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

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome

稀少遺伝性自己炎症性疾患: OTULIN関連自己炎症症候群の新たな病態を解明～既報の疾患に新たな視点を追加し、未診断患者の診断や炎症・細胞死研究の進展に期待～. 京都大学プレスリリース. 2024-04-25.OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis

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

Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation

CDC42-C末端異常症に於ける炎症病態を解明 --ゴルジ体への異常蓄積がパイリンインフラマソーム形成を過剰促進--. 京都大学プレスリリース. 2022-05-02.Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β–blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42[R186C], we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42[R186C] protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42[*192C*24] that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells

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

Receptor Activity Modifying Protein RAMP Sub-Isoforms and Their Functional Differentiation, Which Regulates Functional Diversity of Adrenomedullin

AM knockout (AM-/-) and RAMP2 knockout (RAMP2-/-) proved lethal for mice due to impaired embryonic vascular development. Although most vascular endothelial cell-specific RAMP2 knockout (E-RAMP2-/-) mice also died during the perinatal period, a few E-RAMP2-/- mice reached adulthood. Adult E-RAMP2-/- mice developed spontaneous organ damage associated with vascular injury. In contrast, adult RAMP3 knockout (RAMP3-/-) mice showed exacerbated postoperative lymphedema with abnormal lymphatic drainage. Thus, RAMP2 is essential for vascular development and homeostasis and RAMP3 is essential for lymphatic vessel function. Cardiac myocyte-specific RAMP2 knockout mice showed early onset of heart failure as well as abnormal mitochondrial morphology and function, whereas RAMP3-/- mice exhibited abnormal cardiac lymphatics and a delayed onset of heart failure. Thus, RAMP2 is essential for maintaining cardiac mitochondrial function, while RAMP3 is essential for cardiac lymphangiogenesis. Transplantation of cancer cells into drug-inducible vascular endothelial cell-specific RAMP2 knockout mice resulted in enhanced metastasis to distant organs, whereas metastasis was suppressed in RAMP3-/- mice. RAMP2 suppresses cancer metastasis by maintaining vascular homeostasis and inhibiting vascular inflammation and pre-metastatic niche formation, while RAMP3 promotes cancer metastasis via malignant transformation of cancer-associated fibroblasts. Focusing on the diverse physiological functions of AM and the functional differentiation of RAMP2 and RAMP3 may lead to the development of novel therapeutic strategies

Volcanic temperature changes modulated volatile release and climate fluctuations at the end-Triassic mass extinction

Emplacement of the Central Atlantic Magmatic Province (CAMP) is thought to have triggered global environmental changes and the end-Triassic mass extinction (ETE). However, the mechanisms linking volcanism and environmental change are unclear. Here we provide new insight into these linkages by measuring the abundance of both sedimentary five- to six-ring polycyclic aromatic hydrocarbons (PAHs) and mercury from strata deposited in shallow marine environments across the ETE at the GSSP Kuhjoch section in Austria and St. Audrie's Bay section in the UK. To contextualize these data, we report results from laboratory experiments measuring the production of SO2 and CO2 during heating of limestone and mudstone. ETE sediments record parallel enrichments of mercury and five- to six-ring PAHs, which could have been produced by intrusive magma (mainly sills) and lava flows during the early stage of the CAMP emplacement; these data indicate a direct link between massive gas emission from sill contact metamorphism and the ETE. The fraction of coronene – a highly condensed six-ring PAH that requires greater energy to form compared to smaller PAHs – accumulated in the sediments during these initial volcanic events is low, and it coincides with the terrestrial plant turnover and initial marine extinction. Coronene increases to medium values coinciding with the final marine extinction level. Our heating experiments of typical carbonate and mudstone materials show that relatively low temperature heating (>350 °C) by sills releases massive amounts of SO2 on a 100 yr time scale, whereas higher temperature heating (500–600 °C) forms more CO2 on the same time scale. The combination of our end-Triassic geochemical data and laboratory results implies that low heating by sills caused SO2-dominated gas emission to the stratosphere and low CO2 emission, inducing global cooling that could have precipitated the mass extinction. The subsequent increase in coronene content indicates higher volcanic temperature that would have volatilized CO2 rich gas; the consequence was a switch to greater CO2 release and long-term (>105 yr) global warming