Shear Stress Modulation of Smooth Muscle Cell Marker Genes in 2-D and 3-D Depends on Mechanotransduction by Heparan Sulfate Proteoglycans and ERK1/2

During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms.Exposure to 8 dyn/cm(2) laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of alpha-smooth muscle actin (alpha-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH(2)O, approximately 0.05 dyn/cm(2), 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of alpha-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of alpha-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs).The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation

Cleavage of heparan sulfate glycosaminglycans (HS-GAGs) by heparinase.

<p>SMCs were grown on the plate for 2 days, and then incubated with 6.7 IU/L heparinase III (Hep) for 1 h followed by immunostaining for HS-GAGs. The surfaces of SMCs present abundant HS-GAGs (blue), which was successfully cleaved by heparinase III. Cell nuclei were stained by propidium iodide shown in red.</p

PD98059 and heparinase suppress both laminar flow and interstitial flow-induced ERK1/2 activation.

<p>Cells in 2-D or 3-D were pretreated with ERK1/2 inhibitor PD98059 (PD) or heparinase III (Hep) and then exposed to laminar flow or interstitial flow for 0 to 30 min. Cells were lysed and proteins were extracted for Western blotting. Gel panels were representative Western blots from three independent experiments, where similar results were found.</p

Effects of interstitial flow on SMC marker gene expression in 3-D.

<p>Interstitial flow attenuates expression of SM-MHC, smoothelin, and calponin, but promotes expression of α-SMA and SM22 in both SMCs and MFBs in 3-D. SMCs (A) and MFBs (B) in collagen I gels were exposed to interstitial fluid flow driven by 1 cmH₂O pressure differential (∼0.05 dyn/cm² shear stress <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012196#pone.0012196-Shi1" target="_blank">[5]</a>) for 6 h. Gene expression was analyzed by RT-qPCR and normalized to its own No-Flow control case. All the data are presented as mean ± SEM. * P<0.05 vs corresponding No-Flow control; n = 4–6.</p

Primer sequences for rat SMC marker genes.

<p>Primer sequences for rat SMC marker genes.</p