Morphology Modified HUVEC Regulated The Inflammatory Response Of THP-1 Through Exosomes Containing MicroRNA

Atherosclerosis is the most serious cardiovascular disease affecting people's health and quality of life.Vascular inflammation participates in the atherosclerosis pathological process,and vascular cells also involve in the process,including endothelial cells(ECs)and smooth muscle cells.Studies have found that the anti-inflammatory microRNAs from endothelial cells(EC)can inhibit the activation of monocytes through exosome,which can affect the inflammation in blood vessels.Up to date,vascular tissue engineering is one of the most promising treatments for vascular diseases such as atherosclerosis.As far as the vascular materials was concerned,major researches focus is on the optimization of the chemical components of the materials or the addition of various growth factors.Recent studies suggested that physical factors,such as surface topology,stiffness,viscosity,elasticity,shear stress and so on,play an important role in regulating cell behaviors.Our previous research has demonstrated that cell morphology was regulated by the surface micro-topology.Even the directed differentiation of the stem cells could be modulated by material surface topology.Since the regulatory effect of endothelial cells on vascular inflammation is a very important process,how material surface micro-topology may affect this process is a very important question in designing tissue engineering materials,it is of great value and interest to clarify this issue.The hypothesis of this project is that the microtopography of biomaterials can regulate the communication between the ECs and the immune cells to affect the inflammatory response.In order to verify our hypothesis,we designed the following research systems:(1)A commonly used artificial blood vessel material,polydimethylsiloxane(PDMS),was prepared as cell culture substrate.The surface of the membrane has parallel microgrooves(microgroove,G).Flat PDMS membranes(flat,F)without any pattern was used as a control.(2)Human umbilical vein endothelial cells(HUVEC)was used as an in vitro artery EC model.(3)The THP-1 cell line activated by lipopolysaccharide(LPS)was used to represent the inflammatory cells around the early sclerosis plaque.THP-1 was co-cultured with HUVEC to study the regulatory relationship between the two cells.On the above research systems,we studied whether the parallel microgroove structure of the PDMS can affect HUVEC exosomes secretion and gene expression.Moreover,whether these changes of HUVEC can pass on to the activated monocytes,and further affecting their inflammatory reaction.The results are as follows:(1)As the basis of my research,the morphology of HUVEC cultured on the PDMS membrane with 10 ?m wide and 3 ?m deep groove structure was changed significantly.The cells grew along the direction that parallel to the microgrooved structure and showed much more elongated shape compared with the polygonal morphology of the cells cultured on the flat PDMS membrane.We also found that miRNAs level was affected,especially inflammation associated miRNAs,among which the expression levels of miR-10 a,miR-21,miR-146 a and miR-181 b were upregulated significantly(P<0.001),and the corresponding target miRNA gene expression were suppressed(P<0.05).(2)Further study revealed that morphology modified HUVEC could secrete more exosome compared with the HUVEC cultured on the flat surface(P<0.05).At the same time,we analysed the anti-inflammation microRNAs in the exosomes from HUVEC,and found the level of miR-10 a was significantly increased in the exosomes(P< 0.01).(3)In order to explore the biological significance of the up-regulation of exosomal miR-10 a,when LPS-activated THP-1 co-cultured with morphology modified HUVEC,we also found that miR-10 a was significantly increased(P<0.05)in THP-1.In agree with that,miR-10 a target gene MAP3K7 in THP-1 was down-regulated(P<0.01).Meanwhile,the expression of the inflammatory cytokines secreted by activated THP-1,such as IL-6,IL-1? and TNF-? were significantly decreased(P<0.001).Therefore,morphology modified HUVEC showed more anti-inflammatory effect on activated THP-1.In addition,the expression of adhesion molecules(VCAM-1,ICAM-1,E-selectin)and chemokine MCP-1 in HUVECs was significantly decreased,and the adhesion of THP-1 was reduced in accordance.Moreover,the expression of immune regulation genes such as TGF-?1,IL-10 and MRC1 were upregulated,which demonstrated that morphology modified HUVEC may also involved in the transformation of monocytes to macrophages,and directing the polarization of macrophages towards M2 phenotype.(4)To further exclude the possibility that in the co-culture system,THP-1 can also secret exosomes or other secreted proteins to interact with HUVEC,we used the trans-medium system to verify the above conclusions.The results were consistent with that from the co-culture system,that miR-10 a in THP-1 was significantly upregulated(P<0.05).The above results indicated that HUVEC can inhibit the inflammatory response of THP-1 through the paracrine pathway and microRNAs.In order to further verify that the exosomes from HUVEC supernatant was the main factor acting on THP-1,an exocytosis inhibitor was added to HUVEC in the trans-medium system to inhibit the production of the exosomes.The FACS results showed that there was a significant reduction in the number of exosomes(P<0.05)in both surfaces,confirming the treatment effect of the inhibitor.With the reduction of the exosomes quantity,miR-10 a level was significantly down-regulated in both exosomes and THP-1(P<0.001),suggesting that exosomes played an important role in the communication of HUVEC with THP-1.In summary,we demonstrated that the surface microtopography of biomaterial could regulate vascular inflammation.As a physical variable of the material,surface microtopography information of the PDMS membrane passed to HUVEC and inflammatory effector cells by exosomes.The microRNA carried in the exosomes regulated the inflammatory response.This study provides new insights to the pathogenesis of As,as well as new ideas for the development and optimization of biomedical materials.