ONLINE FIRST This is a provisional PDF only. Copyedited and fully formatted versión will Be made available at final puBliCation ISSN: 0213-3911 e-ISSN: 1699-5848 HISTOPATHOLOGY Submit your article to this Journal (http://www.hh.um.es/Instructions.htmmanuscript)) Membrane TraffickingAND and Exocytosis are Upregulated in Port Wine Stain Blood Vessels Authors: Rong Yin, Shawn J. Rice, Jinwei Wang, Lin Gao, Joseph Tsai, Radean T. Anvari, Fang Zhou, Xin Liu, Gang Wang, Yuxin Tang, Martin C. Mihm, Jr., Chandra P. Belani, Dong bao Chen, J. Stuart Nelson and Wenbin Tan (non-edited DOI: 10.14670/HH-18-051 Article type: ORIGINAL ARTICLE HISTOLOGYAccepted: 2018-10-10 Epub ahead of print: 2018-10-10 This artiCle has Been peer reviewed and puBlished immdediately upon acCeptanCe. ArtiCles in “Histology and Histopathology” are listed in PuBmed. Pre-print author´s version 1 Membrane Trafficking and Exocytosis are Upregulated in Port Wine Stain 2 Blood Vessels 3 Running Title: membrane trafficking and exocytosis in PWS 4 Rong Yin1,2,9, Shawn J. Rice3, Jinwei Wang1,8, Lin Gao5, Joseph Tsai1, Radean T. Anvari1, Fang 5 Zhou1,8, Xin Liu3, Gang Wang5, Yuxin Tang8, Martin C. Mihm, Jr.6, Chandra P. Belani3,4, Dong- 6 bao Chen10, J. Stuart Nelson1,7 and Wenbin Tan1,2* 7 1Department of Surgery, Beckman Laser Institute and Medical Clinic, University of California, 8 Irvine, Irvine, California 92617, USA 9 2Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, 10 Columbia, South Carolina 29209, USA 11 3Penn State Cancer Institute, Hershey, PA 17033HISTOPATHOLOGY 12 4Department of Medicine, Penn State College of Medicine,manuscript) Hershey, PA 17033 13 5Department of Dermatology,AND Xijing Hospital, Xi'an, 710032, China. 14 6Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, 15 Massachusetts 02115, USA 16 7Department of Biomedical Engineering, University of California, Irvine, Irvine, California 17 92617, USA (non-edited 18 8Department of Urology, the Xiangya 3rd Hospital, Xiangya School of Medicine, Central South 19 University, Changsha, Hunan 412000, China HISTOLOGY20 9Department of Dermatology, the Second Hospital of Shanxi Medical University, Taiyuan 21 030001, China 1 22 10Department of Obstetrics and Gynecology, University of California, Irvine, Irvine, California 23 92617, USA 24 Correspondence: Wenbin Tan, Department of Cell Biology and Anatomy, University of South 25 Carolina School of Medicine, Columbia, South Carolina 29209, USA; Department of Surgery, 26 Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, California 27 92617, USA 28 Email: [email protected] or [email protected] Phone: 803-216-3816 Fax: 803-216- 29 3846 30 Conflict of Interest: Disclosure: None Declared 31 Disclaimer Any views expressed here represent personal opinion and do not necessarily reflect 32 those of the United States Department of Health and Human Services or the federal government. HISTOPATHOLOGY 33 Prior Publication: None of the material in this manuscriptmanuscript) has been published or is under 34 consideration for publication elsewhere, including the Internet. 35 Funding statement: This workAND was supported by grants from the National Institutes of Health 36 (AR063766 and AR073172 to WT, HL70562 to DBC, AR47551 and AR59244 to JSN), the 37 Department of Defense/CDMRP (W81XWH1810096 to WT) and National Natural Science 38 Foundation of China (non-edited(81571432 to YT). 39 Acknowledgments: Institutional support was provided by the Arnold and Mabel Beckman 40 Foundation and the David and Lucile Packard Foundation. We greatly appreciate the Sue & Bill HISTOLOGY41 Gross Stem Cell Research Center at the University of California, Irvine, for their assistance with 42 the histology image acquisition process experiments. 43 Words: 2404; References: 34; Figures: 3; Table: 1 2 44 Abstract: 211 words 45 HISTOPATHOLOGY manuscript) AND (non-edited HISTOLOGY 3 46 ABSTRACT 47 Introduction: Port wine stain (PWS) is characterized as a progressive dilatation of immature 48 venule-like vasculatures which result from differentiation-impaired endothelial cells. In this 49 study, we aimed to identify the major biological pathways accounting for the pathogenesis of 50 PWS. 51 Methods: Sequential windowed acquisition of all theoretical fragment ion mass spectra 52 (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed 53 by confirmative studies with immunohistochemistry, immunoblot and transmission electron 54 microscopy (TEM). 55 Results: 107 out of 299 identified proteins showed differential expressions in PWS lesions as 56 compared to normal skin, mainly involvingHISTOPATHOLOGY the functions of biosynthesis, membrane trafficking, 57 cytoskeleton and cell adhesion/migration. The confirmativemanuscript) studies showed that expressions of 58 membrane trafficking/exocytosisAND related proteins such as VAT1, IQGAP1, HSC70, clathrin, 59 perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared 60 to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, 61 TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from 62 PWS blood vessels as(non-edited compared to control. 63 Conclusions: The biological process of membrane trafficking and exocytosis is enhanced in 64 PWS blood vessels. Our results imply that the extracellular vesicles released by lesional HISTOLOGY65 endothelial cells may act as potential intercellular signaling mediators to contribute to the 66 pathogenesis of PWS. 67 4 68 Key words: Port wine stain, vascular malformations, endothelial cells, extracellular vesicle, 69 exocytosis, SWATH-MS 70 71 Introduction 72 Port wine stain (PWS) is a congenital vascular malformation resulting from differentiation- 73 impaired endothelial cells (ECs) in human skin with a progressive dilatation of immature venule- 74 like vasculatures (Tan et al., 2017). The prevalence is estimated at 3-5 children per 1,000 live 75 births (Pratt, 1953, Jacobs and Walton, 1976). PWS initially appears as flat red macules in 76 childhood; lesions tend to darken progressively to purple with soft tissue hypertrophy and, by 77 middle age, they often become raised as a HISTOPATHOLOGYresult of the development of vascular nodules which 78 are susceptible to spontaneous bleeding or hemorrhagemanuscript) (Lever and Schaumburg-Lever, 1990, 79 Geronemus and Ashinoff, 1991). PWS is a significant clinical problem that results in loss of self- 80 esteem since most malformationsAND occur on the face (Kalick, 1978, Heller et al., 1985, Malm and 81 Carlberg, 1988). The pulsed dye laser (PDL) is the treatment of choice for PWS but the regrowth 82 of pathologic blood vessels post-PDL treatment is a major clinical barrier that needs to be 83 overcome (Tan et al., 2012, Gao et al., 2014). The pathogenesis of PWS remains incompletely 84 understood. Recent (non-editedstudies have suggested that the sporadic somatic mutation of guanine 85 nucleotide-binding protein, G alpha subunit q (gnaq) (c.548G>A), is linked to PWS (Shirley et 86 al., 2013, Lian et al., 2014). The gnaq (c.548G>A) is primarily present in PWS blood vessels HISTOLOGY87 (Couto et al., 2016, Tan, Nadora , et al., 2016). In addition, PWS have sustained activation of 88 mitogen-activated protein kinases in the infantile stage and, particularly, activation of PKCα and 89 PI3K pathways in hypertrophic and nodular lesions (Tan et al., 2014, Yin et al., 2017). We have 5 90 recently found that PWS blood vessels are immature capillary vasculatures with aberrant 91 expressions of stem cell markers CD133 and CD166, venous marker EphB1 and arterial marker 92 Ephrin B2 (EfnB2). The disruption of normal EC-EC interactions by co-existence of EphB1 and 93 EfnB2 contributes to progressive dilatation of PWS vasculatures (Tan et al., 2017). 94 In this study, we aimed to identify differentially expressed (DE) proteins in PWS lesions as 95 compared to normal skin using a proteomics approach, namely sequential windowed acquisition 96 of all theoretical fragment ion mass spectra (SWATH-MS). We then performed a further 97 investigation to determine their expression patterns and cellular localizations in PWS 98 hypertrophic lesions and nodules as compared to control. We found that PWS blood vessels have 99 increased expressions of cell membrane trafficking/exocytosis related molecules and that the 100 exocytosis of extracellular vesicles (EVs) isHISTOPATHOLOGY enhanced in PWS blood vessels. 101 manuscript) AND (non-edited HISTOLOGY 6 102 Materials and Methods 103 104 Patients and Tissue Samples 105 This study was approved by the Institutional Review Board at the University of California, Irvine 106 (UCI) (#2013-9396) and Xijing Hospital. A total of 21 PWS biopsies and 6 adjacent normal skin 107 samples were obtained from 13 subjects and de-identified for this study. The clinical 108 characteristics of the subjects and biopsy samples have been described previously (Tan et al., 109 2014, Yin et al., 2017). 110 111 SWATH-MS HISTOPATHOLOGY 112 The formalin-fixed paraffin-embedded (FFPE) sectionsmanuscript) were used for SWATH-MS. There were 113 two groups of specimens, e.g., control (n=3) and PWS (n=6). The protocols of protein extraction 114 from FFPE sections and peptideAND library preparation have been previously reported (Ostasiewicz 115 et al., 2010). All samples were duplicated during the mass spectrometry studies. Mass spectra