5‐Fluorouracil Induces Vascular Calcification in Rat Aortic Smooth Muscle Cells

Abstract

Background 5-Fluorouracil (5-FU) is a commonly prescribed chemotherapy used to treat malignancies of the gastrointestinal tract, breast, and head/neck. Despite its high clinical efficacy in treating cancer, it is associated with late occurring cardiotoxicity, specifically increases in arterial stiffness along with a heightened risk for developing heart failure and cardiovascular disease. Because increases in arterial stiffness and vessel calcification can occur concurrently and are associated with cardiovascular disease outcomes, we determined if 5-FU treated aortic smooth muscle cells adopted a calcified phenotype. We hypothesized 1) aortic smooth muscle cells resected from 5-FU treated rats would have a faster calcification onset when challenged with inorganic phosphate media and 2) a single in vitro dose of 5-FU in healthy cells would result in a calcified phenotype. Methods Primary aortic smooth muscle cell culture (ASMC) was performed via resection of rat thoracic aorta from 6-month-old Sprague Dawley rats (n= 8) that were treated with a clinically relevant continuous infusion of 5-FU (50mg/kg and 270mg/kg) or saline equivalent volume. After treatment and resection, ASMC were cultured using explant methods until cells were identifiable via smooth muscle actin staining. Cells derived from each group were cultured with normal media (NM) or media including a pathological concentration (3.2 mmol) of inorganic phosphate media (PI) for 5, 8, or 10 days to determine differences in calcification onset between conditions. In a second experiment, healthy rat aortic smooth muscle cells were incubated in NM or media containing 7x10-3 5-FU (NM + 5-FU) to determine if a single dose, in vitro, directly results in calcification. Calcification was quantified using an Alizarin Red staining assay and colorimetric calcium assay. Data are expressed as Alizarin Red Stain concentration (mM) and total calcium (mg/dL). Results In experiment 1, 5-FU+PI presented with significantly higher concentration of Alizarin Red at days 8 (.043 vs .031; p=.048) and 10 (.068 vs .017mM; p<0.0001) compared to 5-FU+NM whereas no differences were found between CON+PI and CON+NM at any timepoint (all p>0.05). These data were confirmed via Calcium assay in both 5-FU (.346 vs.235 mg/dl; p =0.0039) and CON (.293 vs .269 mg/dl; p=0.69) at the 8-day timepoint. Experiment 2 indicated that healthy rat aortic smooth muscle cells treated directly with 5-FU for 72 h expressed significantly greater calcium absorbance compared to CON (5-FU = .3347 mg/dl, CON = .2362 mg/dl; p=0.02). Conclusion After treatment with 5-FU, ASMCs from rat thoracic aorta explants are prone to a calcification phenotype compared to saline control treated cells. This predisposition with treatment with 5-FU is an ongoing investigation into the possible molecular mechanisms involved in oxidative stress signaling.