title: Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within three-dimensional cocultures.
abstract: Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.
authors.affiliation: 1 Department of Chemical and Biomedical Engineering, Cleveland State University , Cleveland, Ohio.
authors.forename: Phillip
authors.initials: P
authors.lastname: Simmers
authors.forename: Arsela
authors.initials: A
authors.lastname: Gishto
authors.forename: Narendra
authors.initials: N
authors.lastname: Vyavahare
authors.forename: Chandrasekhar R
authors.initials: CR
authors.lastname: Kothapalli
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology*; D040761:Biomimetic Materials / Q000494:pharmacology; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques / Q000379:methods*; D004549:Elastin / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D000071837:Fibrillins; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D044085:Microfluidics; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism; D051381:Rats; D026422:S-Nitrosoglutathione / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
chemical_list: D000071837:Fibrillins; D006025:Glycosaminoglycans; D008840:Microfilament Proteins; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D004549:Elastin; D008249:Protein-Lysine 6-Oxidase; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9