In this project we develop a label free technique based on three dimensional (3D) holographic stimulated Raman scattering (SRS) combined with microfluidics and the patch clamp technique.
The goal is to measure and to understand the functioning of pulmonary arteries during oxygen shortage and to learn more about the biomolecular and mechanical actions of pulmonary arterial smooth muscle cells (PASMCs) during hypoxic pulmonary vasoconstriction (HPV) and pulmonary arterial hypertension (PH). Pulmonary hypertension (PH) is equivalent with increased blood pressure within the blood vessels of the lung. This life-threatening and progressive disease has only very limited treatment and there is currently no cure [1, 2].
The World Health Organization (WHO) classifies PH into five groups, whereby Group III (PH associated with hypoxia and/or lung disease) is regarded as the second common cause of elevated blood pressure in the pulmonary arteries. Risk factors for this form of PH include chronic obstructive pulmonary disease (COPD), sleep apnea, but also chronic exposure to high altitude. This severe disorder relies - at least in part - on Hypoxic Pulmonary Vasoconstriction (HPV). HPV is a physiological mechanism that tightens (constricts) pulmonary arteries due to the shortening of PASMCs in response to oxygen shortage (Hypoxia) in the lung [3, 4]. Thereby, HPV optimizes gas exchange and maintains optimal arterial oxygenation during regional ventilation disturbances .
The impact of this vital mechanism becomes evident in lung failure as it currently occurs in the Covid-19 pandemic: insufficient gas exchange due to disturbed HPV often leads to death during lung infections which are either caused by bacteria or viruses, such as Sars-CoV-2. Moreover, widespread and chronic hypoxia in the lungs, e.g. due to chronic lung diseases, lead to PH due to HPV and subsequent vascular remodeling processes in pulmonary arteries [5, 6]. As a result, the right ventricle of the heart has to compensate for the increased vascular resistance, which can ultimately - due to the constant work-overload - result in heart failure . Thus, understanding the underlying mechanisms of HPV is the prerequisite to develop treatment strategies for life-threatening lung diseases which impairs HPV.
PASMCs are the key sensor and effector cells underlying hypoxia-induced pulmonary hypertension and other forms of PH . They are located in the wall of precapillary lung vessels and have the ability to sense acute oxygen shortage in the lung’s alveoli. Under these circumstances, they immediately constrict (HPV) which tightens the lung vessels - thereby redirecting the blood flow to areas of higher oxygen content. HPV is unique for the pulmonary circulation and is essential to maintain arterial oxygenation. The oxygen sensing and signaling mechanisms underlying HPV are hitherto not entirely clarified. Under acute HPV - that generally lasts for seconds to minutes – HPV seems largely to depend on release of reactive oxygen species (ROS) mediated by different signaling cascades, crucially involving mitochondrial redox reactions . The multifactorial nature of HPV, depicted in Fig. 1, is believed to have several hypoxia-sensitive components contributing to HPV [4, 9]. In contrast, chronic hypoxic pulmonary responses that last for several minutes to hours operate by distinct mechanisms.
Since HPV is triggered by oxygen deprivation, an experimental setup must include a system that enables control over rapid shifts in oxygen concentration in the vicinity of PASMCs. We have designed a gas-tight microfluidic system, see Fig. 2a. The design supports multimodal measurements with the Patch Clamp technique and optical probing, while tracking the oxygen concentration . A perfusion system delivers a constant flow of nutrients in biological buffers to the sample in the chamber. Further, the perfusion system can create seamless shifts between normoxic and hypoxic solutions - resulting in a step response due to shifts in oxygen availability. The design of the system was supported by simulations of the chamber, Fig. 2b. It was also crucial to determine that the patch pipette channel would not overflow, because it would render measurements impossible. The simulations revealed that the fluid buildup in the patch pipette channel was low while maintaining a constant flow in the chamber, Fig 2c. The simulation of the fluidic system was done in ANSYS in collaboration with Fluid Mechanics at LTU.
The Patch Clamp technique is an electrophysiological method used to measure the activity of ion channels in the cell membrane . Cells use ion channels in order to communicate with their surroundings or to fuel biochemical reactions. PASMCs respond to oxygen deprivation by creating an inflow of calcium ions through the membrane, which binds to the protein calmodulin. By attaching a pipette to the membrane of a PASMC, changes in ionic concentration can be detected through potential differences and the dynamics of the ion channels can be traced. An overview of a patch pipette that is forming a seal with the membrane of a PASMC, can be seen in Fig. 3.
Raman spectroscopy is a label free spectroscopic tool that gives information on the molecular vibrations of a sample. A Raman spectrum is unique for any given molecule; it shows the energy difference (in inverse centimeters) between incoming excitation photons and the inelastically Raman scattered photons. Raman spectroscopy is extremely sensitive and informative regarding type, state, ordering, and abundance of molecules in a sample. Raman spectroscopy has been widely used to study the redox state of the mitochondria, the power station of all living systems [12-15]. The Raman effect is very weak which renders the method slow since long acquisition times have to be applied. In recent years, coherent time resolved stimulated Raman scattering (SRS) has become a reliant tool for time resolved live cell analysis. With this method video speed capabilities can be achieved. In SRS two laser beams (a pump and a Stokes beam) are required; for targeted imaging they should have an energy difference equal to a Raman shift of the biomarker/molecule of interest. Upon interaction with the molecule, photons are transferred from the pump beam to the Stokes beam through the process of stimulated emission. The result is a decrease in intensity of the pump beam (Stimulated Raman Loss) and a corresponding intensity amplification of the Stokes beam (Stimulated Raman Gain). To achieve holographic imaging, the Stokes laser is divided into two paths; a Stokes and a reference beam. The Stokes beam hits the sample while the reference beam goes directly to the camera. By smart signal acquisition, the phase, and hence the holographic information, can be retrieved. A schematic of the multimodal setup with holographic stimulated Raman set up can be seen in Fig. 4.
Fig. 5 shows the membrane potential due to the charge from calcium ions in the patch pipette from measurements on PASMCs in our gas-tight microfluidic system. A significant increase of the membrane potential occured (black line) as the oxygen was decreased (red line). The membrane potential was restored as soon as oxygen was returned to the sample.
Fig. 6 displays the difference Raman spectra of PASMCs in the normoxic state (21% oxygen) minus the hypoxic state (2-4% oxygen) and from the hypoxic state back to ambient oxygen content. The biomarkers are highlighted by dashed lines in different colors; blue NADH, red NAD+, yellow ferrous cyt c and green ferric cyt c. The differences are small but correlate well with the expected results. The biomarkers - highlighted in different colors - show, as expected, opposite directions for the two cases, i.e. the redox state markers increase or decrease depending on the situation.
First SRS holograms have been recorded and phase images have been retrieved in the transmission mode . The results show that the presence and motion of a specific molecule can be simultaneously traced, see Fig. 7.
To proceed with the holographic SRS setup it is important to achieve SRS in the reflection mode on the biomarkers of the PASMCs. First results that were achieved in transmission can be seen in Fig. 8.
We are currently working on the development of holographic SRS in the reflection mode in order to record holographic stimulated Raman images of PASMCs at the ECCPS in Giessen, Germany.
So far, the studies have concerned single PASMCs; in order to truly understand how the PASMCs communicate and function experiments on whole PASMCs populations and on endothelial-denuded pulmonary arteries, i.e. so called arteries on a chip, will be performed. Unfortunately, the patch clamp technique cannot support monitoring of distributed electrophysiological activity in networks of cells. Therefore, we propose to use microelectrode arrays (MEAs) as a more suitable alternative, tailored so that they can be integrated within the multi-modal lab-on-chip devices. This together with holographic 3D SRS images will create a vast amount of data that - by deep learning - will be transferred to 3D rotatable videos where the signals from the different biomarkers and signalling agents can be turned on/off as desired. We envision a multimodal setup and experimental approach which will serve as a platform for the development of novel therapeutic strategies against PH.
We would like to thank our partners at the Justus Liebig University, Giessen, Germany. Special thanks go th Dr. rer. nat. Fenja Knöpp and Prof. Dr. Norbert Weissmann who are a great access to the project. We would also like to thank Dr. Anders Andersson at Fluids Mechanics, LTU for the simulations on the microfluidic system. For financial support we would like to thank the Swedish research council (Grant 2016-04220), as well as the Kempe Foundation and LTU Labbfond.
- Bogaard, H.J., et al., The Right Ventricle Under Pressure: Cellular and Molecular Mechanisms of Right-Heart Failure in Pulmonary Hypertension. Chest, 2009. 135(3): p. 794-804.
- Sommer, N., et al., Current and future treatments of pulmonary arterial hypertension. British Journal of Pharmacology. n/a(n/a).
- Sylvester, J.T., et al., Hypoxic Pulmonary Vasoconstriction. Physiological Reviews, 2012. 92(1): p. 367-520.
- Sommer, N., et al., Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing. Circ Res, 2017. 121(4): p. 424-438.
- Sommer, N., et al., Oxygen sensing and signal transduction in hypoxic pulmonary vasoconstriction. European Respiratory Journal, 2016. 47(1): p. 288-303.
- Savai, R., et al., Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension. Nat Med, 2014. 20(11): p. 1289-300.
- Barberà, J.A., V.I. Peinado, and S. Santos, Pulmonary hypertension in chronic obstructive pulmonary disease. European Respiratory Journal, 2003. 21(5): p. 892-905.
- Sommer, N., et al., Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing. Circulation Research, 2017. 121(4): p. 424-438.
- Sommer, N., et al., Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing. ScienceAdvances, 2020. in press.
- Knoepp, Fenja, et al. "Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions." Sensors 18.10 (2018): 3238.
- Sakmann, Bert, and Erwin Neher. "Patch clamp techniques for studying ionic channels in excitable membranes." Annual review of physiology 46.1 (1984): 455-472.
- Erjavec, N., G. Pinato, and K. Ramser, Raman spectroscopy as a tool for detecting mitochondrial fitness. Journal of Raman Spectroscopy, 2016. 47(8): p. 933-939.
- Denton, M.L., et al. Redox reactions of cytochrome c in isolated mitochondria exposed to blue or red lasers using resonance Raman spectroscopy. in Progress in Biomedical Optics and Imaging - Proceedings of SPIE. 2018.
- Kitt, J.P., et al., Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability. Journal of the American Chemical Society, 2017. 139(10): p. 3851-3860.
- Perry, D.A., et al., Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest. Science Translational Medicine, 2017. 9(408).
- Amer, E., Gren, P., Ramser, K., & Sjödahl, M. (2018). Measurement of selective species concentration using spectroscopic holography. In Speckle 2018 : VII International Conference on Speckle Metrology (Vol. 10834). https://doi.org/10.1117/12.2319386