In today’s INSPIRE weekly post, I present the “circle of life”. This graph shows the volume and pressure relationship during a single heart beat in the left ventricle presented as a loop. A heart beat can be described in 4 phases displayed in the image (A-D). The cardiac cycle starts with the so-called isovolumetric relaxation of the heart (A). This means that the heart muscle cells relax while the volume of blood in the chambers is constant. The second phase (B) is the ventricular filling where blood enters the heart chamber and the pressure in the heart increases. After a certain threshold is reached the heart contracts and builds up pressure (C), which leads to ejection of the blood (D) from the heart into the aorta. After the ejection of the blood, the cardiac cycle starts again with phase A.
These phases can be simplified as diastole (phase A and B) and systole (phase C and D). Diastole describes the relaxation and filling of the heart. Whereas systole the ejection and contraction includes.
In my project I will determine pressure volume loops via in invasive catheter in mice treated with known cancer drugs to precisely investigate changes in systole and diastole.
The use of animals in experiments is something very important to all of us, scientists or not. In the past decades, a lot of progress has been made to replace lab animals where possible and a lot of work is still ongoing nowadays. In general, scientists operate following the rules known as the "3Rs", which are hierarchically organized:
- Replacement = Use a model that is not an animal if possible
- Reduction = Use the least amount of animal labs that still allows getting significant data
- Refinement = Give the animals the best conditions possible
In the image here you find a gross representation of the proportion of the animals used in scientific research and here there is a table with the exact percentages .
- Mice: 2,92%
- Fish: 14,61%
- Rats: 5,05%
- Birds: 4,21%
- Other mammals: 2,36%
- Reptiles : 0,003%
- Amphibians: 0,28%
- Primates: 0,09%
- Cats: 0,005%
- Dogs: 0,13%
- Horses: 0,30%
- Robinson et al. (2019), The current state of animal models in research: A review, International Journal of surgery, https://doi.org/10.1016/j.ijsu.2019.10.015
Myocardial infarction (MI) is a common cause of heart failure (HF). Therefore, induction of an MI is often used to study the resulting HF in mice. To induce MI in mice, several surgical methods have been developed over the past decades. Surgical ligation of the left anterior descending coronary artery (LAD) is the most commonly used method. Sicklinger et al. recently published a new method, which employs a cardiac ultrasound for LAD localization and a coagulator to induce a MI.
The method of Sicklinkger et al. uses a high frequency (HF) electrical impulse to coagulate the LAD. To this end, a micromanipulator-controlled needle is inserted into the chest and navigated to the LAD under ultrasound guidance. This new technique allows real-time assessment of cardiac function, infarct size and improves stratification post-procedure. In addition, the duration of the procedure is reduced from 20–30 minutes to 6–8 minutes and, importantly, the procedure is minimally invasive and improves recovery of the mice.
During my PhD, we will set-up and use this method in our lab to induce HF in a colon cancer mice model previously shown to have enhanced cancer growth in the presence of HF. Next, we will study the role of endothelial-derived growth factors as a potential integrator of the pathophysiology of HF and cancer.
Electrophysiology is the branch of physiology investigating into the flow of ions (ion currents) using electrical recording techniques that enable the measurement of these currents. In my laboratory practice, I am using a common tool of electrophysiologists, a patch clamping experiment, which is an assay to evaluate ion flows of the cell. We are using this assay as the heart cells (cardiomyocytes) for beating require ion currents to interexchange between inner and outer parts of the cellular membrane, where the most important currents are potassium (K+), sodium (Na+) and calcium (Ca2+) . A number of transporters for each of the ion currents are intercalated into the membrane serving as a gate opening upon change of the membrane’s electrical charge.
Therefore, to study the gating ability of ion channels in cells exposed to a drug, we apply a voltage range and measure the occurring response as an ion flow. For the electrical impulse and subsequent recording, two electrodes are used. One is in the microscope’s bath with the cells and another is situated in the glass micropipette, with a tip diameter of < 1 micrometre, which you can see approaching the cell on the photo. Here, I am going to record an ion flow in Chinese hamster ovary (CHO) cells in response to a drug causing side effect on human cardiomyocytes. Thereby, during my PhD project (‘’Personalized safety pharmacology against drug-evoked proarrhythmia’’), I would like to investigate the mechanisms when this off-target effect occurs.
- Grant AO. Cardiac ion channels. Circ Arrhythm Electrophysiol. 2009 Apr;2(2):185-94.
To monitor the spread of the SARS-CoV-2 (COVID-19) and the number of infections, Health and Sanitary organizations have adopted several measures to counteract the pandemic, including testing patients for their positivity to the virus with qPCR tests. The quantitative Polymerase Chain Reaction (qPCR) is a laboratory technique that allows to detect genetic material from a specific organism, such as DNA- or RNA- viruses. But how it really works? Firstly, RNA transcripts are converted by reverse transcription into their complementary DNAs (cDNA); then, DNA molecules are amplified by 3 repeating steps: denaturation of the double strand DNA into single strands thanks to heat; annealing with the DNA’s primers; elongation, in which DNA polymerase extends the 3′ end of each primer along the template strands, until the complete formation of a new dsDNA. In this way, at every cycle, it is possible to exponentially increase the number of the targeted DNAs.
During my PhD, I am going to use the qPCR technique to investigate new biomarkers of cardiotoxicity - as, for example, the expression of miRNAs - on in vitro cultures of human induced Pluripotent Stem Cells - derived Cardiomyocytes (hiPSC-CMs), when exposed to some cardiotoxic drugs. Using qPCR, we will amplify selected miRNAs from hiPSC-CM and compare their expression to the control.
Just over 15 years ago, the first induced pluripotent stem cells (iPSCs) were generated using mouse fibroblasts 1. Shortly after, induction of pluripotency in human fibroblasts was also proven possible 2, opening a whole new chapter of human based research paradigms.
Concurrent refinement of differentiation protocols paved the way for cell types such as iPSC-derived cardiomyocytes (iPSC-CMs) to routinely serve as disease models, platforms for drug testing, and enter clinical trials for cell therapies 3-5.
However, iPSC-CMs still resemble an embryonic phenotype compared to adult cardiomyocytes, leaving maturation as one of the most pressing challenges in the field. Approaches aiming to mimic biochemical and -mechanical cues that drive heart development in vivo have shown success in approximating the adult cardiac cellular phenotype. Methods such as applying electromechanical stimuli, modifying extracellular matrix, introducing nanotopology, 3D culture and adjusting medium composition also lead to a certain extent of structural and functional maturity, however, several limitations persist 6, 7.
As part of my project, I will be working on testing various platforms promoting iPSC-CM maturation with compatibility to high throughput screening.
- Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
- Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
- Lian, X., et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A, 2012. 109(27): p. E1848-57.
- Burridge, P.W., et al., Chemically defined generation of human cardiomyocytes. Nat Methods, 2014. 11(8): p. 855-60.
- Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011. 8(12): p. 1037-40.
- Ahmed, R.E., et al., A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes. Front Cell Dev Biol, 2020. 8: p. 178.
- Karbassi, E., et al., Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nature reviews. Cardiology, 2020. 17(6): p. 341-359.
The use of telemetry technology to measure cardiovascular parameters is a core principle during safety pharmacology studies. Most of these studies are performed with laboratory animals kept in single cages, which is a well-known stress factor. It has also been shown that group-housing has some beneficial effects on the cardiovascular system1,2. Therefore, safety pharmacology strives to advance future studies from single to group house environments to stimulate safer drug development.
A novel Stellar Telemetry implant with integrated micro-GPS functionality, of which its development and validation is the main objective of my PhD project, offers a new technology for researcher to analyse cardiovascular parameters and behavioural traits simultaneously in socially interacting animals, which has been neglected in safety pharmacology so far. This technology ensures the possibility to keep laboratory animals in groups, which reflects their natural habitat and thereby minimizes stress.
Beyond the development of innovative technologies, TSE Systems designs group-housed environments in which such experiments can be performed and combined with automated behavioural phenotyping. The PhenoWorld (picture) is a new concept which raises animal housing and experimentation onto the next level by combining different measurement compartments with highly enriched living environments3.
Future laboratory animal experiments performed in such multilayer quarters will contribute to elucidate possible improvements on animal welfare aspects and on data quality, which could – for example – lead to the development of new and safer drugs.
- Xing, G., Lu, J., Hu, M., Wang, S., Zhao, L., Zheng, W., ... & Skinner, M. (2015). Effects of group housing on ECG assessment in conscious cynomolgus monkeys. Journal of pharmacological and toxicological methods, 75, 44-51. https://doi.org/10.1016/j.vascn.2015.05.004
- Späni, D., Arras, M., König, B., & Rülicke, T. (2003). Higher heart rate of laboratory mice housed individually vs in pairs. Laboratory animals, 37(1), 54-62. https://doi.org/10.1258/002367703762226692
- Castelhano-Carlos, M. J., Baumans, V., & Sousa, N. (2017). PhenoWorld: addressing animal welfare in a new paradigm to house and assess rat behaviour. Laboratory animals, 51(1), 36-43. https://doi.org/10.1177/0023677216638642
This flyer shows an image acquired by echocardiography. It is a comparison of ultrasound images of a heart of mouse (left) and a human (right). *Disclaimer: The images are taken with a different resolution and have a different scale. The heart consists of 4 chambers divided in two small- (atria) and two large chambers (ventricles). The blood enters the heart via the atrium. Next, the blood is collected in the ventricle (diastole) and pumped out with every heart beat (systole).
These types of images are routinely used to examine the heart of patients, since it presents safe, convenient and non-invasive way to investigate both anatomy (B-mode) and function (M-mode) of the heart. In a comparison the four-chamber-view of the two hearts is shown. This view is used to specific investigate the pumping behavior including the blood flow of all chambers and gives therefore insights about the diastolic and systolic function.
As seen in the image, the four-chamber view of the mouse does not always show all 4 chambers. In the mouse heart there is often just the left ventricle and atrium visible. This is caused by a shadow from the breast bone covering the right side of the mouse heart. However, the parameters measured by this method good concordance between mice and human. Consequently, I will use, among other techniques, high-resolution ultrasound imaging method in my project to examine the function of the left ventricle in mice during cancer therapy.
What do you think scientists observe on a daily basis? The picture above reproduces the view from a microscope loaded with the animal cells. These are the cells of ovarian epithelium derived from Chinese hamsters (CHO). The hamsters are easy to breed, yet once the cells were isolated in 1957 by Theodor Puck 1, there is no need anymore to keep the living hamsters for cell production. The isolated CHO cells, like any other mammalian cells, have an ability to grow and divide while being maintained in a liquid medium containing all the essential nutrients such as glucose as carbohydrate, amino acids for protein production and vitamins for the activity of enzymes within the cell. As the cells are commonly arranged into tissues, such as epithelial ovarian tissue in case of CHO cells, they retain a tendency to arrange into groups and attach to a substrate. So that these CHO cells simply grow on the bottom of a plastic Petri dish filled up with the nutrient medium. The simplicity of maintenance, cellular line stability and similarity of produced proteins to human, made CHO cells a well-known system for the production of recombinant proteins for industrial and research purposes 2. Recombinant proteins are produced by the methods of genetic engineering and incorporate the genetic material from different sources. For instance, on the picture, CHO cells were cloned (i.e. transfected) with a gene originated from jellyfish producing green fluorescent protein (GFP) 3, the glow of which is not visible under the light microscope, but you can observe its fluorescence in the next post. Another transfected gene was the gene of an ion channel. Since CHO cells do not express any cardiac-related ion transporters 1, it is a convenient system to study an ion channel of interest without any influence of other channels present in human heart tissue.
Thus, as a part of my PhD project, I am using CHO cells as a model organism to study the pharmacological effects of drugs on the cellular level. I examine these cells using a patch-clamp technique, about which I will elaborate in the next post.
- Gamper N, Stockand JD, Shapiro MS. The use of Chinese hamster ovary (CHO) cells in the study of ion channels. Journal of Pharmacological and Toxicological Methods. 2005 May; 51(3):177-85
- Kim JY, Kim YG, Lee GM. CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol. 2012 Feb;93(3):917-30
- Remington SJ. Green fluorescent protein: a perspective. Protein Sci. 2011 Sep;20(9):1509-19
In the poster, you see pressure signals obtained from in silico modelling in different conditions, i.e. in a compliant (healthy) and stiff (aged) blood vessel. The picture shows P0 (blue), which is the pressure wave over time measured close to the beginning of a tube, while P1 (orange) is the pressure measured further down the vessel, assuming a cylindrical elastic tube set-up. The left picture corresponds to an aged or diseased blood vessel with a Young modulus of 2.5 ∙106 dyn·cm−2 . The right graph shows a healthy blood vessel with a Young modulus of 0.5 ∙106 dyn·cm−2. The Young modulus represents the stiffness of the vessel and has been correlated to arterial ageing and cardiovascular disease, such as arterial calcification.
Mathematical modelling and numerical simulation help to obtain insight in the relation between wall parameters such as the Young’s modulus and pulse wave behavior. Moreover, increased pulse wave velocity (PWV) as well as deviation in the morphology are risk factors for cardiovascular disease. As such, mathematical models provide a useful tool for safety pharmacologist to better understand the possible impact of drug-induced changes in hemodynamic parameters and how these translate into clinical risk depending on patient-specific characteristics.
Ultrasound imaging is a common practice in clinical setups which has been around since the late 1950’s1. This technology is frequently used in pregnant women to gain insight into anatomical imaging2. However, our lab has implemented this technique for functional imaging of the cardiovascular system in rodents. The spatial resolution of conventional ultrasound imaging systems used in the clinic provides a barrier for pre-clinical imaging of rodents (rat or mice) given their small size. To overcome this challenge we will make use of transducers which have a much higher frequency to produce higher resolution images3. More specifically when investigating the aortic regions where the diameters (captured by B-Mode imaging) and the velocities of the blood flow (captured by Doppler imaging) will be recorded as presented in the images above. These images are then respectfully plotted side by side in order to calculate the pulse wave velocity which will provide us valuable insight into the local stiffness of the vasculature. The main goal and importance of my PhD project is for this technique to be used as a valuable tool in safety pharmacology testing for the screening of existing as well as potential new drug candidates.
- Sprawls P. The Physical Principles of Medical Imaging, 2nd Ed. 1995, Medical Physics Pub. (Madison, Wis).
- Liff I, Bromley B. Fetal Anatomic Imaging Between 11 and 14 Weeks Gestation. Clin Obstet Gynecol. 2017 Sep;60(3):621-635. doi: 10.1097/GRF.0000000000000296. PMID: 28742595.
- Moran, C. M. and A. J. W. Thomson (2020). Preclinical Ultrasound Imaging—A Review of Techniques and Imaging Applications.Frontiers in Physics 8(124).
In this image, you can see a blood vessel segment mounted on two glass micro-cannulae. In this set-up, the environment of the vessel is maintained under physiologically relevant conditions. Acidity and temperature of the fluid around the vessel can be adjusted and the pressure both inside and outside of the vessel can be regulated. After the vessel is secured and the environment is prepared, this technique, called pressure myography, allows measurement of dynamic changes in the diameter of the vessel.1 Drugs can be tested, either in the bathing solution or intra-luminally; their vasoactive properties may cause a contraction of the vessel, so the diameter of the vessel segment will decrease, or cause a vasodilation, meaning the diameter will increase.
Vascular Endothelial Growth Factor (VEGF) inhibitors are powerful drugs to stop tumour growth, but they are known to cause an elevated blood pressure in patients taking them.2 During my PhD, I will study the mechanisms by which these VEGF-inhibitors might change the diameter of the isolated vessel. If these drugs cause a decrease in the vessel diameter, this could be an explanation for the increased blood pressure in patients. In this way, our knowledge on the mechanism behind the blood pressure raise will develop further, giving us more information on how to avoid this in the future.
- Schjørring, O. L., Carlsson, R. & Simonsen, U. Pressure Myography to Study the Function and Structure of Isolated Small Arteries. Methods in Molecular Biology, vol. 1339, 277–285 (2015).
- Ferrara, N. & Adamis, A. P. Ten years of anti-vascular endothelial growth factor therapy. Nat. Rev. Drug Discov. 15, 385–403 (2016).
On average, every year around 37 new drugs are launched on the market with a cost of 1.5 billion dollars each. However, attrition rates remain high, allowing only 2% of potential candidates to enter clinical trials¹. The discipline of Safety Pharmacology aims to detect safety liabilities and adverse events early-on. While safety evaluation requires animal experimentation for certain steps, significant progress has been made to perform initial screenings on cellular assays. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative, established in 2013 by pharmaceutical industry and regulatory bodies is showing the value of cellular (“in vitro”) assays and computer models (“in silico”) to evaluate cardiotoxicity of drugs in the early stages of development, avoiding economical and time wastes ²,³. Moreover, based on the results of the CiPA initiative, a dialogue has been started to change the regulatory guidelines for non-clinical and clinical cardiac safety evaluation of new drugs.
This picture shows the system we are using at UCB Pharma to evaluate drug-induced cardiotoxicity on in vitro human induced pluripotent stem cell - derived cardiomyocyte (hiPSC-CM) cultures. The xCELLigence Real-Time Cell Analysis (RTCA) CardioECR System is a platform that provides powerful means to record cells in real time and in a non-invasive way. It allows to combine field potential recording and impedance for the measurement of integrated electrophysiology and contractility of cardiomyocytes, enabling the detection of changes in morphology, cell adherence and viability. Thanks to this system, we are able to quantify and predict drug cardiotoxicity in a 48-well format in the early phase of its development. The aim of my PhD project is to investigate the sensitivity of different cell models when treated with cardiotoxicants and to explore additional molecular biomarkers to further improve the predictive value.
- IFPMA. IFPMA-Facts-And-Figures-2017. Int Fed Pharmacutical Manuf Asoc. 2017;
- Blinova K, Stohlman J, Vicente J, Chan D, Johannesen L, Hortigon-Vinagre MP, et al. Comprehensive translational assessment of human- induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias. Toxicol Sci. 2017;
- Blinova K, Dang Q, Millard D, Smith G, Pierson J, Guo L, et al. International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment. Cell Rep. 2018
Nowadays, most people use wireless systems in their homes for connecting to the internet and other data transportation. However, this technique can be used for cardiovascular data acquisition and other physiological data evaluation.
Wireless monitoring with implantable telemetry microchip allows us to remotely collect animals' physiological data, such as cardiovascular parameters, anytime during undisturbed movement and social interaction during group housing. This technique increases animal welfare by allowing a continuous recording of their physiological and ECG parameters. The telemetry system consists of two major components:
- An implantable unit
- A receiver/antenna linked to a computer
During my Ph.D. project, I will investigate real-time telemetry data to qualify, cluster, and validate cardiovascular data in combination with video tracking to understand animals' social traits to reduce data variance.
- Home. (n.d.). Retrieved February 03, 2021, from https://www.tse-systems.com/
This image presents a Mass Spectrometry Imaging (MSI) section of a mouse heart. MSI enables to visualise biomolecules or chemical compounds on tissue sections. During the last decade, MSI has emerged as a powerful technique in drug development.
MSI can be used to investigate spatial distribution of a drug and its metabolites. In this context, we performed an image acquisition of hearts dosed with doxorubicin. Doxorubicin is an efficient anti-cancer drug that may cause dose-dependent cardiotoxicity. The image (top slide) shows three hearts of which two were collected 3 (middle) and 24 hours (right) post-doxorubicin injection. A colour scale of intensity (from blue-minimum to white-maximum) represents the concentration of doxorubicin. In a next step, this workflow can be complemented by MSI-guided proteomics and metabolomics approaches to estimate the impact of the drug within the tissue.
During my PhD-project, I will employ MSI and spatial -omics strategies to study drug distribution and the associated tissue response in the field of cardiovascular diseases, safety pharmacology and toxicology.
This Stellar telemetry device developed by TSE Systems allows researcher to collect cardiovascular data in laboratory animals. It can be implanted in small as well as in larger animals. Its unique internal data storage capability gives the researcher the freedom to move the animals away from their usual home cage environment while maintaining data acquisition. This ensures simultaneous data collection of vital signs while performing phenotypical, physiological, pharmacological, behavioural, metabolic and inhalation studies.
During my PhD project, I will help to further improve the current system by adding a micro-GPS function to additionally measure behaviour and locomotor activity. This technology will advance research in socially housed animals, in which human interaction will be kept to a minimum. This in turn improves the relevance and quality of the collected data and - very importantly - the well-being of the animals. 1,2
This small telemetry device will have a huge impact on the flexibility to perform a broad range of social animal experiments, especially in the context of developing new and safer drugs.
- Jennings, M., Prescott, M. J., & Joint Working Group on Refinement (Primates). (2009). Refinements in husbandry, care and common procedures for non-human primates: Ninth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. Laboratory Animals, 43(1_suppl), 1-4 https://doi.org/10.1258/la.2008.0071437.
- Ellegaard, L., Cunningham, A., Edwards, S., Grand, N., Nevalainen, T., Prescott, M., & Schuurman, T. (2010). Welfare of the minipig with special reference to use in regulatory toxicology studies. Journal of pharmacological and toxicological methods, 62(3), 167-183. https://doi.org/10.1016/j.vascn.2010.05.006
This picture shows a rat heart isolated and perfused according to the Langendorff method. This technique permits to study the mechanical activity of isolated mammalian heart, allowing us to measure cardiac contractility (left ventricle pressure or LVP) by means of a latex balloon inserted into the left ventricle and connected to a pressure transducer, as well as coronary perfusion pressure (CPP), recorded by a pressure transducer placed in the inflow line 1. The electrocardiogram (ECG) is also recorded, providing information about the electrical activity of heart.
Unanticipated cardiovascular toxicities have shed light on the necessity to identify cardiovascular safety of drugs in a preclinical setting. In this context, the isolated Langendorff heart model is a useful method to evaluate the effect of drugs on haemodynamic, as well as on electrophysiological parameters. The significant sensitivity and specificity of such approach make it a method with high translational value 2.
During my PhD, I will use the Langendorff technique to assess cardiovascular safety liabilities of novel anti-cancer therapies, specifically receptor tyrosine kinase inhibitors (RTKI) targeting vascular endothelial growth factor (VEGF) signalling pathway 3. This model will complement my in vivo experiments in order to clearly identify the cardiovascular complications due to these drugs, as well as to investigate the mechanisms underlying such toxicities.
- Bell, R.M., Mocanu, M.M. & Yellon, D.M. Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion. Journal of Molecular and Cellular Cardiology 50, 940-950 (2011).
- Authier, S., Pugsley, M.K. & Curtis, M.J. Haemodynamic Assessment in Safety Pharmacology. Handb Exp Pharmacol 229, 221-241 (2015).
- Poster SPS meeting 2019, Barcelona
Safety Pharmacology and Arterial Stiffness
Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable properties of a drug primarily prior to clinical trials. Successful screening strategies to detect cardiovascular liabilities have been implemented, but there is room for further refinement 1. In this perspective, arterial stiffness is an interesting, blood pressure-independent factor to predict the risk of cardiovascular disease, but has not been widely considered in safety pharmacology. Historically, arterial stiffness was considered to reflect (passive) biomechanical properties of the artery wall, such as the ratio of compliant elastin versus stiffer collagen fibers. Our PHYSPHAR research group has developed a unique and proprietary organ set-up, as seen in the image (i.e., the Rodent Oscillatory Tension Set-up to study Arterial Compliance, ROTSAC) that enables ex vivo determination of intrinsic arterial stiffness, independent of confounding factors, such as blood pressure or heart rate 2. This novel technological platform holds potential to directly evaluate acute effects of chosen drugs, untangling their underlying mechanisms.
Could this be the tool us scientists have been waiting for? Join us on our journey to find out!
- Guns, P. D., Guth, B. D., Braam, S., Kosmidis, G., Matsa, E., Delaunois, A.,Valentin, J. P. (2020). INSPIRE: A European training network to foster research and training in cardiovascular safety pharmacology. J Pharmacol Toxicol Methods, 106889. doi:10.1016/j.vascn.2020.106889
- Le loup, A. J. A., Van Hove, C. E., De Moudt, S., De Meyer, G. R. Y., De Keulenaer,G. W., & Fransen, P. (2019). Vascular smooth muscle cell contraction and relaxation in the isolated aorta: a critical regulator of large artery compliance .Physiol Rep, 7(4), e13934. doi:10.14814/phy2.13934
The calculation of adequate sample size is crucial in any safety pharmacology study. Power calculation it is the process by which we calculate the optimum number of animals required to arrive at ethically and scientifically valid results. Power calculations uses mathematics/statistical equations, which describe the relationship between the expected effect size of biological interest, standard deviation (SD) of the data, significance level (usually p <0.05 ), desired power, sample size, and the alternative H0 (null hypothesis). A study with too small sample size may produce inconclusive and unreliable results.
As part of my PhD project, I will develop data analysis workflows that enable efficient exploration of values of control groups reported in historical safety studies. Moreover, we believe that a better understanding of factors inducing variation in historical studies may help to improve future study designs.
- Fitzpatrick, R. (n.d.). Why is sample Size important? Retrieved February 03, 2021, from https://blog.statsols.com/why-is-sample-size-important/
- Kadam, P., & Bhalerao, S. (2010, January). Sample size calculation. Retrieved February 03, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876926/
The picture shows an electrical signal
recorded from human heart muscle cells (cardiomyocytes). Interestingly, these
cardiomyocytes have been derived from human inducible Pluripotent Stem Cells
(hiPSC), a specific type of human stem cell obtained after reprogramming of
human skin cells (this topic will later be explained by ESR1 in another INSPIRE
Weekly contribution). hiPSC-based assays are used in drug development studies,
particularly for cardiovascular safety evaluation. Drug toxicity to the heart
could induce specific changes in the amplitude or shape of the signal, thereby
reflecting underlying mechanisms. However, manually analysing these signal data
is challenging given the very large amount of data generated by these
My PhD research will focus on automatically analysing the large datasets from hiPSC-cardiomyocytes assays and developing computer-based prediction models (using artificial intelligence and machine learning) to detect and classify drug effects.
- Raphel F, De Korte T, Lombardi D, Braam S, Gerbeau JF (2020). A greedy classifier optimization strategy to assess ion channel blocking activity and pro-arrhythmia in hiPSC-cardiomyocytes. PLOS Computational Biology 16(9): e1008203. https://doi.org/10.1371/journal.pcbi.1008203
Blood pressure is a physiological parameter that can easily be measured and that correlates to one’s cardiovascular risk. Blood pressure is also a key factor to evaluate the cardiovascular safety of new drug candidates. Typically, blood pressure has a pulsatile pattern resulting in systolic (high) and diastolic (low) pressure values. An additional haemodynamic parameter includes pulse wave velocity (PWV), which is estimated based on pressure measurements at different locations in the body (e.g. arm versus foot). The PWV is a proxi of the stiffness (or reduced elasticity) of one’s larger arteries and is a good predictor of cardiovascular risk, independently of blood pressure. Further, using biophysics it is possible to derive from PWV the intrinsic arterial stiffness that may reflect pathological or drug-related changes of the vascular wall.
As part of my PhD, I will improve parameter estimation of biophysical quantities that cannot be measured directly in patients or lab animals. In fact, by combining pressure measurements and mathematical modelling as well as numerical simulation, we can estimate quantities like the Young modulus of the artery wall (or modulus of elasticity in tension). The Young modulus is a mechanical property that measures the tensile stiffness of a solid material and that will inform whether a disease (e.g. vascular calcification that occurs in dialysis patients) or a drug (cf. safety pharmacology) affects the biomechanical properties of our vascular system. Moreover, this detailed information is important to carefully assess and investigate the haemodynamic impact of changes in intrinsic stiffness.
- Gerbeau, J., Lombardi, D., & Tixier, E. (2018). How to choose biomarkers in view of parameter estimation. Mathematical Biosciences, 303, 62-74. https://doi.org/10.1016/j.mbs.2018.06.003
This picture shows some cardiovascular variables obtained through a method to determine blood flow called pulsed Doppler flowmetry and recorded using a computer-based system (IdeeQ). This technique allows us to make measurements of blood flow in three different vascular beds, while at the same time measuring heart rate and blood pressure, in conscious, freely moving rats. In order to evaluate these changes in the dynamics of the blood flow (haemodynamic), Doppler flow probes need to be surgically placed on the studied vessels. Several parameters will then be recorded using a computer-based system 1,2. Because the experiments are performed in a live rat, this method also permits us to measure haemodynamic changes in different parts of the vascular system, with reflex systems intact; these are often affected by anesthetics.
During my PhD project I will use the pulsed Doppler flowmetry to investigate changes in mesenteric, renal and hindquarters vascular conductance, as well as heart rate and mean arterial pressure, in rats treated with anticancer drugs targeting vascular endothelial growth factor (VEGF) signalling. Moreover, this approach will also provide essential information to clearly define the mechanism of action underlying cardiovascular impairment due to such novel targeted therapies used in cancer treatment. This project will give us more detailed information about the safety pharmacology issues associated with these treatments 3.
- Haywood, J.R., Shaffer, R.A., Fastenow, C., Fink, G.D. & Brody, M.J. Regional blood flow measurement with pulsed Doppler flowmeter in conscious rat. American Journal of Physiology-Heart and Circulatory Physiology 241, H273-H278 (1981).
- Gardiner, S.M., Compton, A.M., Bennett, T. & Hartley, C.J. Can pulsed Doppler technique measure changes in aortic blood flow in conscious rats? American Journal of Physiology-Heart and Circulatory Physiology 259, H448-H456 (1990).
- Poster SPS meeting 2019, Barcelona
The measurement of blood pressure gives us valuable information on the health of a patient’s heart and blood vessels. The blood pressure is recorded as a waveform, a regularly repeating signal over time. Typically, the maximum and minimum value of the waveform are evaluated. In a healthy individual, these values are approximately 120/80 mmHg. In many diseases, the blood pressure is raised.
However, besides this maximum and minimum value, there is considerably more information captured in the recording of a blood pressure, that is often not taken into account. Analysis of the shape of the waveform and variability of this signal may give us additional information on how the heart and blood vessels are affected, particularly after drug treatment. In order to look at subtle changes in these waveform features, a new model was developed, called the attractor reconstruction. This mathematical model converts the blood pressure waveform (on the left-hand side of the flyer) into a 2D image (as shown on the right-hand side). By analysing different features of this 2D image, such as colour or length of the loops, we can extract detailed information on the effects of drugs on the heart and blood vessels. During my project, I will apply this attractor reconstruction on blood pressure waveforms and blood flow waveforms, to see how a number of anticancer drugs affect the cardiovascular system. In this way, I will explore the mechanism and safety of these medicines.
- Nandi, M., & Aston, P. J. (2020). Extracting new information from old waveforms: Symmetric projection attractor reconstruction: Where maths meets medicine. Experimental Physiology, 105(9), 1444–1451. https://doi.org/10.1113/EP087873
- Nandi, M., Venton, J., & Aston, P. J. (2018). A novel method to quantify arterial pulse waveform morphology: Attractor reconstruction for physiologists and clinicians. Physiological Measurement, 39(10). https://doi.org/10.1088/1361-6579/aae46a
This image presents a usual workflow for data analysis by MALDI-MSI. MSI, standing for mass spectrometry imaging, is a label free technique allowing visualization of biomolecules on tissue sections. The image on the flyer shows heart sections obtained from an ischemia mouse model to study peptide distribution differences between ischemic (highlighted in black) and healthy regions. An overlay (right) of a conventional histology image (left) and a MSI image provides a better understanding of the molecular distribution within the tissue.
Segmentation analysis (middle) clustered the peptide spectra into different groups represented by different colors based on their molecular similarity, revealing a specific peptide signature in the ischemic area. This workflow is usually followed by MALDI-MSI guided proteomics for in depth protein identification.
During my PhD-project, I will employ MSI and spatial -omics strategies to study protein and glycan regulation in the field of cardiovascular diseases and toxicology.
Historically, cancer and cardiovascular diseases were seen as separate entities only sharing several common risk factors, e.g. smoking, obesity and genetic background.1 Recent evidence, however, demonstrates that heart failure as a result of a heart attack promotes cancer development in mice.2 In the field of cardio-oncology, the toxic effects of anti-cancer drugs on the heart, eventually leading to heart failure or other cardiovascular adverse events, are being investigated extensively. However, the possibility of heart failure causing or worsening cancer growth is a novel discovery and is leading to the rise of the ‘reversed cardio-oncology’ field.
How does heart failure promote cancer growth? Several studies point towards proteins that are secreted by the damaged heart.2,3 These proteins are secreted by several biological processes with the aim to heal injuries, similar to wound healing. The ‘wound healing’ processes that are favorable in the heart, however, might promote cancer growth. During my PhD I will study the link between heart failure and cancer with a specific focus on endothelium-derived growth factors.
- Avraham, S., Abu-Sharki, S., Shofti, R., Haas, T., Korin, B., Kalfon, R., Friedman, T., Shiran, A., Saliba, W., Shaked, Y., & Aronheim, A. (2020). Early Cardiac Remodeling Promotes Tumor Growth and Metastasis. Circulation, 142(7), 670–683. https://doi.org/10.1161/CIRCULATIONAHA.120.046471
- Meijers, W. C., Maglione, M., Bakker, S. J. L., Oberhuber, R., Kieneker, L. M., De Jong, S., Haubner, B. J., Nagengast, W. B., Lyon, A. R., Van Der Vegt, B., Van Veldhuisen, D. J., Westenbrink, B. D., Van Der Meer, P., Silljé, H. H. W., & De Boer, R. A. (2018). Heart failure stimulates tumor growth by circulating factors. Circulation, 138(7), 678–691. https://doi.org/10.1161/CIRCULATIONAHA.117.030816
- Moslehi, J., Zhang, Q., & Moore, K. J. (2020). Crosstalk between the heart and cancer: Beyond drug toxicity. In Circulation (Vol. 142, Issue 7, pp. 684–687). Lippincott Williams and Wilkins. https://doi.org/10.1161/CIRCULATIONAHA.120.048655
This image shows a size comparison of a regular pen and a pressure volume catheter used for precise analysis of the heart function in mice. This useful but tiny device can be inserted via a blood vessel inside the neck into the left ventricle (left chamber) to determine simultaneously the pressure and volume in the beating heart of a mouse (in vivo experiment). The catheter is connected to a computer allowing real time assessment of each heart beat generating pressure volume loops (PV-loops). PV-loops can be used to assess various parameters that characterize heart function.
Due to the small size of the mice and especially the mice heart, precise assessment of the cardiac function remains challenging. Nowadays, several imaging technologies are used to provide information regarding cardiac function in mice (e.g. ultrasonic or magnetic resonance imaging). However, only PV-loops provide the precise, real time and simultaneous measurement of chamber pressure and volume in mice heart. Also a great advantage of PV-loops is that they have already been well established in humans, thereby enhancing translation of the results to humans.1 During my PhD project I will use PV-loops to study the toxic effects of anti-cancer drugs in mice hearts.2 This gives a better insight into the side effects of anti-cancer drug and helps to develop new strategies to reduce them.