(H-292) Sodium Nitroprusside mediates ECM regeneration by diseased human aortic SMCs

Introduction:: Aortic Aneurysms (AAs) are a common vascular condition wherein the vessel walls become weakened and dilated, leading to an enlarged balloon-like expansion. This irreversible expansion is primarily attributed to the breakdown of structural proteins such as collagen and elastic fibers by enzymes such as matrix metalloproteases (MMPs). Consequently, leukocyte infiltration and the elevated release of pro-inflammatory cytokines contribute to chronic inflammation and tissue damage in AAs. At the mechanistic level, reduction in nitric oxide (NO) signaling and endothelial dysfunction contribute to irreversible expansion of aortic wall and transformation of contractile vascular smooth muscle cells (VSMCs) to a synthetic phenotype. We hypothesize that restoring NO levels might coax VSMCs to revert to a contractile phenotype, awaken the NO-dependent signaling pathways, and facilitate matrix remodeling by inducing elastogenesis in SMCs, ultimately leading to slow down of AA progression.

Materials and Methods:: Human aortic aneurysmal smooth muscle cells isolated from donors (AASMCs) were cultured in varying concentrations of sodium nitroprusside (SNP; 50 – 500 nM). Cell survival was assessed using Live/Dead assay. The following assays were performed: (1) Extracellular matrix proteins released into media and deposited as matrix by AASMCs in 4-week cultures were quantified using LC-MS/MS analysis. Samples are compared using two measures including the spectral counts and the normalized LFQ intensities. (2) Supernatant media collected over the 4-week culture was used to quantify the secretome composition using human cytokines/chemokines 71-Plex Discover assay (HD71), human MMP and TIMP Discovery assay (HMMP/TIMP-S, P), and TGFB 3-Plex Assay (TGFβ1, TGFβ2, TGFβ3). (3) Metabolomic analysis using semi-targeted liquid chromatography-mass spectrometry (LC-MS) analytic technique to obtain signaling pathways (glycolysis, oxidative phosphorylation, amino acid pathways) affected by exogenous NO in both cell types. (4) Changes in biophysical and biomechanical characteristics of live SMCs were quantified using a nanoindentation assay. A MFP-3D-Bio atomic force microscope (AFM; Oxford Instrument) mounted on an inverted epi-fluorescence microscope (Nikon Eclipse Ti) was used to measure elastic modulus, tether forces, membrane tension, surface adhesion, and surface roughness of cells. (5) RNA sequencing was done to determine changes at the transcription level. Tapestation was used to check RIN score and Qubit was used to measure concentration of RNA samples. Following quality control, all the samples are pooled and molarity for sequencing was determined via qPCR. The pooled libraries were sequenced using Novaseq 6000 platform with Illumina's sequencing reagents.

Results, Conclusions, and Discussions:: Cell survival over the 28-days with SNP exposure remained similar to that in controls (90% ± 3.2%). Secretome analysis suggested a possible connection between NO deficiency and chronic overexpression of the elastolytic MMPs (MMPs-2, 9). NO addition inhibited MMPs release with a concomitant increase in TIMPs-2,4. Metabolomic analysis revealed overexpression of glycolysis pathways, and downregulation of oxidative pathways and ROS pathways. AFM indentation of live cells showed a significant reduction in young’s modulus and surface adhesive forces as a function of increasing SNP dose, indicating that AASMCs became softer with SNP treatment. Tether forces, which is dependent on membrane adhesion, as well as membrane tension were significantly reduced with increasing SNP dosage. Proteomics analysis suggested decreased cytoskeletal density in AASMC cultures, correlating to biomechanical outcomes. Transcriptomic analysis is currently ongoing, and the outcomes will be correlated with the changes noted in proteomic analysis and metabolomic analysis.

This study aims to investigate the benefits of exogenously delivering NO donors such as SNP to induce changes in human AASMC phenotype, genotype, metabolism, matrix remodeling, and biophysical/ biomechanical characteristics. Results so far attest to the dose-dependent benefits of SNP in this regard. Ongoing studies will help us identify the pathways that are involved in this process and the translational benefits of NO donors to restoring homeostasis in aneurysmal vessel walls.

Acknowledgements (Optional): : The authors acknowledge partial support from the National Science Foundation under grants 1927602 and 1337859.

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