Substrate Stiffness Regulates Vsmc Phenotype And Extracellular Matrix Via Tgf-β Signaling Pathway

Cardiovascular disease has the highest fatality rate worldwide,and atherosclerosis has been proven to be its most common pathological feature.Atherosclerotic lesion is characterized by patchy subintimal thickening of the middle and large arteries,and the pathological process is accompanied by the changes of the stiffness of the blood vessel walls.Smooth muscle cell(SMC)is a major component that maintains the integrity of the blood vessel wall and angiotasis.The functional changes in vascular SMC plays an important role in vascular pathology,which is a typical sign of the initiation and development of atherosclerosis.Studies have shown that vascular SMC can sense physical signals such as substrate stiffness and respond to the stiffness variations through behavioral changes,and demonstrates significant difference between deteriorated and normal vessel.In recent years,tissue engineered blood vessel has become more important than ever in the treatment of cardiovascular diseases due to the limited sources of autogenous and allogeneic blood vessels.Most researches focused on the preparation method and the chemical composition.Medicines or growth factors were also commonly used to improve the performance of vascular tissue engineering materials.Few study has considered to modify the physical properties of the material such as surface mophorlogy and stiffness.However,latest researches have already demonstrated that the physical properties of the material has great impact on cell behaviors.The recent work in our laboratory has already shown that micro or nano patterns on material surface have exerted effects on cell morphology,cell-cell communication and stem cell differentiation.Therefore,it is reasonable to hypothesize that substrate stiffness can participate in vascular remodeling by regulating phenotypic transformation of SMC and expression of the extracellular matrix.In order to further validate this hypothesis,polyacrylamide gel(PA)was chosen to simulate the stiffness of blood vessels under different conditions.PA has been tested to have good biocompatibility and controllable surface modulus.SMC seeded onsubstrates with different stiffness were compared.The designed in vitro research system is as follows:(1)Polyacrylamide gels(PA)with stiffness of 1 kPa,40 kPa and 100kPa were prepared by adjusting the ratio of Acrylamide and Bis-acrylamide.(2)Arterial SMCs were simulated in vitro using human umbilical artery smooth muscle cells(HUASMC).Based on the above research system,this study focuses on whether the stiffness of the substrate can affect SMC cell phenotype and extracellular matrix.In addition,how the substrate stiffness affects SMC and the role of substrate stiffness in the process of vascular remodeling were investigated.The research results is concluded as follows:(1)PA substrates with different stiffness have no effect on the proliferation of SMC,but the cell morphology.On soft substrate(1 kPa),the cell spreading area and nuclear area were relatively smaller,only 2871μm~2 and 191μm~2.With the increase of substrate stiffness,the cell spreading area and nuclear area increased gradually.When the substrate stiffness was 100 kPa,the cell spreading area and the nucleus area reached4974μm~2 and 302μm~2,respectively.The cells spread out with tensive cytoskeleton network.(2)Further studies showed that substrate stiffness was closely related to the expression of phenotypic markers of the SMCs.The expression of synthetic phenotypic markers OPN and EREG decreased with the increase of stiffness,whereas the expression of the contractile markers SMTN and CNN increased with the increase of substrate stiffness.At the same time,both the gene and protein expression level of MMP-2 and FN1,which regulate extracellular matrix of SMC,showed a negative correlation with the substrate stiffness.In addition,the gene expressions of TGF-β,Smad4and MAPK,all related to TGF-βsignaling pathway,were also increased with the increase of substrate stiffness,especially Smad2/3,the main signal molecule of TGF-βsignaling pathway.The concentration of phosphorylated Smad2/3(p-Smad2/3)increased with the increase of substrate stiffness,suggesting that TGF-βsignaling pathway was activated by substrate stiffness and participate in the regulation of SMC function.(3)In order to further determine the effect of TGF-βsignaling pathway on the regulation of SMC phenotype and extracellular matrix in response to substrate stiffness,RepSox,a TGF-βreceptor inhibitor was used to block the TGF-βsignaling pathway in SMC.The expression of related genes and proteins were examined by qPCR,Western Blot and immunofluorescence staining.The results showed that the concentration of p-Smad2/3 in SMC did not change with the change of substrate stiffness when TGF-βsignaling pathway was inhibited.The effects of substrate stiffness on SMC phenotypic markers SMTN,EREG,CNN,OPN and extracellular matrices related proteins MMP-2 and FN1 also diminished.These results suggested that TGF-βsignaling pathway was involved in the regulation of SMC phenotype and extracellular matrix.In summary,this study demonstrated that SMC sensed the change in substrate stiffness and responsed.By activating of TGF-βsignaling pathway,along with the phenotype switch,SMC could alsoregulate its extracellular matrix to counterplay the change of stiffness and participate in the remodeling of blood vessels.The results of this study will provide new insights for the design of tissue engineering vessle materials.It also provides a useful in vitro model for atherosclerosis related mechanism study and drug screening.