Synthesis And Characterization Of N Nitroso Nitric Oxide Donors And Their Effect On Human Aortic Smooth Muscle Cells Proliferation PDF Download

Abstract: Vascular diseases such as atherosclerosis and aneurysms 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. Recent tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency and matrix deposition yields. In this study, the benefits of exogenous nitric oxide cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within 3D biomimetic cultures have been explored. The first experiment utilized a microfluidic platform with two adjacent, permeable 3D culture chambers, to enable paracrine signaling between vascular cell cocultures. Healthy HA-SMCs were cultured in these devices within 3D collagen hydrogels, either alone or in the presence of human aortic endothelial cell (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. However, 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 total protein amount synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. On a per cell basis, 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. The matrix yields of these proteins reached almost 40 - 51 % within selective cocultures receiving GSNO. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages, although MMP-2 levels remained attenuated. These quantitative assay data were strongly supported by immunofluorescence images under respective conditions. Based on the optimized dosage (100 nM) deduced from first experiment, we tested the utility of NO cues within diseased HA-SMC (derived from aneurysmal human aortic segment) cultures, under both 2D and 3D culture conditions. Results showed that cell proliferation was higher on 2D substrates compared to that within 3D cultures. In accordance to the trends noted within healthy SMC cultures, there was a decrease in cell proliferation in both 2D and 3D, with increasing GSNO dose. Similarly, the presence of GSNO stimulated elastin, glycosaminoglycans, hyaluronic acid, lysyl oxidase crosslinking enzyme (LOX), MMP-2, 9 and TIMP-1 synthesis and release by aneurysmal SMCs, although the matrix deposition levels were lower compared to their healthy counterparts. Both experiments results attest to the tremendous benefits of delivering nitric oxide 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.