Ingenium 2018
into smooth muscle cells (SMCs) with both behaviors being linked to elastogenesis [10]. Additionally, previous work conducted by our lab has shown that periadventitial delivery of adipose-derived MSCs preserves elastic fibers in a murine elastase-induced abdominal aortic aneurysm model [11]. This finding opens a new possible avenue for non-surgical treatment of elastic fiber disorders. This study sought evidence for a paracrine mechanism in MSC-mediated preservation of aortic elastin. In short, we investigated whether the secreted factors from MSCs could induce that elastin-preservative effect on their own. We utilized a three-dimensional in vitro fibrin gel culture to investigate the effect of MSC secreted factors (MSCSF) on SMC elastic fiber formation. We hypothesized that application of MSCSF would upregulate elastic fiber components (tropoelastin, fibrillin-1, and fibulin-5) in healthy aortic SMCs, yielding a higher quantity of mature elastic fibers.
2. Methods
every 48-72 hours. All constructs were cultured in the presence of a lysinemimicking fibrinolysis inhibitor, aminocaproic acid (ACA) at 15 mM. “MSCSF” treatment refers to a 1:1 ratio of SMC growth-supplemented medium and collected MSC secreted factors, plus 15mM ACA. “Non-Treated (NT)” refers to SMC growth-supplemented medium, plus 15mM ACA. 2.3 Real-Time Quantitative Polymerase Chain Reaction (qPCR) Constructs (NT n = 2, MSCSF n = 2) were frozen in liquid nitrogen and then crushed. RNA was extracted using an RNAeasy Mini Kit (Qiagen). cDNA was synthesized by priming for 5 minutes at 25 °C, reverse transcription for 20 minutes at 46 °C, and reverse transcriptase inactivation for 1 minute at 95 °C. Tropoelastin, fibulin-4, fibulin-5, and lysyl oxidase (LOX) were amplified using purchased primers (Sigma, Table 1). GAPDH was the reference gene to which all samples were normalized.
2.1 Cell Culture Human aortic SMCs were purchased from ATCC (adult human aortic SMC, #PCS-100-012), and cultured in Cell Applications’ Human Smooth Muscle Cell Growth Medium (#311K-500). MSCs were collected from the adipose tissue of nondiabetic, non-smoking female patients under the age of 45 (provided by Dr. J. Peter Rubin, Department of Plastic Surgery, UPMC). MSC were cultured using a 50/50 mixture of DMEM (ThermoFisher Scientific, #12100046) and DMEM + F12 media (ThermoFisher Scientific, #12500096), supplemented with 10% fetal bovine serium, 1% fungizone, 1% penicillin/streptomycin, and 0.2mM dexamethasone. To collect conditioned media, MSC were grown to 60% confluence, given fresh media, which was collected after 24-72 hours of conditioning. All MSC were used between passages 0 and 2. Conditioned media was stored at -80 °C, and thawed immediately before treatments. 2.2 Fibrin-Based Tissue Constructs Three-dimensional fibrin-based tissue constructs were prepared as previously described from a solution of fibrinogen (33.3 mg/ml, Sigma), thrombin (25 U/ml, Sigma), and the cells of interest (1x105 cells/construct) [12]. Gelation of constructs occurred within sterile 5/16” (7.94mm) heat-stamped circles on tissue culture plastic. Constructs were cultured for 20 days (37 °C, 5% CO2) under each treatment condition, with media changes
Table 1. Forward and reverse primers for targeted genes in qPCR. All primers are reported 5' to 3'.
2.4 Indirect Immunofluorescence Constructs (NT n = 8, MSCSF n = 8) were fixed using ice-cold methanol and blocked in a 0.1% Tween-20 (Sigma) and 0.1% cold water fish gelatin solution (Aurion, #900-033). Target antigens were elastin (polyclonal rabbit anti-human, Elastin Products Company, #PR533, 1:1000), fibrillin-1 (rabbit polyclonal antiserum against human fibrillin-1, #PaB9543, 1:100), and fibulin-5 (monoclonal mouse anti-human, R&D Systems #MAB3095, 1:1000). Alexa Fluor secondary antibodies were targeted to elastin (mouse anti-rabbit 546) and the target matricellular protein (rabbit anti-mouse 488) (ThermoFisher). Nuclei were stained with Hoechst dye (Sigma). Mounted constructs were imaged as z-stacks using a Zeiss LSM 700 confocal microscope with a 20x oil-immersion lens. Final images are brightest pixel projections of z-stacks as produced by ImageJ (NIH) software. 2.5 Insoluble Elastin Ninhydrin Assay Insoluble elastin content as a fraction of total protein in each construct was determined (NT n = 8, MSCSF n
Undergraduate Research at the Swanson School of Engineering
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