title: Three-dimensional multilayers of smooth muscle cells as a new experimental model for vascular elastic fiber formation studies.
abstract: OBJECTIVE
  Elastic fiber formation is disrupted with age and by health conditions including aneurysms and atherosclerosis. Despite considerable progress in the understanding of elastogenesis using the planar culture system and genetically modified animals, it remains difficult to restore elastic fibers in diseased vessels. To further study the molecular mechanisms, in vitro three-dimensional vascular constructs need to be established. We previously fabricated vascular smooth muscle cells (SMCs) into three-dimensional cellular multilayers (3DCMs) using a hierarchical cell manipulation technique, in which cells were coated with fibronectin-gelatin nanofilms to provide adhesive nano-scaffolds. Since fibronectin is known to assemble and activate elastic fiber-related molecules, we further optimized culture conditions.
  
  
  RESULTS
  Elastica stain, immunofluorescence, and electron microscopic analysis demonstrated that 3DCMs, which consisted of seven layers of neonatal rat aortic SMCs cultured in 1% fetal bovine serum (FBS) in Dulbecco's modiﬁed Eagle's medium, exhibited layered elastic fibers within seven days of being in a static culture condition. In contrast, the application of adult SMCs, 10% FBS, ε-poly(lysine) as an alternative adhesive for fibronectin, or four-layered SMCs, failed to generate layered elastic fiber formation. Radioimmunoassay using [(3)H]valine further confirmed the greater amount of cross-linked elastic fibers in 3DCMs than in monolayered SMCs. Layered elastic fiber formation in 3DCMs was inhibited by the lysyl oxidase inhibitor β-aminopropionitrile, or prostaglandin E2. Furthermore, infiltration of THP-1-derived macrophages decreased the surrounding elastic fiber formation in 3DCMs.
  
  
  CONCLUSIONS
  3DCMs may offer a new experimental vascular model to explore pharmacological therapeutic strategies for disordered elastic fiber homeostasis.
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Ishiwata
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. Electronic address: utako@yokohama-cu.ac.jp.
authors.forename: Utako
authors.initials: U
authors.lastname: Yokoyama
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Michiya
authors.initials: M
authors.lastname: Matsusaki
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Asano
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kadowaki
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ichikawa
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Masanari
authors.initials: M
authors.lastname: Umemura
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Fujita
authors.affiliation: Department of Cell Physiology, Jikei University School of Medicine, Tokyo, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Minamisawa
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimoda
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akashi
authors.affiliation: Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. Electronic address: yishikaw@med.yokohama-cu.ac.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ishikawa
mesh_terms: D000629:Aminopropionitrile / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D015232:Dinoprostone / Q000494:pharmacology; D004547:Elastic Tissue / Q000378:metabolism*; D005353:Fibronectins; D005780:Gelatin; D020869:Gene Expression Profiling; D008264:Macrophages / Q000166:cytology; D009024:Morphogenesis; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D049329:Nanostructures; D036103:Nanotechnology; D011107:Polylysine / Q000494:pharmacology; D061251:Primary Cell Culture; D051381:Rats; D017208:Rats, Wistar; D018874:Spheroids, Cellular; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods; D054457:Tissue Scaffolds
chemical_list: D005353:Fibronectins; D000629:Aminopropionitrile; D011107:Polylysine; D005780:Gelatin; D015232:Dinoprostone
keywords: Blood vessels; Elastic fibers; Fibronectin; Nanotechnology; Smooth muscle cells; Tissue engineering