Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor–binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.
Susumu Sakimoto, Valentina Marchetti, Edith Aguilar, Kelsey Lee, Yoshihiko Usui, Salome Murinello, Felicitas Bucher, Jennifer K. Trombley, Regis Fallon, Ravenska Wagey, Carrie Peters, Elizabeth L. Scheppke, Peter D. Westenskow, Martin Friedlander
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