Novel Adipokine, FAM19A5, Inhibits Neointima Formation After Injury Through Sphingosine-1-Phosphate Receptor 2
Authors: Pingzhang Wang
Wei Kong
Yan Zhang
Youfei Guan
Nan Xie
Can Zheng
Yingbao Wang
Baihui Ma
Shiyang Huang
Bing Yu
Dalong Ma
Songyang Zhang
Lu Zhang
Ziyi Liu
Xiaoning Mo
Yi Fu
Zeyu Cai
Guizhen Zhao
Xian Wang
Dixin Chen
Ying Wang
Journal: Circulation (2018)
Discipline: CLINICAL MEDICINE
Keywords: FAM19A5; S1PR2; VSMC; adipokine; neointima formation.
Affiliations: Department of Physiology and Pathophysiology (Y.W., D.C., S.Z., B.Y., L.Z., G.Z., B.M., Z.C., N.X., Z.L., X.W., Y.F., W.K.)., Department of Immunology, Key Laboratory of Medical Immunology of Ministry of Health (D.C., Y.Z., P.W., C.Z., S.H., D.M., Y.W.), School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China., Center for Human Disease Genomics, Peking University, Beijing, China (P.W., X.M., D.M., Y.W.)., Advanced Institute for Medical Sciences, Dalian Medical University, Liaoning, China (Y.G.)., Department of Immunology, Key Laboratory of Medical Immunology of Ministry of Health (D.C., Y.Z., P.W., C.Z., S.H., D.M., Y.W.), School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China  ., Department of Physiology and Pathophysiology (Y.W., D.C., S.Z., B.Y., L.Z., G.Z., B.M., Z.C., N.X., Z.L., X.W., Y.F., W.K.)  .

Abstract:  Background: Obesity plays crucial roles in the development of cardiovascular diseases. However, the mechanisms that link obesity and cardiovascular diseases remain elusive. Compelling evidence indicates that adipokines play an important role in obesity-related cardiovascular diseases. Here, we found a new adipokine-named family with sequence similarity 19, member A5 (FAM19A5), a protein with unknown function that was predicted to be distantly related to the CC-chemokine family. We aimed to test whether adipose-derived FAM19A5 regulates vascular pathology on injury. Methods: DNA cloning, protein expression, purification, and N-terminal sequencing were applied to characterize FAM19A5. Adenovirus infection and siRNA transfection were performed to regulate FAM19A5 expression. Balloon and wire injury were performed in vivo on the rat carotid arteries and mouse femoral arteries, respectively. Bioinformatics analysis, radioactive ligand-receptor binding assays, receptor internalization, and calcium mobilization assays were used to identify the functional receptor for FAM19A5. Results: We first characterized FAM19A5 as a secreted protein, and the first 43 N-terminal amino acids were the signal peptides. Both FAM19A5 mRNA and protein were abundantly expressed in the adipose tissue but were downregulated in obese mice. Overexpression of FAM19A5 markedly inhibited vascular smooth muscle cell proliferation and migration and neointima formation in the carotid arteries of balloon-injured rats. Accordingly, FAM19A5 silencing in adipocytes significantly promoted vascular smooth muscle cell activation. Adipose-specific FAM19A5 transgenic mice showed greater attenuation of neointima formation compared with wild-type littermates fed with or without Western-style diet. We further revealed that sphingosine-1-phosphate receptor 2 was the functional receptor for FAM19A5, with a dissociation constant (Kd) of 0.634 nmol/L. Inhibition of sphingosine-1-phosphate receptor 2 or its downstream G12/13-RhoA signaling circumvented the suppressive effects of FAM19A5 on vascular smooth muscle cell proliferation and migration. Conclusions: We revealed that a novel adipokine, FAM19A5, was capable of inhibiting postinjury neointima formation via sphingosine-1-phosphate receptor 2-G12/13-RhoA signaling. Downregulation of FAM19A5 during obesity may trigger cardiometabolic diseases. 

Retracted: No

PubPeer comments:
 Readers should note a recent preprint that could not reproduce part of the results obtained in Wang et al., study. From the preprint :  In the peripheral tissues, but not the brain, FAM19A5 protein levels in adipose and reproductive tissues were below detectable limits for analysis by Western blot. (...) In contrast to the previously published study, our data showed that FAM19A5 does not interact with S1PR2 to induce receptor-mediated G-protein activation and u03b2-arrestin recruitment that leads to receptor internalization. We showed that FAM19A5 can bind to and be internalized by HEK293 cells independent of S1PR2. Furthermore, the binding and internalization events were inhibited by pretreatment with ECM molecules. FAM19A5 interacts with ECM moleculesin either a competitive or antagonistic manner, which can contribute to functional modulation of the ECM under pathological conditions. , 
 We noticed the contradictory results from the recent preprint on bioRxiv. In terms of the adipose FAM19A5 expression, we applied TaqMan Realtime PCR to analyze FAM19A5 expression in human tissues by using of human cDNA library and verified its adipose expression by immunohistochemistry staining in human, rat and mouse adipose tissue by commercial available antibody (R D Systems). We also measured secreted FAM19A5 in supernatant of cultured adipose tissue cell and in the plasma of WT transgenic mice with or without HFD by using of ELISA and Cytometric Bead Assay respectively. We did not compare FAM19A5 transcription levels in various mice tissue. For the receptor characterization, we have applied radio-ligand binding assay (a gold standard for determining the receptor-ligand interaction), co-immunoprecipitation, receptor internalization, Ca2+ mobilization, G12 13 RhoA activation, and VSMCs functional assay to cumulatively demonstrate that the FAM19A5 is the ligand of the S1PR2 in VSMCs. Second, we have found that the FAM19A5 elicited the S1PR2-G12 G13 signaling in VSMCs whereas the referred manuscript is assaying Gi activity using a Gqi chimera in HEK293 to draw a conclusion that FAM19A5 did not activate G-protein activation. Surprisingly, the manuscript mentioned that they used a His-tag at N-terminal of FAM19A5 for protein purification, antibody preparation, and subsequent receptor assay, which is unusual since the N termini of chemokines like factors is key signaling domain and that modification of the N termini may affect its secretion, conformation, signaling or receptor interactions. This may lead to the inconsistent data with ours. In particular, the manuscript did not include the detail information regarding how to incorporate the His-tag at N terminus. There is no data in the preprint showing the amino acids sequence and bioactivity of purified FAM19A5. Additionally, in their experiment procedure, the digestions with AcTEV protease overnight at 30u00b0C may compromise the activity of purified protein based on our experience. ,