Reconstruction Of The Mechanotransduction Networks On Vsmc Proliferation Induced By Cyclic Strain: Phosphoproteomic Based Analysis

It had been revealed that mechanical strain, especially the cyclic strain, plays an important role in vascular homeostasis maintenance and pathological mechanism of vascular remodeling. Hence, exploring the mechanotransduction network induced by cyclic strain, investigating the biological functions of underlying key molecules are of great importance to investigate the physiological as well as the pathological mechanism of cardiovascular diseases.Reversible protein phosphorylation, one of the important protein posttranscriptional modifications, is involved in regulating almost every aspect of life activities. However, only less than 10% proteins among the total undergo such reversible phosphorylation process, and they are hard to be revealed by classical proteomics analysis. To tackle these difficulties, phosphoproteomics approaches, consisting the stable isotope labelling by amino acid in cell culture(SILAC), TiO2 affinity chromatography and mass spectrum(MS), were used in our research to explore the mechanotransduction networks in VSMCs induced by cyclic strain.VSMCs isolated from rat arota were labelled by SILAC, then exposed to 10% physiological cyclic strain at 1.25 Hz for 0 min, 15 min, 30 min, 1 h and 6 h respectively. Combined with TiO2 affinity chromatography and MS, the temporal phosphoproteome profiles and phosphorylation sites were detected. Then, bioinformatics approaches such as DAVID, IPA analysis and fuzzy c-means clustering(FCM) were applied to analyze the dynamic patterns of differentially expressed phosphoproteins, main functions as well as possible signaling pathways. It indicated that Rho-associated coiled-coil containing protein kinase(ROCK) and protein kinase C(PKCs) family may involved in modulating VSMC functions induced by cyclic strain.In order to investigate the role of ROCK and protein kinase family in VSMC functions induced by cyclic strain, the protein expressions of ROCK1,p-PKCθ, p-PKCα/β, p-PKCζ/λ, p-PKCδ/θ, p-PKD/PKCμ Ser916, p-PKD/PKCμ Ser744/748 and p-Akt in VSMCs were detected by WB after exposed to 10%-1.25 Hz cyclic strain for 0 min(regarded as static), 15 min, 30 min, 1 h, 6 h, 12 h and 24 h respectively. Then RNA interference was applied to further investigate the role of ROCK1 in such modulating mechanism. Results showed that: 1) 10% physiological strain remarkably enhanced the viability of VSMCs; 2) the expression of ROCK1 was down-regulated upon the application of strain; 3) two activations characterized the expression profile of p-PKCθ during cyclic strain application, one in the short time-phase(15 min & 30 min), the other in the long time-phase(6 h & 12 h &24 h); while cyclic strain had no effect on PKCα/β, PKCζ/λ, PKCδ/θ; 4) the activations of p-PKD/PKCμ Ser744/748 and p-Akt induced by physiological cyclic strain were an immediate and short term effect, while the activation of PKD/PKCμ on Ser916 residue was a little time lagged compared with PKD/PKCμ Ser744/748; 5) A decrease in ROCK1 expression by using target siRNA transfection resulted in increased expressions of p-PKCθ, p-PKD/PKCμ Ser916 and p-PKD/PKCμ Ser744/748, and a decreased expression of p-Akt.In summary, by combining phosphoproteomics with bioinformatics and biological analysis, we demonstrated that physiological strain exerts a protective role to vascular system by enhancing the viability of VSMCs. ROCK1 may involve in those mechanotransduction networks by regulating the phosphorylation of PKCθ and Akt. Our study provides novel insights into investigating the molecular mechanism of vascular remodeling induced by mechanical cyclic strain, thus may contributing to better understanding of cardiovascular diseases.