[1]肇莉莉,王萍,孙连义,等.HTRA3基因对脉络膜新生血管和M2型巨噬细胞极化的影响[J].眼科新进展,2024,44(4):275-281.[doi:10.13389/j.cnki.rao.2024.0054]
　ZHAO Lili,WANG Ping,SUN Lianyi,et al.Effect of HtrA serine peptidase 3 gene on choroidal neovascularization and M2 macrophage polarization[J].Recent Advances in Ophthalmology,2024,44(4):275-281.[doi:10.13389/j.cnki.rao.2024.0054]
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HTRA3基因对脉络膜新生血管和M2型巨噬细胞极化的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
44卷
期数:
2024年4期
页码:
275-281
栏目:
实验研究
出版日期:
2024-04-05

文章信息/Info

Title:
Effect of HtrA serine peptidase 3 gene on choroidal neovascularization and M2 macrophage polarization
作者:
肇莉莉; 王萍; 孙连义; 马为梅; 张乐; 喻磊
710004　陕西省西安市，西安市人民医院（西安市第四医院），陕西省眼科医院，西北大学附属人民医院眼科（肇莉莉，王萍，孙连义，马为梅，喻磊）；710003　陕西省西安市，西北妇女儿童医院眼科（张乐）
Author(s):
ZHAO Lili1; WANG Ping1; SUN Lianyi1; MA Weimei1; ZHANG Le2; YU Lei1
1.Department of Ophthalmology,Xi’an People’s Hospital (Xi’an Fourth Hospital),Shaanxi Eye Hospital,the People’s Hospital Affiliated of Northwest University,Xi’an 710004,Shaanxi Province,China
2.Department of Ophthalmology,Northwest Women’s and Children’s Hospital,Xi’an 710003,Shaanxi Province,China
关键词:
湿性年龄相关性黄斑变性; 脉络膜新生血管; HtrA丝氨酸肽酶3; M2型巨噬细胞极化
Keywords:
wet age-related macular degeneration;  choroidal neovascularization;  HtrA serine peptidase 3;  M2 macrophage polarization
分类号:
R774.5
DOI:
10.13389/j.cnki.rao.2024.0054
文献标志码:
A
摘要:
目的　探讨HtrA丝氨酸肽酶3（HTRA3）基因对脉络膜新生血管（CNV）和M2型巨噬细胞极化的影响。
方法　收集30例湿性年龄相关性黄斑变性（wAMD）患者（wAMD组）和30例同期健康体检者（健康组）的空腹静脉血，通过qRT-PCR检测血清HTRA3 mRNA水平。将RF/6A细胞随机分为对照组、NC-sh组和HTRA3-sh组，使用Lipofectamine2000将NC-shRNA和HTRA3-shRNA慢病毒载体分别转染到NC-sh组和HTRA3-sh组RF/6A细胞中，通过qRT-PCR和Western blot检测HTRA3的转染情况。将RF/6A细胞随机分为N组、H组、H+NC-sh组和H+HTRA3-sh组，细胞转染后，N组RF/6A细胞在完全RPMI 1640培养基中进行常氧培养，其他组细胞在添加200 mmol·L-1氯化钴（CoCl2）的RPMI 1640培养基中进行低氧培养，使用Matrigel测定小管形成。将C57BL/6J小鼠随机分为对照组、CNV组、CNV+NC-sh组和CNV+HTRA3-sh组，每组12只。对照组为未建模的小鼠，其他组为激光诱导的CNV模型小鼠。向CNV+NC-sh组和CNV+HTRA3-sh组小鼠玻璃体内分别注射1 μL滴度为1×1011 TU·mL-1的NC-shRNA和HTRA3-shRNA慢病毒载体。对照组和CNV组小鼠注射PBS。注射后7 d，对小鼠进行荧光素眼底血管造影（FFA）检测和眼球苏木精伊红（HE）染色。通过qRT-PCR检测RF/6A细胞或各组小鼠脉络膜组织中HTRA3、类几丁质酶3样蛋白3（Ym-1）、精氨酸酶 1（Arg-1）、诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）和血管内皮生长因子（VEGF）mRNA水平。通过Western blot检测RF/6A细胞或脉络膜组织中HTRA3、VEGF和细胞核核因子κB（NF-κB） p65的蛋白表达水平。
结果　与健康组比较，wAMD组患者的血清HTRA3 mRNA水平升高（t=11.804，P<0.001）。与对照组和NC-sh组比较，HTRA3-sh组RF/6A细胞的HTRA3 mRNA和蛋白表达水平降低（P<0.05）。与N组比较，H组RF/6A细胞的闭合管腔数量、HTRA3和VEGF的mRNA和蛋白表达水平均增加（均为P<0.05）。与H+NC-sh组比较，H+HTRA3-sh组RF/6A细胞的闭合管腔数量、HTRA3和VEGF的mRNA和蛋白表达水平均减少（均为P<0.05）。与对照组比较，CNV组小鼠的HTRA3的mRNA和蛋白表达水平增加，CNV相对荧光强度升高，Ym-1和Arg-1 mRNA水平升高，iNOS和COX-2 mRNA水平降低，细胞核NF-κB p65蛋白表达水平升高（均为P<0.05）。与CNV+NC-sh组比较，CNV+HTRA3-sh组小鼠的HTRA3的mRNA和蛋白表达水平减少，CNV相对荧光强度降低，Ym-1和Arg-1 mRNA水平降低，iNOS和COX-2 mRNA水平升高，细胞核NF-κB p65蛋白表达水平降低（均为P<0.05）。
结论　下调HTRA3可抑制CNV形成和M2型巨噬细胞极化，HTRA3可能是防治wAMD的重要潜在靶点。
Abstract:
Objective　To investigate the effect of the HtrA serine peptidase 3 (HTRA3) gene on choroidal neovascularization (CNV) and M2 macrophage polarization.
Methods　Fasting venous blood was collected from 30 patients with wet age-related macular degeneration (wAMD group) and 30 healthy subjects (normal group). The serum HTRA3 messenger ribonucleic acid (mRNA) level was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RF/6A cells were randomly divided into the control group, NC-sh group and HTRA3-sh group. Lentiviral vectors of NC-shRNA and HTRA3-shRNA were transfected into RF/6A cells in the NC-sh group and HTRA3-sh group by Lipofectamine2000. HTRA3 transfection was detected by qRT-PCR and Western blot. Then, the RF/6A cells were randomly divided into the N group, H group, H+NC-sh group and H+HTRA3-sh group. After cell transfection, RF/6A cells in the N group were cultured in a RPMI 1640 complete medium at a normoxia state, and cells in other groups were cultured in a RPMI 1640 medium with 200 mmol·L-1 CoCl2 at a hypoxia state. Tubule formation was measured by Matrigel. The C57BL/6J mice were divided into the control group, CNV group, CNV+NC-sh group and CNV+HTRA3-sh group, with 12 mice in each group. Mice in the control group were unmodeled mice, and mice in the other groups were laser-induced CNV model mice. NC-shRNA and HTRA3-shRNA lentiviral vectors with a titer of 1×1011 TU·mL-1 were administered to mice in the CNV+NC-sh group and CNV+HTRA3-sh group via intravitreal injection. Mice in the control group and CNV group were injected with phosphate buffered saline. After 7 days of treatment, the mice were examined by fundus fluorescein angiography, and the eyeballs received hematoxylin & eosin staining. The mRNA levels of HTRA3, chitinase-like protein 3 (Ym-1), arginase 1 (Arg-1), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in RF/6A cells or choroidal tissues were detected by qRT-PCR. The protein expression levels of HTRA3, VEGF and nuclear factor kappa B (NF-κB) p65 in RF/6A cells or choroidal tissues were detected by Western blot.
Results　Compared with the normal group, serum HTRA3 mRNA level of patients in the wAMD group increased (t=11.804, P<0.001). Compared with the control group and NC-sh group, the expressions of HTRA3 mRNA and protein in RF/6A cells in the HTRA3-sh group decreased (all P<0.05). Compared with the N group, the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF in RF/6A cells in the H group increased (all P<0.05). Compared with the H+NC-sh group, the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF decreased in RF/6A cells in the H+HTRA3-sh group (all P<0.05). Compared with the control group, the mRNA and protein expression levels of HTRA3 increased, the relative fluorescence intensity of CNV increased, the mRNA levels of Ym-1 and Arg-1 increased, the iNOS and COX-2 mRNA levels decreased, and the NF-κB p65 protein expression level increased in mice of the CNV group (all P<0.05). Compared with the CNV+NC-sh group, the mRNA and protein expression levels of HTRA3 decreased, the relative fluorescence intensity of CNV decreased, the mRNA levels of Ym-1 and Arg-1 decreased, the mRNA levels of iNOS and COX-2 increased, and the NF-κB p65 protein expression level decreased in mice of the CNV+HTRA3-sh group (all P<0.05).
Conclusion　Down-regulation of HTRA3 can inhibit the formation of CNV and the polarization of M2 macrophages. HTRA3 may be an important potential target for the prevention and treatment of wAMD.

参考文献/References:

［1］　MITCHELL P,LIEW G,GOPINATH B,WONG T Y.Age-related macular degeneration［J］.Lancet,2018,392(10153):1147-1159.
［2］　STAHL A.The diagnosis and treatment of age-related macular degeneration［J］.Dtsch Arztebl Int,2020,117(29/30):513-520.
［3］　AMBATI J,FOWLER B J.Mechanisms of age-related macular degeneration［J］.Neuron,2012,75(1):26-39.
［4］　AMMAR M J,HSU J,CHIANG A,HO A C,REGILLO C D.Age-related macular degeneration therapy:a review［J］.Curr Opin Ophthalmol,2020,31(3):215-221.
［5］　YANG S,ZHAO J,SUN X.Resistance to anti-VEGF therapy in neovascular age-related macular degeneration:a comprehensive review［J］.Drug Des Devel Ther,2016,10:1857-1867.
［6］　SNIEGOWSKI M,MANDAVA N,KAHOOK M Y.Sustained intraocular pressure elevation after intravitreal injection of bevacizumab and ranibizumab associated with trabeculitis［J］.Open Ophthalmol J,2010,4:28-29.
［7］　BAKRI S J,LARSON T A,EDWARDS A O.Intraocular inflammation following intravitreal injection of bevacizumab［J］.Graefes Arch Clin Ophthalmol,2008,246(5):779-781.
［8］　AMBATI J,ATKINSON J P,GELFAND B D.Immunology of age-related macular degeneration［J］.Nat Rev Immunol,2013,13(6):438-451.
［9］　CAO X,SHEN D,PATEL M M,TUO J,JOHNSON T M,OLSEN T W,et al.Macrophage polarization in the maculae of age-related macular degeneration:a pilot study［J］.Pathol Int,2011,61(9):528-535.
［10］　XU N,BO Q,SHAO R,LIANG J,ZHAI Y,YANG S,et al.Chitinase-3-like-1 promotes M2 macrophage differentiation and induces choroidal neovascularization in neovascular age-related macular degeneration［J］.Invest Ophthalmol Vis Sci,2019,60(14):4596-4605.
［11］　NIE G Y,HAMPTON A,LI Y,FINDLAY J K,SALAMONSEN L A.Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3),characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2［J］.Biochem J,2003,371(Pt 1):39-48.
［12］　TOCHARUS J,TSUCHIYA A,KAJIKAWA M,UETA Y,OKA C,KAWAICHI M.Developmentally regulated expression of mouse HtrA3 and its role as an inhibitor of TGF-beta signaling［J］.Dev Growth Differ,2004,46(3):257-274.
［13］　YANG Z,CAMP N J,SUN H,TONG Z,GIBBS D,CAMERON D J,et al.A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration［J］.Science,2006,314(5801):992-993.
［14］　DEWAN A,LIU M,HARTMAN S,ZHANG S S,LIU D T,ZHAO C,et al.HTRA1 promoter polymorphism in wet age-related macular degeneration［J］.Science,2006,314(5801):989-992.
［15］　刘培源.两种视网膜下新生血管小鼠模型基因表达谱的RNA-Seq研究［D］.汕头:汕头大学,2016.
LIU P Y.RNA-Seq study on gene expression profiles of two mouse models of subretinal neovascularization［D］.Shantou:Shantou University,2016.
［16］　JI C,SUN L S,XING F,NIU N,GAO H L,DAI J W,et al.HTRA3 is a prognostic biomarker and associated with immune infiltrates in gastric cancer［J］.Front Oncol,2020,10:603480.
［17］　BIRD A C,BRESSLER N M,BRESSLER S B,CHISHOLM I H,COSCAS G,DAVIS M D,et al.An international classification and grading system for age-related maculopathy and age-related macular degeneration［J］.Surv Ophthalmol,1995,39(5):367-374.
［18］　ZURAWA-JANICKA D,SKORKO-GLONEK J,LIPINSKA B.HtrA proteins as targets in therapy of cancer and other diseases［J］.Expert Opin Ther Targets,2010,14(7):665-679.
［19］　TOSI G M,CALDI E,NERI G,NUTI E,MARIGLIANI D,BAIOCCHI S,et al.HTRA1 and TGF-β1 concentrations in the aqueous humor of patients with neovascular age-related macular degeneration［J］.Invest Ophthalmol Vis Sci,2017,58(1):162-167.
［20］　OWEN L A,SHIRER K,COLLAZO S A,SZCZOTKA K,BAKER S,WOOD B,et al.The serine protease HTRA-1 is a biomarker for ROP and mediates retinal neovascularization［J］.Front Mol Neurosci,2020,13:605918.
［21］　SCHLINGEMANN R O.Role of growth factors and the wound healing response in age-related macular degeneration［J］.Graefes Arch Clin Exp Ophthalmol,2004,242(1):91-101.
［22］　张敬法,赵珍珍.湿性年龄相关性黄斑变性发病机制及治疗［J］.眼科新进展,2022,42(2):85-98.
ZHANG J F,ZHAO Z Z.Pathogenesis and treatment of wet age-related macular degeneration［J］.Rec Adv Ophthalmol,2022,42(2):85-98.
［23］　陶方方,亢泽峰,陈水龄,褚文丽,刘健,周志豪.加减驻景方对脉络膜新生血管动物模型缺氧信号因子表达的影响［J］.中国中医眼科杂志,2021,31(5):309-315.
TAO F F,KANG Z F,CHEN S L,CHU W L,LIU J,ZHOU Z H.The effects of modified Zhujing formula on the expression of hypoxia signal factors in choroidal neovascularization animal model［J］.CHN J Chin Ophthalmol,2021,31(5):309-315.
［24］　HADDOCK L J,KIM D Y,MUKAI S.Simple,inexpensive technique for high-quality smartphone fundus photography in human and animal eyes［J］.J Ophthalmol,2013,2013:518479.
［25］　NIE G,LI Y,HE H,FINDLAY J K,SALAMONSEN L A.HtrA3,a serine protease possessing an IGF-binding domain,is selectively expressed at the maternal-fetal interface during placentation in the mouse［J］.Placenta,2006,27(4/5):491-501.
［26］　JENSEN L D,HOT B,RAMSKLD D,GERMANO R F V,YOKOTA C,GIATRELLIS S,et al.Disruption of the extracellular matrix progressively impairs central nervous system vascular maturation downstream of β-catenin signaling［J］.Arterioscler Thromb Vasc Biol,2019,39(7):1432-1447.
［27］　YOSHIDA K,KUSAMA K,AZUMI M,YOSHIE M,KATO K,TAMURA K.Endoplasmic reticulum stress-regulated high temperature requirement A1 (HTRA1) modulates invasion and angiogenesis-related genes in human trophoblasts［J］.J Pharmacol Sci,2022,150(4):267-274.
［28］　PALLEN M J,WREN B W.The HtrA family of serine proteases［J］.Mol Microbiol,1997,26(2):209-221.
［29］　GHOSH S,SHANG P,YAZDANKHAH M,BHUTTO I,HOSE S,MONTEZUMA S R,et al.Activating the AKT2-nuclear factor-κB-lipocalin-2 axis elicits an inflammatory response in age-related macular degeneration［J］.J Pathol,2017,241(5):583-588.
［30］　NAGAI N,IZUMI-NAGAI K,OIKE Y,KOTO T,SATOFUKA S,OZAWA Y,et al.Suppression of diabetes-induced retinal inflammation by blocking the angiotensin II type 1 receptor or its downstream nuclear factor-kappaB pathway［J］.Invest Ophthalmol Vis Sci,2007,48(9):4342-4350.
［31］　KARIN M.Nuclear factor-kappaB in cancer development and progression［J］.Nature,2006,441(7092):431-436.
［32］　LIN L W,WANG S W,HUANG W C,HUYNH T K,LAI C Y,KO C Y,et al.Melatonin inhibits VEGF-induced endothelial progenitor cell angiogenesis in neovascular age-related macular degeneration［J］.Cells,2023,12(5):799.
［33］　HIKAGE F,LENNIKOV A,MUKWAYA A,LACHOTA M,IDA Y,UTHEIM T P,et al.NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice［J］.Exp Cell Res,2021,403(1):112581.
［34］　HOU Y,LIN H,ZHU L,LIU Z,HU F,SHI J,et al.Lipopolysaccharide increases the incidence of collagen-induced arthritis in mice through induction of protease HTRA-1 expression［J］.Arthritis Rheum,2013,65(11):2835-2846.

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备注/Memo

备注/Memo:
陕西省自然科学基础研究计划项目（编号：2021JM-547）；陕西省卫生健康科研基金项目（编号：2022D036）
更新日期/Last Update: 2024-04-05