UVA induces retinal photoreceptor cell death via receptor interacting protein 3 kinase mediated necroptosis

Ultraviolet light A (UVA) is the only UV light that reaches the retina and can cause indirect damage to DNA via absorption of photons by non-DNA chromophores. Previous studies demonstrate that UVA generates reactive oxygen species (ROS) and leads to programmed cell death. Programmed cell death (PCD) has been implicated in numerous ophthalmologic diseases. Here, we investigated receptor interacting protein 1 and 3 (RIPK1 and RIPK3) kinases, key signaling molecules of PCD, in UVA-induced photoreceptor injury using in vitro and ex vivo models. UVA irradiation activated RIPK3 but not RIPK1 and mediated necroptosis through MLKL that lie downstream of RIPK3 and induced apoptosis through increased oxidative stress. Moreover, RIPK3 but not RIPK1 inhibition suppresses UVA-induced cell death along with the downregulation of MLKL and attenuates the levels of oxidative stress and DNA fragmentation. In conclusion, these results identify RIPK3, not RIPK1, as a critical regulator of UVA-induced necroptosis cell death in photoreceptors and highlight RIPK3 potential as a neuroprotective target.This work was supported by the Yeatts Family Foundation (D.G.V.); Monte J. Wallace (D.G.V.); 2013 Macula Society Research Grant Award (to D.G.V.); a Physician Scientist Award (to D.G.V.); unrestricted grant from the Research to Prevent Blindness Foundation (to J.W.M. and D.G.V.); National Eye Institute (NEI) R21EY023079-01/A1 (to D.G.V.); NEI Grant EY014104 (Massachusetts Eye and Ear Infirmary Core Grant) (to D.G.V.); Loeffler Family Fund (D.G.V.); R01EY025362-01 (to D.G.V.); ARI Young Investigator Award (to D.G.V.); Foundation Lions Eye Research Fund (D.G.V.); NIH NEI Core Grant P30EY003790 (to D.G.V.); Loeffler Family Fund (to D.G.V.); ARI Young Investigator Award (to D.G.V.). Shenzhen Science and Technology Program (Grant No. JCYJ20220530153607015); Shenzhen Science and Technology Program (Grant No. JCYJ20220531094004010); Shenzhen-Hong Kong Co-financing Project (Grant No. SGDX20190920110403741); Guangdong Basic and Applied Basic Research Foundation (No. 2022A1515012326); Shenzhen Science and Technology Program (Grant No. JSGG20201102174200001); Shenzhen Key Medical Discipline Construction Fund (No. SZXK038); Sanming Project of Medicine in Shenzhen (No. SZSM202011015); Shenzhen Fund for Guangdong Provincial High level Clinical Key Specialties (No. SZGSP014); National Natural Science Foundation of China (No. 82271103)

Endogenous CGRP protects against neointimal hyperplasia following wire-induced vascular injury

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第942号）・楊 磊Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after percutaneous coronary intervention. Calcitonin gene-related peptide (CGRP) is produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. Originally identified as a strongly vasodilatory neuropeptide, CGRP is now known to be a pleiotropic peptide widely distributed in various organs and tissues. Our aim was to investigate the possibility that CGRP acts as an endogenous vasoprotective molecule. We compared the effect of CGRP deficiency on neointimal formation after wire-induced vascular injury in wild-type and CGRP knockout (CGRP-/-) mice. We found that neointimal formation after vascular injury was markedly enhanced in CGRP-/- mice, which also showed a higher degree of oxidative stress, as indicated by reduced expression of nitric oxide synthase, increased expression of p47phox, and elevated levels of 4HNE, as well as greater infiltration of macrophages. In addition, CGRP-deficiency led to increased vascular smooth muscle cell (VSMC) proliferation within the neointima. By contrast, bone marrow-derived cells had little or no effect on neointimal formation in CGRP-/- mice. In vitro analysis showed that CGRP-treatment suppressed VSMC proliferation, migration, and ERK1/2 activity. These results clearly demonstrate that endogenous CGRP suppresses the oxidative stress and VSMC proliferation induced by vascular injury. As a vasoprotective molecule, CGRP could be an important therapeutic target in cardiovascular disease. (C) 2013 Elsevier Ltd. All rights reserved.ArticleJOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY. 59(0):55-66 (2013)journal articl

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM-RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor beta (TGF beta) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-gamma receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM-RAMP2 signaling for development of LSECs. (C) 2011 Elsevier Inc. All rights reserved.ArticlePEPTIDES. 32(9):1855-1865 (2011)journal articl

Vascular Endothelial Adrenomedullin-RAMP2 System Is Essential for Vascular Integrity and Organ Homeostasis

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第935号）・小山 晃英Background-Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. Methods and Results-We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Conclusions-Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage. (Circulation. 2013;127:842-853.)ArticleCIRCULATION. 127(7):842-853 (2013)journal articl

Endogenous CGRP protects against neointimal hyperplasia following wire-induced vascular injury

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第942号）・楊 磊Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after percutaneous coronary intervention. Calcitonin gene-related peptide (CGRP) is produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. Originally identified as a strongly vasodilatory neuropeptide, CGRP is now known to be a pleiotropic peptide widely distributed in various organs and tissues. Our aim was to investigate the possibility that CGRP acts as an endogenous vasoprotective molecule. We compared the effect of CGRP deficiency on neointimal formation after wire-induced vascular injury in wild-type and CGRP knockout (CGRP-/-) mice. We found that neointimal formation after vascular injury was markedly enhanced in CGRP-/- mice, which also showed a higher degree of oxidative stress, as indicated by reduced expression of nitric oxide synthase, increased expression of p47phox, and elevated levels of 4HNE, as well as greater infiltration of macrophages. In addition, CGRP-deficiency led to increased vascular smooth muscle cell (VSMC) proliferation within the neointima. By contrast, bone marrow-derived cells had little or no effect on neointimal formation in CGRP-/- mice. In vitro analysis showed that CGRP-treatment suppressed VSMC proliferation, migration, and ERK1/2 activity. These results clearly demonstrate that endogenous CGRP suppresses the oxidative stress and VSMC proliferation induced by vascular injury. As a vasoprotective molecule, CGRP could be an important therapeutic target in cardiovascular disease. (C) 2013 Elsevier Ltd. All rights reserved.ArticleJOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY. 59(0):55-66 (2013)journal articl

Complete regression of branching vascular network in polypoidal choroidal vasculopathy by ranibizumab and photodynamic therapy, two case reports

Abstract Background Polypoidal choroidal vasculopathy (PCV) consists of polyps that potentially cause massive subretinal hemorrhage and their branching vascular network (BVN) of feeder vessels. Although conventional indocyanine green angiography (IA) has shown anti-vascular endothelial growth factor (VEGF) agents and/or photodynamic therapy (PDT) to successfully induce polyp closure, the BVN appears resistant to these therapies and serves as the origin of recurrent active polyps. Recently introduced optical coherence tomography angiography (OCT-A) enables more frequent angiographic evaluation of polyps and the BVN than does conventional IA since it does not require intravenous fluorescent dye injection and is thus considered non-invasive. Case presentation Case 1. A 70-year-old male with PCV in his left eye suffered from vision deterioration (20/40) due to persistent subretinal fluid despite 42 intravitreal injections of ranibizumab (IVRs) over 5 years and 7 months. PDT was performed as an adjunct therapy 3 days after the 43rd IVR. IA at 3 months after PDT showed successful polyp closure but persisting BVN. However, more frequent evaluation with OCT-A starting at 1 week after PDT demonstrated complete regression of both the BVN and polyp. OCT-A at every subsequent outpatient visit depicted gradual re-perfusion of the BVN and the restoration of most of its original network at 3 months, which was compatible with IA findings. Neither OCTA nor IA revealed polyp recurrence at 3 months. Case 2. A 65-year-old female suffering from left vision deterioration due to PCV underwent 5 intravitreal injections of aflibercept. Since her subretinal fluid persisted, the treatment was switched to a combination of IVR and PDT. OCT-A revealed marked regression of the BVN and polyp at 2 weeks, but the BVN had regained its original shape at 2 months without any sign of polyp recurrence. Conclusions Differently from previous observations obtained by IA alone, more frequent non-invasive OCT-A examination revealed complete but transient regression of the BVN just after combination therapy with IVR and PDT

Marked biochemical difference in amyloid proportion between intra- and extraocular tissues in a liver-transplanted patient with hereditary ATTR amyloidosis

<p>In order to elucidate the pathomechanism of ocular amyloid formation in a liver-transplanted patient with hereditary ATTR amyloidosis, we investigated detailed biochemical features of ocular amyloid. The patient was a 49-year-old woman with V30M transthyretin (TTR) variant (p.TTRV50M), who underwent ophthalmectomy due to corneal rupture 10 years after liver transplantation (LT). The amyloid was selectively isolated from several portions in intra- and extraocular tissues using a laser microdissection (LMD) system and analyzed by liquid chromatography–tandem mass spectrometry to determine the composition percentage of wild-type and variant TTR in the isolated amyloid. Biochemical analysis revealed that the amyloid consisted mainly of variant TTR in intraocular tissues with a percentage > 80%. On the contrary in the extraocular muscles, wild-type TTR was the main component of the amyloid with a percentage of ∼70%. Our data indicate that intraocular amyloid formation strongly depends on locally synthesized variant TTR and the contribution of wild-type TTR to amyloid formation is quite limited.</p

Vascular Endothelial Adrenomedullin-RAMP2 System Is Essential for Vascular Integrity and Organ Homeostasis

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第935号）・小山 晃英Background-Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. Methods and Results-We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Conclusions-Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage. (Circulation. 2013;127:842-853.)ArticleCIRCULATION. 127(7):842-853 (2013)journal articl