Abstract
Endothelial proliferation, which is an important process in vascular homeostasis, can be regulated by the extracellular microenvironment. In this study we demonstrated that proliferation of endothelial cells (ECs) was enhanced on hydrogels with high stiffness (HSG, 21.5 kPa) in comparison to those with low stiffness (LSG, 1.72 kPa). ECs on HSG showed markedly prominent stress fibers and a higher RhoA activity than ECs on LSG. Blockade of RhoA attenuated stress fiber formation and proliferation of ECs on HSG, but had little effect on ECs on LSG; enhancement of RhoA had opposite effects. The phosphorylations of Src and Vav2, which are positive RhoA upstream effectors, were higher in ECs on HSG. The inhibition of Src/Vav2 attenuated the HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs on LSG. Septin 9 (SEPT9), the negative upstream effector for RhoA, was significantly higher in ECs on LSG. The inhibition of SEPT9 increased RhoA activation, Src/Vav2 phosphorylations, and EC proliferation on LSG, but showed minor effects on ECs on HSG. We further demonstrated that the inactivation of integrin αvβ3 caused an increase of SEPT9 expression in ECs on HSG to attenuate Src/Vav2 phosphorylations and inhibit RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation.
Highlights
Many pathological conditions, such as vessel injury or tumor formation, alter the extracellular microenvironment for cells and influence cellular functions including proliferation and migration
These results suggest that 21.5 kPa may have already reached the plateau for substrate stiffness-mediated Endothelial cells (ECs) proliferation
The results showed that the actin level was evenly distributed across the cell body of ECs on High Stiff-Gel 10 (HSG) (Fig. 1B right panel) and that a preferential distribution of actin in the peripheral cortical layer was observed in ECs on Low Stiff-Gel 5 (LSG) (Fig. 1B, right panel)
Summary
Many pathological conditions, such as vessel injury or tumor formation, alter the extracellular microenvironment for cells and influence cellular functions including proliferation and migration. EC spreading and focal adhesion formation are attenuated in ECs co-cultured with smooth muscle cells (SMCs, with the SMC layer serving as a soft substrate) and in ECs seeded on soft polyacrylamide gels in comparison to ECs on hard surfaces [9]. Such EC-SMC co-cultures serve as a model of the healthy vessel wall to inhibit the TNF-a-induced EC inflammation [10]. ECs derived from tumor stromas with high stiffness show distinct differences in structure, signaling, and function when compared with ECs from normal tissues [11]
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