Matrix Stiffness Regulates Endothelial Cell Inflammation And Adherens Junctions Through PIEZO1

Atherosclerosis(AS)is the major cause of cardiovascular diseases,which is a severe threat to human health and life quality.In the early stage of atherosclerosis,lipoproteins accumulate under the endothelium,and endothelial cells(ECs)are activated to show an inflammatory response,which induces monocytes in the blood circulation to adhere to it and enter the tissue through the vascular endothelium.As the inflammatory response intensifies,the cell-cell junctions among the ECs are weakened,and monocytes are more likely to enter the subendothelial area.Although studies have shown that the lipoproteins and their modifications accumulating under the endothelium can activate ECs,but the accumulation of lipoproteins also softens the blood vessels.Therefore,this study hypothesizes that the decrease in blood vessel stiffness can directly activate ECs,causing them to undergo an inflammatory response,and further cause damage to the cell-cell junction among the ECs.In order to verify this hypothesis,we prepared polyacrylamide hydrogels with the stiffness of 1 kPa and 20 kPa as an in vitro culture system for ECs.One kPa represents the vascular substrate with lipid accumulation,and 20 kPa represents healthy blood vessel stiffness.Human umbilical vein endothelial cells(HUVECs)were used as the representative of ECs.The ECs were cultured to reach confluent before the inflammatory response of ECs was analyzed.We found that on the 1 kPa substrate,the m RNA and protein expression levels of the adhesion molecules,VCAM1 and ICAM1,increased significantly,and the amount of the monocytes adhered to ECs also increased significantly.In addition,compared to the ECs on the 20 kPa substrate,the cytoplasmic IκBα level decreased and the NF-κB nuclear translocation increased in ECs on 1 kPa substrate.These results indicate that1 kPa substrate could directly induce inflammatory responses in ECs.As for the study of the AJs among ECs,immunostaining of VE-cadherin and β-catenin showed AJs displayed punctuated distribution between ECs in the endothelial monolayer on 1 kPa substrate.Although the protein levels of VE-cadherin in the ECs cultured on 1 and 20 kPa PA hydrogels did not show significant difference,the protein levels of β-catenin demonstrated a significant decrease in ECs on soft substrate.In short,soft substrate could lead to disrupted AJs between ECs.On this basis,we further explored the molecular mechanism of the regulation of substrate stiffness on ECs inflammatory response.PIEZO1 is a newly identified mechano-transduction ion channel.To explore whether it is involved in regulating the inflammatory response of ECs induced by substrate stiffness,we examined its expression level and activity in ECs on different substrate stiffness.The results showed that the protein level of PIEZO1 in ECs on 1 kPa substrate was significantly higher than that on 20 kPa.By quantitation of ATP in the ECs culture supernatant,we found that 1kPa matrix increased the PIEZO1 activity on the cell membrane of ECs compared to 20 kPa matrix.Besides,by knocking down PIEZO1 using PIEZO1 specific si RNA,cytoplasmic IκBα protein in ECs on 1 kPa matrix increased,while the protein level of VCAM1 and the amount of the adhered monocytes decreased.These results suggested that the inflammatory response of ECs induced by soft substrate stiffness was mediated by PIEZO1.Similarly,after treated with si PIEZO1,β-catenin protein level in ECs cultured on the 1 kPa substrate increased to a level similar to that in ECs on the 20 kPa substrate.This result showed that PIEZO1 was also involved in the regulation of AJs between ECs induced by soft substrate.In summary,this study found for the first time that PIEZO1 can participate in the regulation of ECs inflammatory response and impaired AJs between ECs induced by soft substrate stiffness.These findings further clarify the mechanism of AS and can provide ideas for the treatment of AS.