Investigating the behavior of endothelial cells and monocytes under disturbed flow conditions within a microfluidic system

Cells are capable of sensing and responding to the mechanical forces exerted by their environment. Reception of physical stimuli is followed by transmission and conversion of the signal into biological activity in a process defined as mechanotransduction. Mechanical signals combine with biochemical signals to produce multiple responses within a cell. Cumulative regulation in response to mechanical cues by a population of cells in close proximity can maintain important tissue functions. Disturbance in physiological patterns of mechanical forces may lead to abnormalities and disease. Blood vessels are particularly regulated via mechanical stimulation. Endothelial cells that line arterial, venular and capillary inner walls are under continuous strain by the hemodynamic forces as a result of blood flow. Alterations in the mechanical signals received by endothelial cells are frequent. The endothelium provides a bridge and a barrier between the bloodstream and the underlying vascular tissue. Consequently, mechanical signaling at the endothelium has an influence on overall vascular maintenance; mostly regulating the level of vessel dilation via the production of nitric oxide. The most studied mechanical force on endothelial cells is shear stress, the parallel frictional force caused by the flow of a fluid. Studies on shear stress have revealed that it has a major role in maintaining endothelial barrier and in preventing vascular diseases. Hemodynamic shear stress have a regular pattern that can locally variate in case of a damaged vessel, or in sites of branching geometries such as bifurcations and curvatures. In the branching geometries, laminar blood flow is disrupted and separated into secondary flows of oscillatory or reversing behavior, creating low shear stress in the local area. Mechanobiology is the discipline that studies how mechanical forces can affect cellular functions. Studying vascular mechanobiology is particularly challenging in that it requires establishment of the three-dimensional vascular microenvironment and generation of fluid flow. Recently, microfluidic channels have come into use to enable creating such research environments. Culturing of endothelial cells in microdevices of unique channel geometries allows to study cell behavior under controlled shear environments. Atherosclerosis, formation of fatty plaques in arteries, draws much attention because it is a common disease that has been linked to local alterations in shear stress. The cytoskeleton is at the core of mechanotransduction. Elements of the cytoskeleton are connected to each other and to membrane proteins, providing the balance and transmission of tensional forces. The shearing force initially deforms the cell surface and activates membrane ion channels, followed by transmission of the signal by secondary messengers assisted by the cytoskeleton. Then the message is converted into biochemical activity and is carried to distant regions of the cell. Transduction of the signal activates intracellular signaling pathways to promote gene transcription or regulates cell adhesion interactions. Upon exposure to laminar shear stress, endothelial cells elongate and align their cytoskeleton in the direction of flow. Low shear stress has been shown to increase the expression of endothelial adhesion molecule ICAM-1 (intercellular adhesion molecule 1), promoting endothelial permeability to leukocytes. Studies on human aortas suggest that monocytes accumulate greatly at the endothelium in the outer walls of bifurcations that are susceptible to disturbed shear stress patterns. Monocytes are phagocytic innate immune cells that remove bacteria, viruses and toxic metabolites. Monocytes circulate in the bloodstream, but they enter tissues via diapedesis and differentiate into macrophages in order to participate in homeostasis or inflammation via phagocytosis and antigen presentation. Monocytes play a central role in long-term, tissue-damaging inflammatory conditions such as atherosclerosis. Atherosclerosis is a chronic inflammatory response to endothelial injury characterized by formation of fat-laden plaques in artery walls. Progression and perchance rupture of atherosclerotic plaques can lead to cardiovascular disease. The occurrence of atherosclerosis depends on the nature of local blood flow as well as the presence of biochemical risk factors. While high laminar shear stress in straight blood vessels is atheroprotective, low shear stress and oscillatory/reversing blood flow in vessel curvatures bring about atheroprone sites. In addition, the blood vessel becomes narrower as the atherosclerotic plaque grows, changing the local shear stress at the plaque site. The significance of physiological shear stress and the response of endothelial cells to alterations in shear stress have been well studied to date. However, no studies yet have analyzed the biological effects of disturbed flow on endothelial cells applying a model that can mimic the atherosclerotic plaque microenvironment with high-to-low alteration of shear stress. In this study, a unique design of cavitated microfluidic channel mimicking the atherosclerotic plaque geometry was used for the first time to study the effects of disturbed flow on HUVECs (Human Umbilical Vein Endothelial Cells). The first part of the study describes methods to obtain a confluent endothelial monolayer within straight and cavitated microfluidic channels and a method to apply shear stress on cells. In the preliminary studies, disturbed shear effects on endothelial F-actin network and ICAM-1 expression were investigated. Low-level disturbed shear stress was shown to cause F-actin accumulation at the cell cortex and F-actin reorganization in the direction of flow. This effect was higher in the narrower channel regions where shear stress was relatively high. The more random alignment of HUVECs in the low shear-harboring cavities may be related to the local disturbance of flow within the region. In addition, low-level disturbed shear stress was found to upregulate ICAM-1 mRNA expression, although a less increase in the expression was obtained when compared to TNFα activation. Taken together, disturbed shear stress at atheroprone regions may contribute to monocyte recruitment at the endothelium via upregulation of the endothelial adhesion molecule ICAM-1, driving the effect further with local presence of TNFα. Since this study involved only static cells as a reference, further studies may include application of laminar shear stress on endothelial cells in straight channels as an additional control. The individual effect of shear stress on monocyte behavior has not been studied to date. In this study, THP-1 monocytic cells were subjected to sequential high and low shear stress in cavitated channels at an average of 4 Pa as normal, 15 Pa as high or 45 Pa as very high shear stress and changes in F-actin levels were analyzed by fluorescence microscopy and flow cytometry. High level (15 Pa) disturbed shear stress was determined to increase F-actin content, whereas very high (45 Pa) level of disturbed shear stress was concluded to decrease the F-actin content in the long term in THP-1 monocytes. Furthermore, the effect of disturbed shear on F-actin in shorter time intervals may be assessed to fully characterize time-dependent changes in the F-actin.