Project: EV-miRNAs as Diagnostic Biomarkers for Lipedema

Principal Investigator: Susanne Wolbank, PhD
Co-Principal Investigator: Eleni Priglinger, PhD
Ludwig Boltzmann Institute for Traumatology
Vienna, Austria

Summary

Lipedema is a largely under-diagnosed disease due to a lack of definitive diagnostic tests. This research aims at this unmet clinical need, novel diagnostic biomarkers for Lipedema. MicroRNAs are small molecules that are released from cells into biofluids and regulate a plethora of physiological and pathophysiological processes. We have previously shown that cells in Lipedema adipose tissue secrete specific microRNAs inside small extracellular vesicles (EV-miRNAs) and protein complexes.

In the present project, we want to investigate if EV-miRNAs may serve as diagnostic biomarkers. For this, we will analyze hundreds of microRNAs by next generation sequencing in samples from three different cohorts: individuals with Lipedema vs obese vs healthy individuals. Since patients’ safety, scalability, and cost-effectiveness are important requirements for diagnostic tools, we will focus on peripheral blood plasma as the main biomarker source. In addition, we plan a thorough characterization of the isolated vesicles to clarify their cellular origin. We hence hypothesize that EV-miRNAs could be utilized as diagnostic biomarkers and that extracellular vesicles can provide insight into the pathophysiology of Lipedema.

Background

For Lipedema diagnosis, there are currently no blood-based biomarkers available for clinical use. Our research focuses on cells of the diseased tissue, especially the so called stromal vascular fraction (SVF), which can easily be obtained during liposuction. It is known that cells release microRNAs, within extracellular vesicles (EVs) or protein complexes, for intercellular communication. Since the molecular load of EVs reflects the physiological status of the cells, EVs can serve as valuable sources for biomarkers. We have previously identified 1 extracellular and 7 EV-miRNAs differently regulated in SVF of Lipedema versus healthy individuals. These microRNAs are involved in processes such as adipogenesis, angiogenesis, inflammation, and fat metabolism, all of which are relevant in Lipedema. Based on these highly promising results, we hypothesize that EV-miRNAs could be utilized as trustworthy diagnostic biomarkers for Lipedema. Importantly, we extend our study to blood plasma samples to establish a non-invasive, circulating biomarker.

Methodology

Within this study we aim to identify miRNA biomarker-profiles from lipoaspirate and blood from individuals voluntarily undergoing liposuction. Lipedema patients will be compared to obese and healthy individuals, selected by preset inclusion/exclusion criteria. A specific cell population, the stromal vascular fraction (SVF) is isolated from lipoaspirates taken from subcutaneous adipose tissue (hips, outer thighs to the knee). The small extracellular vesicles (sEVs) are isolated, enriched and stored from these cells and from peripheral blood (platelet poor plasma (PPP)). SVF quality and composition will be characterized by flow cytometry and in vitro assays, isolated sEVs by nanoparticle tracking analysis. On the generated samples an unbiased quantitative analysis of small non-coding RNAs (sEV RNA and total extracellular RNA) will be performed. This method will be monitored by exogenous spike-in-controls for absolute quantification in the RNA samples.

To verify the obtained results, we will perform the gold standard method (RT-qPCR) for targeted analysis of selected miRNAs. This platform comparison is important for selection of robust biomarker candidates, which will independently confirmed by analyzing further samples for final selection of a biomarker panel. To complete the picture, we will characterize the molecular surface composition (cell-specific and EV-specific) of the obtained sEVs (from SVF and PPP) via a newly established modality (multi-laser QUATT NTA). The novel method will be compared to a well-established methodological control (FT-FC). To confirm the identified cellular contribution, biopsies from lipoaspirate tissue-fragments will be immune-stained using the same markers.

Expected outcomes

We hypothesize that sEV-miRNAs are suited as trustworthy diagnostic biomarkers to distinguish between Lipedema and other conditions such as obesity. We further hypothesize that in addition to cells (SVF) isolated from lipoaspirate, blood plasma samples could be used to identify specific sEV-miRNA profiles. While lipoaspirates are easily accessible during liposuction procedures, non-invasive, circulating Lipedema biomarkers will facilitate clinical trials and hence contribute to narrowing the treatment gap. Importantly, readily available systems for blood-based testing (RT-qPCR) will accelerate translation into clinical practice.

Specifically, we expect that sEV preparations can be obtained in sufficient amount and quality from lipoaspirate-derived SVF and peripheral blood plasma for miRNA profile comparison of 3 different cohorts: Lipedema – obese – healthy individuals. Furthermore, we hypothesize that Lipedema patients carry a unique EV-miRNA profile in lipoaspirate-derived SVF and/or peripheral blood plasma, which can be utilized for diagnosis of Lipedema patients through distinction from obese and non-obese individuals (healthy controls).

Additionally, we expect that multi-laser fluorescent NTA will allow discriminating particles from vesicular structures carrying EV-specific and cell-specific surface markers to determine the origin of the sEVs from all three cohorts.

Practical implementations of results

A molecular diagnostic marker for Lipedema would largely help to reliably identify this disease in patients, who are still largely under- or misdiagnosed in clinical practice. The investigation of sEV content and origin in Lipedema compared to healthy adipose tissue and other conditions such as obesity might provide further knowledge on cellular mechanisms in Lipedema and a better understanding of its etiology. The focus on blood samples to obtain a non-invasive biomarker based on an established method for biomarker characterization such as RT-PCR reflects our focus on the translational aspect of this project to support future clinical studies by providing a reliable circulating biomarker.

Our Research Grantees by:   Topic | Recent Projects | Past Projects

LF37_21