Mikrofluidiksystem zur Charakterisierung der endothelialen Permeabilität und der transendothelialen Leukozytentransmigration
Pharmakologie
Zusammenfassung der Projektergebnisse
The project was established to investigate permeability regulation of confluent pulmonary endothelial cells and transendothelial migration of leucocytes spatially and time-resolved under physiological flow conditions in a microfluidic system. A combination of optical and electrical impedance spectroscopy (EIS) based measurements was used to monitor the confluent endothelial cell layers. With this system, we were able to study the relevance and function of metalloproteinases and adhesion molecules in regulation of endothelial cell survival and transmigration. These molecules are potential target molecules for new therapeutic approaches and therefore an easy and fast electrical measurement of their functions on endothelial cells can accelerate drug development in inflammation research. Four types of microfluidic cell cultivation devices were developed as two chamber systems separated by a membrane. The version used at last consists of ibidi® sticky-Slide I luer slides with a perforated polyimide membrane in-between. The fabrication of perforated polyimide membranes with microelectrodes on both sides was really challenging. The whole fabrication process comprised 56 process steps and requires typically 13 processing days. The difficulties in fabrication were not foreseeable in the project proposal. At least, only 20 polyimide membranes with integrated electrodes were successfully fabricated during the whole project. An impedance spectrometer was granted for the project. In corporation with the selling company, it took about 2 years intensive troubleshooting to eliminate different failures in soft- and hardware made by the company. Cell culture conditions were first tested using the commercial ibidi® system consisting of a µ-slide perfused via the ibidi® pump system. Human umbilical vein endothelial cells (HUVEC) were cultured under physiological flow conditions acquired an elongated shape and aligned in the direction of flow. The regulation of inflammatory mediators, adhesion molecules and proteases by flow was further analyzed as described below. First EIS measurements of endothelial cells cultured under flow conditions were performed. HUVECs were seeded into the upper channel and cell attachment was monitored for 2.5 h by EIS demonstrating that it was possible to culture and detect HUVECs on the polyimide membrane. The developed setup can also be used to measure changes in morphology and permeability of the endothelial cell layer by electrical impedance spectroscopy. To study transcriptional responses, protein expression, leukocyte adhesion and endothelial survival/apoptosis, we focused on the transmembrane chemokine fractalkine (also termed CX3CL1), which is known to promote atherosclerosis development in the carotid and aorta. We noted that low shear stress typically seen at atherosclerosis prone regions promotes the induction of endothelial CX3CL1 and thereby induces monocytic cell recruitment, whereas physiological shear stress counteracts this inflammatory activation and suppresses monocytic cell adhesion to endothelial cells. We next studied proteinases of the ADAM family that have been implicated in the regulation of CX3CL1 and other surface molecule on endothelial cells. Transcriptomic and proteomic analysis indicated that ADAM15 within this family is considerably upregulated when primary endothelial cells are exposed to shear stress. We found that this induction is mediated by the transcription factor KLF2 and that ADAM15 induction by flow contributes to endothelial survival whereas the absence of ADAM15 at low shear stress or static conditions led to increased endothelial damage. Thus, endothelial measurements on CX3CL1-mediated leukocyte adhesion and ADAM15 mediated cell survival was successful with the established ibidi® system. It was our aim to also detect ADAM15-mediated changes of endothelial permeability under flow conditions. The developed microfluidic setup with the gold-electrode embedded polyimide membrane can be used to detect profound permeability changes. However, the microfluidic device was not yet optimized in fabrication for reproducibly and sensitively detecting subtle ADAM15 dependent changes in permeability. In addition, successful seeding growth and stimulation of endothelial cell cultures in the device was also challenging at the beginning of the project and led to several redesigns of the layout. In summary, the concept of characterization the influence of shear flow on the behaviour of confluent cell layers by microfluidic devices is possible but needs further improvements for increasing the sensitivity.
Projektbezogene Publikationen (Auswahl)
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(2019) The metalloproteinase ADAM15 is upregulated by shear stress and promotes survival of endothelial cells. Journal of molecular and cellular cardiology 134 51–61
Babendreyer, Aaron; Molls, Lisa; Simons, Indra M.; Dreymueller, Daniela; Biller, Kristina; Jahr, Holger; Denecke, Bernd; Boon, Reinier A.; Bette, Sebastian; Schnakenberg, Uwe; Ludwig, Andreas
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Shear Stress Counteracts Endothelial CX3CL1 Induction and Monocytic Cell Adhesion. Mediators Inflamm. 2017:1515389
Babendreyer A, Molls L, Dreymueller D, Uhlig S, Ludwig A
