Investigation on the effects of mechanical forces on endothelial and monocytic cell behaviour by using microfluidic systems

In human physiology, it is known that cells can sense not only biochemical signals but also physical factors which employee in tissue functionality such as mechanical forces, geometry and matrix flexibility. Mechanobiology is the field of these physical factors function basically through mechanotransduction process. The mechanotransduction is a physiological process that is needed to sense mechanical forces and the conversion of these forces into electrical and biochemical signals. For instance, tensile strength, compression and shear stress, are the most important mechanical stimulations which have influence on cells in body. These mechanical stimulators playing major role on regulation of cellular functions like gene expression, protein synthesis, cell growth, differentiation and death which are necessary for providing the maintenance of tissue balance by triggering different electrophysiological and biochemical responses. Traction forces formed by sensing extracellular environment and responses to this environment can affect many biological processes like wound healing, angiogenesis and metastasis at cell level. Therefore, characterization of these mechanical forces in terms of magnitude and direction could be meaningful in pretending and understanding the physiological and pathological events at tissue and organ level. In tissue and organ level, shear stress which is described as the friction force per unit area in blood has tremendous importance in terms of evolution and physiology over the endothelial cells covering the inner surface of vascular network to maintain the physiological conditions. This shear stress is related to flow rate gradient on the cross section of a tube. While velocity is maximum at center, it is zero near to the inner surface. It is known that the biological responses to shear stress takes a significant part of preventing vascular diseases. Due to non-pulsatile characteristic of venous system, shear stress levels are low and between 0.076-0.76 Pa. In arterial system, flow is laminar and pulsatile with high shear stress levels that are between 1-7 Pa for large arteries and between 2-6 Pa for arterioles. Studying vascular cell biology in laboratory is difficult in terms of mimicking the dynamics in vivo environment and 3D structures. Work done by in vitro is achieved by exposing the endothelial cells into shear force in flow chambers composed of parallel plates. Besides, thanks to the devices produced by microfluidic technology which can be used instead of huge systems such as parallel plate or cone plate flow chambers, the mechanical forces, especially shear stress can be studied in in vitro environment. Because of being cheap, biocompatible and transparent, poly(dimethylsiloxane) is used in production of microfluidic devices which is used usually in cell biology studies. There are some advantages of microfluidic devices produced by poly(dimethylsiloxane) like using small amount of sample or agents, providing cell culture environment with stable liquid flow compared to traditional methods used in cell culture applications and analysis. Also, microfluidic devices are made of transparent materials, have suitability to fluorescence microscope or spectroscopy studies which enable monitoring cell behavior under long-term and high amplification conditions. In this thesis, the microchannels were designed as cavitated to mimic the environment of the atherosclerotic plaque where the shear stress and pressure difference occur depending on the diameter of the vessels and fabricated using MEMS techniques.. HUVEC is endothelial cells which is lining the inner surface of the wall seeded in the straight and cavitated microchannels were subjected to shear stress at 0.25 and 0.75 Pa, separately, for 4 hours by the syringe pump. Also, there are two subgroups as TNF--treated and untreated. The change of F-actin level in HUVEC was investigated under fluorescence microscope and fluorescence intensity of F-actin protein level was examined by Image J 1.50i programme. According to the results, there is a significant increase at 0.25 Pa in straight microchannel group with respect to control counterpart for untreated group. There is a significant decrease at 0.75 Pa groups compared to 0.25 Pa groups but MFI increased only in 0.25 Pa straight microchannel compared to control. Overall, the MFI of F-actin at 0.25 Pa is significantly higher than 0.75 Pa for TNF--untreated group. In TNF--treated group, there is not any significant difference between experimental groups. The change of ICAM-1 mRNA levels in HUVEC cells was investigated by qRT-PCR. For untreated group, there is significant increase at 0.25 Pa in cavitated group. The mRNA expression level at straight groups are lower than cavitated groups. For TNF--treated group, the mRNA expression level at 0.25 Pa is higher than 0.75 Pa for cavitated group. There is a significant difference between straight and cavitated groups at 0.25 Pa. The mRNA expression level at 0.25 Pa cavitated group is higher than control. F-actin synthesis and ICAM-1 mRNA levels have also been investigated in monocyte cells as examined in endothelial cells due to the contribution of monocytes to the process of inflammation in atherosclerotic plaque regions. In this direction, THP-1 cells were exposed to 4, 15 and 45 Pa shear stress, for 0.5 and 3 hours, separately. In addition, since TNF- is known to enhance the synthesis of F-actin polymer and ICAM-1 gene expression, experimental groups were divided into two subgroups, treated and not treated with TNF-. F-actin synthesis in Phalloidin labeled THP-1 cells was examined by fluorescence microscopy and fluorescence intensity was analyzed by Image J 1.50i program. According to the mean fluorescence intensity, F-actin synthesis was observed significantly increased at 4 Pa and 45 Pa shear stresses after 0.5 h, while the greatest increase was observed at 15 Pa. As the duration of exposure increased, a decrease in F-actin polymerization was observed at 45 Pa. Since the increase in F-actin synthesis was seen at a maximum 15 Pa shear stress, the determination of ICAM-1 mRNA level in THP-1 cells was only examined by qRT-PCR at 15 Pa. According to the results, about two-fold increase was observed in the experimental group compared to the control group. The releasing level of IL-8 chemokine molecule from THP-1 cells exposed to shear stress which secreted for leukocyte recruitment from monocyte and endothelial cells in atherosclerotic regions was examined. For this reason, the level of IL-8 in THP-1 cells were exposed 4, 15 and 45 Pa shear stress for 3 hours was examined by ELISA. According to the fluorescence intensity results, a decrease in the experimental groups was observed compared to the control group. Except the control group, the highest level was observed at 15 Pa and the lowest level at 45 Pa. Hoescht 33342 fluorescently labeled THP-1 cells were exposed to 15 Pa shear stress for 0.5 and 3 hours, respectively, were added to the HUVEC cells seeded in the wells and incubated for 1 hour, the florescence images were taken after incubation to investigate how the shear stresses affected the adhesion between the endothelial and monocyte cells. The number of cells adhered to HUVEC cells was analysed with Image J 1.50i programme. According to the results, as the duration of exposure to shear stress increases, the degree of adhesion decreases. Data obtained from these experiments will enable diagnostic, treatment and new medicine designs by revealing the targeted molecular mechanism definitely in atherosclerosis treatment within the scientific area by the future. Furthermore, examination of the changes in the molecules involved in sheared cells by microfluidic systems will greatly contribute to the work to be done in the field of nanotechnology.