Based on cell vesicles and the molecular profiles that researchers encounter in them, the risk of specific cardiovascular diseases can be determined. This is before symptoms even occur. VITO perfected the analysis of these bio-markers in the Interreg TTD project.

More than a hundred Belgians die every day from a cardiovascular (CV) disorder. This means cardiovascular disorders remain one of the most major causes of death in our country. And due to the rising life expectancy, the number of patients with a CV disorder is only expected to rise in the coming decades. That will mean more and more people needing to undergo heart and blood vessel examinations too. However, these examinations take time (and money) and can impact the patient.

Innovative technological platform

The risk of cardiovascular disease cannot be driven down through better prevention and a healthier lifestyle alone, but requires earlier diagnosis – ideally, before the first symptoms appear. The latter lay at the heart of Trans Tech Diagnostics (TTD), a (completed) European Interreg project that centred around the development of an innovative technological platform for efficient and accurate cardiovascular diagnostics, with little impact upon the patient. The project ran from early 2016 to early 2020.

TTD focused upon the early recognition of an elevated risk of CV disorders using new kinds of bio-markers, namely extra-cellular vesicles. These are minuscule bubbles of up to 150 nanometres in size (a nanometre is eighty thousand times smaller than the length of a human hair) that are expelled by the cells in our body, whose composition reflects the health of those cells. This composition can be determined by examining blood samples. The major advantage of these bio-markers is that they provide an accurate prediction and can shed light on CV disorders even before symptoms appear.

Stressed cells

But which vesicles should we zoom in on in a patient's sample exactly, so that a personalised risk of cardiovascular disease can be determined? This was VITO's task in the TTD project. Along with the project partners, there has been a search for new and more refined detection methods for vesicles in recent years. ‘In fact, we were looking for signals in the vesicles that point to symptoms of inflammation in the cells they come from,’ says Inge Nelissen from VITO. ‘In order to pick up those signals, we not only need to be able to detect different kinds of biomolecules, but to “refurbish” the vesicles in vitro so these signals become clearly measurable too.’

In TTD, which came about through a collaboration between the Universities of Hasselt and Maastricht (hence the cross-border nature of this Interreg project), a so-called ‘bio-assay’ was developed, which is a biological testing environment in miniature that allows the potential and concentration of inflammation-related vesicles to be determined from the effect on living cells and tissues. Because cardiovascular diseases are often the result of an early or dormant infection in the blood vessels, this bio-assay was equipped with cells from both human blood vessels and the immune system. The assay was then integrated into a suitable medium for this, a lab-on-a-chip.

VITO (in collaboration with the Universities of Hasselt and Maastricht) used this bio-assay to be able to select the right biomolecules and the molecular profiles that are an indicator of an elevated risk of certain CV disorders. The focus here was on artherosclerosis (hardening of the arteries) and angina pectoris (pain in the chest resulting from a poorly functioning heart muscle). The VITO researchers were looking at endothelial cells that form the basis of the blood vessels, and at monocytes and macrophages – the immune system's first line of defence, lets say.

Unique analytical capacity

The measurement of the vesicles took place using hyper-sensitive flow cytometry, a technique that allows individual particles (such as vesicles) to be detected and identified down to a resolution of 50 nanometres. VITO purchased specialist equipment for this, meaning it now has technology and expertise in-house in terms of flow cytometry that is unique in Flanders. ‘This equipment gives us an analytical capacity that we don't see in any other technology,’ says Nelissen.

Although the flow cytometry technique is not new, standardisation and calibration were still somewhat lacking before TTD commenced – at least for the analysis of vesicles. VITO has perfected this in recent years, also writing protocols to guarantee high-quality analyses. That means that diagnostics based on extra-cellular vesicles are in principle ready to be evaluated in clinical practice. Nelissen: ‘We can now analyse vesicles, both qualitatively and quantitatively, once we've refurbished them from blood or urine samples. Hospital labs will now have to decide whether they want to make the move and develop the diagnostics further in-house.’

VITO would like to continue its research into flow cytometry on vesicles in-house too. ‘For example, we'd like to start working with more comprehensive bio-assays to be able to read several bio-markers from the vesicles at the same time. Alongside this, we'd like to shift in a practical direction, by helping to translate this into clinical applications,’ says Nelissen. VITO will submit an application for a fellowship via the European Marie Curie programme in order to staff this further research. ‘We'd like to set up a full-time post-doc here.’

The portfolio of the public partners in the consortium behind TTD has since been classified into a separate structure: the TTD Open Technology Platform. This serves as the basis for further collaboration with external partners or users. The short-term aim is to enter into accessible collaborations through a number of demo projects. There will be communication from the project partners on this in the run-up to TTD's closing event (which was postponed owing to the corona crisis).

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