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Lysyl Oxidase Inhibition Via Β- Aminoproprionitrile Hampers Human Umbilical Vein Endothelial Cell Angiogenesis and Migration in Vitro

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Lysyl Oxidase Inhibition Via Β- Aminoproprionitrile Hampers Human Umbilical Vein Endothelial Cell Angiogenesis and Migration in Vitro Lysyl oxidase inhibition via β- aminoproprionitrile hampers human umbilical vein endothelial cell angiogenesis and migration in vitro The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Shi, Lin, Ning Zhang, Hetao Liu, Lei Zhao, Jing Liu, Juan Wan, Wenyi Wu, Hetian Lei, Rongqing Liu, and Mei Han. 2018. “Lysyl oxidase inhibition via β-aminoproprionitrile hampers human umbilical vein endothelial cell angiogenesis and migration in vitro.” Molecular Medicine Reports 17 (4): 5029-5036. doi:10.3892/mmr.2018.8508. http://dx.doi.org/10.3892/mmr.2018.8508. Published Version doi:10.3892/mmr.2018.8508 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:35981960 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA MOLECULAR MEDICINE REPORTS 17: 5029-5036, 2018 Lysyl oxidase inhibition via β-aminoproprionitrile hampers human umbilical vein endothelial cell angiogenesis and migration in vitro LIN SHI1*, NING ZHANG1*, HETAO LIU1, LEI ZHAO1, JING LIU1, JUAN WAN2, WENYI WU3, HETIAN LEI3, RONGQING LIU2 and MEI HAN1 1Department of Pathogenic Biology and Immunology, School of Basic Medical Sciences, Ningxia Medical University; 2Department of Rheumatology and Immunology, The General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, P.R. China; 3Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA Received August 13, 2017; Accepted January 17, 2018 DOI: 10.3892/mmr.2018.8508 Abstract. Lysyl oxidase (LOX) is an enzyme that oxidizes lysine and phorbol 12-myristate 13-acetate (PMA). In addition, LOX residues in collagens and elastin. It stabilizes or remodels the inhibition blocked the angiogenesis stimulated by VEGF extracellular matrix and basement membrane of blood vessels. bFGF and PMA, and the inhibition of LOX reduced the migra- Current oncology studies have revealed that LOX is upregu- tion of HUVECs. Furthermore, the microarray and RT-qPCR lated in invasive cancer cells and bolstered cell movement, and revealed that BAPN downregulated myeloid progenitor LOX was observed to promote the angiogenesis and migration inhibitory factor 1, and western blot analysis demonstrated of endothelial cells. In the present study, angiogenesis and that BAPN decreased the phosphorylation of MAPK and Akt, migration were examined in human umbilical vein endothelial suggesting that the specific inhibitor of LOX, BAPN, may cells (HUVECs). Following cell treatment with 0.1-0.4 mM serve as an alternative strategy for preventing angiogenesis. β-aminoproprionitrile (BAPN), a specific inhibitor of LOX, angiogenesis was analyzed with a fibrin gel in vitro angio- Introduction genesis assay kit and migration was examined via a Boyden Chamber assay. Angiogenesis-associated gene expression Angiogenesis is a process by which the primitive vasculature was investigated with a microarray assay and confirmed with is expanded and remodeled to form the networks of large reverse transcription-quantitative polymerase chain reaction and small vessels that have the interconnecting, branching (RT-qPCR). The results showed that HUVEC angiogenesis pattern of the mature vascular system. The neovascularization substantially increased in the presence of vascular endothelial develops from endothelial cells that originate from antecedent growth factor (VEGF), basic fibroblast growth factor (bFGF) vessels (1). Throughout the sprouting process, endothelial cells deteriorate the concealed basement membrane, move into the adjacent tissue, proliferate, and assemble into tubes (2). Soluble factors and cell-cell and cell-matrix interactions are necessary for the established vasculature to acclimate to Correspondence to: Professor Mei Han, Department of Pathogenic varying demands (1,3). Angiogenesis plays a critical role in Biology and Immunology, School of Basic Medical Sciences, physiological and pathological processes, including halting Ningxia Medical University, 1160 Shengli Street, Yinchuan, ischemic damage caused by stroke (4), cancer migration (5) Ningxia 750004, P.R. China and pannus formation in rheumatoid arthritis (6). The lesion E-mail: [email protected] of microvessel basement membrane is critical for the blood Dr Rongqing Liu, Department of Rheumatology and Immunology, brain barrier (BBB) damage that happens following isch- The General Hospital of Ningxia Medical University, 804 Shengli emic stroke (7,8). Well-acknowledged experimental data and Street, Yinchuan, Ningxia 750004, P.R. China clinical results of these microvascular wall changes are due to E-mail: [email protected] edema caused by endothelial integrity loss after extravasations *Contributed equally of noncellular blood elements as haemorrhages (9). Lysyl oxidase (LOX) can oxidize lysine residues in collagens Key words: angiogenesis, migration, lysyl oxidase, human and elastin, catalyse lysine-derived cross-links, and stabilize umbilical vein endothelial cell, myeloid progenitor inhibitory the extracellular matrix and basement membrane of blood factor 1, phospho-mitogen-activated protein kinase, phospho-protein vessels in normal tissues (10,11). Current oncology outcomes kinase B have revealed that LOX is expressed in prolifically spreading tumor cells, and it promoted cancer metastasis (12,13). Clinical and experimental evidence singles out LOX as a possible 5030 SHI et al: LYSYL OXIDASE AND HUVECS ANGIOGENESIS therapeutic target (14). Endothelial cells also express LOX. cells were grown to 80% confluence, they were harvested. One study suggested that the LOX genes upregulated in tumor 600 µl of medium containing 10% FBS without or with endothelial cells (TECs) compared to normal endothelial BAPN (0.1, 0.2, 0.3 and 0.4 mM) was added into each well cells, and LOX could be a TEC marker (15). The lysyl oxidase of the 24-well (?) plate. Then, we placed 1x105 cells in 100 µl inhibitor β-aminopropionitrile (BAPN) impedes tumor serum-free medium with or without BAPN into the insert wells angiogenesis and glioblastoma multiforme development in a of 8 µl pore membrane Boyden Chambers. After incubating mouse orthotopic brain tumor model (16). A previous study for 24 h at 37˚C, the unattached cells were removed. Then, of our group indicated that inhibition of LOX attenuated the cells were fixed by methanol for 5 min, washed once with PBS, microvascular density in the synovial membranes of collagen- and stained with 0.05% Giemsa stain. We quantified the cells induced arthritis rats (17). In the present study we found that moved to the lower sides of the membrane by counting that BAPN inhibited angiogenesis and migration of human in 5 random visual fields (magnification, x200) for each well umbilical vein endothelial cells (HUVECs). under a light microscope. Materials and methods Microarray assay. HUVECs were cultured with or without BAPN (0.4 mM) for 24 h in serum-free medium. Total RNA Cell culture. HUVECs were purchased from the China Center was extracted using Tri Reagent (Molecular Research Center, for Type Culture Collection (Wuhan, China), and cultured in Inc. Cincinnati, OH, USA) based on the manufacturer's Dulbecco's modified Eagle's medium (DMEM) supplemented instructions. The RNA concentration was evaluated by an with 10% fetal bovine serum (FBS), 2 mM L-glutamine, optical density (OD) ratio of 260:280 and with gel electro- 5 U/ml penicillin G, 5 µg/ml streptomycin sulfate (Invitrogen, phoresis. The cDNA was synthesized by reverse transcription Carlsbad, CA, USA). The present study was approved by the of the RNA. The cDNA microarrays were generated with a Ethical Committee of The General Hospital of Ningxia Medical SPBIO2000 robotic arrayer, and it had 1080 cDNA clones. University (Yinchuan, China; registration no. 2016-230). Fluorescence intensities, produced by Cy5 or Cy3 impaired to the target sequences on the microarray slides, were quantified Enzyme-linked immunosorbent assay (ELISA). HUVECs with a ScanArray 5000 laser confocal microscope equipped (1x105/well) were seeded on 96-well plates, incubated for 24 h scanning system (General Scanning, Watertown, MA, USA) to allow for attachment to the surface of plate, then starved by using the necessary excitation and emission filters. The signal incubating another 24-48 h in serum-free DMEM. The cell- from every impaired cDNA target on the microarray slide free supernatants were harvested for measuring the secreted was found, and the expression ratio among the experimental LOX using an ELISA kit according to the manufacturer's and reference samples (Cy5/Cy3 ratio) was established with recommendations (SEC580Ra; Cloud-Clone Corp., Houston, ImaGene version 3.0 software (Biodiscovery, Los Angeles, TX, USA). Optical density values of the samples were used to California, USA). Microarray analysis was conducted in tripli- calculate the concentrations of all of the samples. cate, and the Cy5/Cy3 ratio values are the averages. Fibrin gel angiogenesis analysis. A fibrin gel in vitro Reverse transcription-quantitative polymerase chain reaction angiogenesis assay kit (ECM630; Chemicon, Temecula, CA, (RT-qPCR). Total RNA was isolated using the TRI Reagent USA) was used to evaluate tube formation of HUVECs in from the HUVECs treated with and without BAPN as described 96-well plates. A
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