Abstract 19981: Aortic Adventitial Fibroblast Sensitivity to Mitogen Activated Protein Kinase Inhibitors Depends on Substrate Stiffness
Introduction: Arterial stiffness changes due to pathogenic remodeling or vessel aging alter the mechanical environment of local adventitial cells; however, the effects of altered stiffness on adventitial fibroblasts (AF) and the mechanisms by which AFs respond to stiffness are not well understood. Here, an in vitro hydrogel model was used to examine the effects of matrix stiffness on AF phenotype and cytokine production during selective blockade of mitogen activated protein kinase (MAPK) pathways linked with matrix signaling.
Hypothesis: The MAPK pathways regulating AF phenotype and cytokine secretion vary depending on substrate modulus.
Methods: Human aortic AFs (53 yo male donor) were grown in polyethylene glycol (PEG) hydrogels at 2x105 cells/mL. The MAPK pathways, ERK, p38, and JNK, were inhibited by 10 μM PD98059, SB203580, or SP600125, respectively. Viability, proliferation, α-smooth muscle actin expression (αSMA; fluorescence), MCP1, IL6, VEGF-A, and MMP2 secretion (ELISA), and monocyte recruitment (Boyden chamber) were evaluated. Statistical significance (p<0.05; n=3) was assessed by ANOVA with Tukey-HSD post hoc test.
Results: AFs grown in 5, 7.5, or 10wt% PEG hydrogels, which exhibited stiffness of 0.4, 1.1, and 3.2 kPa, respectively, remained >90% viable. In 5% gels, AFs had higher proliferation rates, elevated MCP1 secretion, and significantly enhanced monocyte recruitment. Significantly more AFs were αSMA positive in 7.5% gels, indicating myofibroblast development. AFs in 10% gels had low proliferation rates but produced high levels of IL6 and VEGF-A. Importantly, the MAPK pathways controlling cytokine production depended on matrix stiffness. For example, ERK and p38 pathways were involved in MCP1 elevation in 5% gels, but were not used in MCP1 production in 10% gels.
Conclusions: Varying hydrogel modulus substantially altered AF phenotype and sensitivity to MAPK pathway blockade. These data indicate that complex cell regulatory changes may occur with altered tissue stiffness and suggest that therapeutics affecting MAPK pathways may have altered effects on AFs depending on arterial stiffness. Further studies resolving relationships between MAPK, matrix stiffness, and cell behavior as they impact arterial status are needed.
Author Disclosures: R.A. Scott: None. P.M. Kharkar: None. K.L. Kiick: None. R.E. Akins: None.
- © 2016 by American Heart Association, Inc.