FIG. 6.

Altered RhoA-related signaling in vSMCs when cultured on the different substrata. (A–C) RhoA upregulation, along with the upregulation of the Rho kinases, ROCK1 and ROCK2, the serine-threonine downstream effectors of RhoA, indicate that the activity of RhoA could be upregulated in the stiff ANFS potentially resulting in overly contractile vSMCs. This phenotype is reminiscent of hypertensive vSMCs and of asthmatic airway smooth muscle cells. (D) Western blots for RhoA and osteopontin (OPN) clearly indicated higher levels of RhoA and OPN for stiff ANFS. The order of the blots indicated by (1–4) is the same as in the other graphs, that is, (1) soft unpatterned, (2) soft ANFS, (3) stiff unpatterned, and (4) stiff ANFS. (E, F) LIM kinase 2 (LIMK2) and cofilin-2, both of which are downstream of RhoA, were significantly upregulated in vSMCs on stiff ANFS. Thus, given that the vSMCs on the stiff ANFS are longer (higher EFF), stain higher for F-actin stress fibers, express high levels of α-SMA and tropomyosin, and as seen in (G) have lowered levels of caveolin-1, it is probable that the phenotype of the vSMCs on the stiff ANFS is distinct from the others. (H) Profilin-2, which has been found to decrease invasiveness and migratory tendencies of cells, was higher in vSMCs cultured on ANFS. Statistically significant difference between nanopatterned and unpatterned substrata (p<0.05) for the same substratum stiffness is indicated with an asterisk. For all other substrata type combinations, the identical signage indicates statistically significant difference for that pair.