Deciphering The Effect Of Nanofiber Alignment Of Electrospinning Material On Huvecs Exosomal Micrornas

In the tissue engineering research,it has been found that the behaviors of the cells seed on the material could be simply regulated by physical cues.In our research group,some preliminary studies have revealed that human umbilical vein endothelial cells(HUVECs)seeded on L-polylactide(PLLA)electrospinning material displayed distinct morphology while the fiber alignment was different.Cells seeded on the aligned fibrous membrane showed a narrow and elongated shape,and the stress fibers,especially actin filaments of the cytoskeleton were parallel to the direction of the nano fibers.In the contrast,cells seed on the random electrospinning membrane showed a regular cobble-stone shape which was similar to those cultured on flat culture plates.Therefore,is it possible that the alignment of nanofiber have further effect on the other physiological functions of the cells.Exosomes are one kind of the extracellular vesicles featuring a diameter of 30-100 nm,carrying abundant mRNA,microRNA(miRNA)and diverse proteins,many of these inclusions enter into the target cells and exert their effects after the fusion of vesicle membrane and cytomembrane.Among them,miRNAs are a class of endogenous,non-coding single-stranded RNA molecules which are 20-24 nt in length,participating regulation of post-transcriptional gene expression in plants and animals.For further study of whether the change of cell morphology can affect the exosome associated signal pathway,we prepared PLLA electrospinning membrane composed of either random or aligned nano fibers and seeded HUVECs on them.Systemetical researches,including intracellular miRNA,target genes in HUVECs,exosome secretion,exosomal miRNA,the corresponding target genes in smooth muscle cells(SMCs)were carried out.The results showed that aligned fiberous membrane could affect a quite large number of miRNAs and their target mRNAs in HUVECs,and increased the quantity of the exosomes secreted and the enclosed miRNAs as well.In particular,the upregulation of miR-143/145 was very consistant in both the HUVECs and the exosomes,which was also affecting the expression levels of their target gene Elk1 and Klf4,respectively.Interestingly,the expression of differentiation related genes-SMA and MYH-11 in the SMCs adjacent to the HUVECs were increased accordingly.In order to confirm the observation that HUVECs transfer the morphology changing signal to SMCs through exosomes,we applied a co-culture system and conditioned medium treatment system in parallel to compare the miRNA levels.The results turned out to be similar in these two different culture systems.In addition,a specific N-Smase inhibitor was used to block the formation of exosomes.We found that aligned surface could not increase exosome secretion any more.As a consequence,miR-143/145 level remained the same,and the expressions of differentiation related genes in SMCs were no longer affected.These results demonstrated that the signal of HUVECs' morphology changes caused by aligned surface of material was indeed transfer to SMCs through exosomes.In summary,this study reveals that HUVECs can sense and respond to the topological signal from the electrospinning membrane,and send out the signal downstream to SMCs via exosomal miRNAs.Especially,this study is the first tissue-engineered blood vessel related research which targeting exosome.The experimental results provided in this thesis offered useful information of the effect of biomaterial topography on cell behaviors,and theoretical basis for optimizing the artificial blood vessel by changing the surface structure of the scaffold material.