Chemie | Biochemie | Medizin
Sebastian Bolli, 2003 | Oberwil, BL
Smooth muscle cells determine the blood pressure by contraction or relaxation. The contraction or relaxation is crucially regulated by vasoactive stimuli which also play a key role in different forms of hypertension and hypotension. In this work we developed a new approach to better classify in vivo vasoactive stimuli by conducting in vitro experiments on a cellular level. We measured the effect of the stimuli on human pulmonary arterial smooth muscle cells using impedance measurements and optical interference spectroscopy. With our impedance measurements we were able to show that endothelin-1, thromboxane analog U46619, serotonin and histamine resulted in a drop in the impedance. We assume that these stimuli act as constrictors. In the case of endothelin-1, thromboxane, and histamine this was confirmed with optical interference spectroscopy. Adrenaline, spermine NONOate and riociguat led to a peak in impedance and therefore an assumed vasodilation. The IP agonist iloprost appeared to constrict the cells in the impedance measurements but showed a cell relaxation in optical interference spectroscopy. We conclude that the combination of the two fundamentally different analytical methods is a powerful approach for the classification of vasoactive stimuli in vitro.
Smooth muscle cells line the lumen of blood vessels, and by contracting or relaxing regulate blood pressure. The interplay between contraction and relaxation is crucially regulated by vasoactive stimuli. To date multiple vasoactive stimuli are known, nine were chosen for this research: endothelin-1, angiotensin II, adrenaline, serotonin, histamine, thromboxane analog U46619, iloprost (Ventavis®), spermine NONOate and riociguat (Adempas®). With these stimuli the goal of this research was to develop an approach to classify the stimuli into vasoconstrictors and vasodilators according to their effect they convey on a cellular level in vitro.
The effect the stimuli convey on human pulmonary arterial smooth muscle cells (hPASMC) were studied by combining two different analytical methods. As a pre-screening the impact of a stimulus on the cell’s impedance was measured with the xCELLigence RTCA® device by Agilent. With this technology concentration-response curves can be measured, and hence stimulus potency and efficacy can be calculated. However, to determine the nature of the effect stimuli were tested with the 3D cell explorer® by Nanolive. Using optical interference spectroscopy, pictures of the cell’s response to the stimuli were generated. With the pictures it is possible to differentiate cell shape changes. According to these changes, the stimuli were classified into vasoconstrictors and vasodilators.
Endothelin-1, thromboxane analog U46619, serotonin and histamine resulted in a drop in the impedance. We assume that a drop reflects a constriction. Pictures of hPASMCs following the stimulation with endothelin-1, thromboxane, and histamine confirmed this assumption. Adrenaline, spermine NONOate and riociguat led to a peak in impedance and therefore an assumed vasodilation. Pictures of the cell’s response to adrenaline supported this thesis. The IP agonist iloprost appeared to constrict the cells in the impedance measurements but showed a cell relaxation in optical interference spectroscopy. Angiotensin II had no relevant impact on the cell in impedance measurements.
For this research we selected nine different natural and synthetic stimuli with a proven effect on blood pressure in vivo. With these stimuli we developed an approach to classify them according to their effect they convey on a cellular level. For this we combined two different analytical methods. We were able to classify the five vasoconstrictors correctly, however, for the precise classification of vasodilators this approach requires further optimisation. A potential improvement may be achieved by precontracting the hPASMCs prior to stimulation with the vasodilator. This makes the effect of the vasodilator more visible.
This research proofs that the response of human blood vessels to natural and synthetic stimuli can be predicted with in vitro experiments. It was demonstrated that the combination of the two fundamentally different analytical methods of impedance measurements and optical interference spectroscopy is a powerful approach to classify various stimuli. This approach has great utility in future discovery and development of cardiovascular medication.
Würdigung durch den Experten
Prof. Dr. Eric Kübler
Sebastian Bolli befasste sich in seiner Arbeit mit der Entwicklung neuer Analysemethoden, um den Effekt von Blutdruck-verändernden Medikamenten in vitro korrekt, schnell und kostengünstig zu quantifizieren. Dazu studierte er den Effekt bekannter Medikamente mittels Impedanzmessungen sowie mittels optischer Interferenzspektroskopie. Dank grossem Einsatz und genauer und sorgfältiger Arbeit konnte er zeigen, dass die von ihm entwickelte Methode es erlaubt, Vasokonstriktoren korrekt zu charakterisieren. Dies ist für die Entwicklung weiterer Medikamente in diesem Bereich von grossem Nutzen.
Sonderpreis «International Summer Science Institute (ISSI)» gestiftet vom Weizmann Institut
Sonderpreis der AO Foundation
Lehrerin: Manuela Koch