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.

Three‐dimensional collagenous niche and azacytidine selectively promote time‐dependent cardiomyogenesis from human bone marrow‐derived MSC spheroids

“…SDS/PAGE Western blot analysis was done for semi-quantitative detection of GSK-3β, βcatenin, DLL4, and notch-1 protein bands with their respective primary antibodies (Simmers, Gishto, Vyavahare, & Kothapalli, 2015). Similarly, samples for Notch1 pathway study include: control group, aza-treated group, γ-secretase inhibitor treated group, and aza + γ-secretase inhibitor treated group (aza exposure for 24 hr followed by its removal and addition of γ-secretase inhibitor).…”

An in vivo study of a gold nanocomposite biomaterial for vascular repair

“…These approaches have involved biomaterials that utilize both chemical [40–42] and topographical modifications [43, 44] to regulate smooth muscle cells (SMCs) over-proliferation and enhance ECM synthesis as well as other innovative biomaterials that are impregnated with stem cells and/or utilize biomechanical stimulation to improve overall biocompatibility [41, 45]. …”

“…In a recent study, a collagen hydrogel construct doped with S-Nitrosoglutathione (GSNO) released exogenous nitric oxide in order to manipulate cell proliferation and matrix deposition by adult human aortic SMCs [40]. Growth factors such as transforming growth factor (TGF-β1) have also been conjugated to fibrin hydrogel vascular grafts and exposed to mechanical stimulation [41] to improve mechanical properties and ECM content.…”

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three‐dimensional collagen hydrogels: Integrating experiments and modelling

“…LOX activity in the cell matrix and in the pooled media was determined using a sensitive fluorescence-based assay (Amplex® Red Hydrogen Peroxide/Peroxidase assay kit, Molecular Probes) that uses the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to detect H 2 O 2 . This assay is based on the principle that LOX amount is proportional to the released H 2 O 2 because LOX oxidatively deaminates alkyl monoamines and diamines (Palamakumbura & Trackman, 2002;Simmers, Gishto, Vyavahare, & Kothapalli, 2015). Absorbance was read at 560 nm, and a linear standard curve was derived to find H 2 O 2 /LOX in the samples.…”