simultaneous-imaging-flow-cytometry-based-detection-multiple

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Johannes Kepler University Linz

Physiologically, following myocardial infarction (MI), retinoid levels elevate locally in the infarcted area. Whereas therapeutic systemic application of retinoids was shown to reduce the progression of ventricular dilatation and the onset of heart failure, the role of acute physiologically increased retinoids in the infarction zone is unknown to date. To reveal the role of local retinoids in the MI zone is the central aim of this study. Using human cell culture and co-culture models for hypoxia as well as various assays systems, lentivirus-based transgene expression, in silico molecular docking studies, and an MI model in rats, we analysed the impact of the retinoid all-trans retinoic acid (ATRA) on cell signalling, cell viability, tissue survival, heart function, and MI-induced death in rats. Based on our results, ATRA-mediated signalling does aggravate the MI phenotype (e.g. 2.5-fold increased mortality compared to control), whereas 5'-methoxyleoligin (5ML), a new agent which interferes with ATRA-signalling rescues the ATRA-dependent phenotype. On the molecular level, ATRA signalling causes induction of TXNIP, a potent inhibitor of the physiological antioxidant thioredoxin (TRX1) and sensitizes cells to necrotic cell death upon hypoxia. 5ML-mediated prevention of ATRA effects were shown to be based on the inhibition of cellular ATRA uptake by interference with the cholesterol (and retinol) binding motif of the transmembrane protein STRA6. 5ML-mediated inhibition of ATRA uptake led to a strong reduction of ATRA-dependent gene expression, reduced ROS formation, and protection from necrotic cell death. As 5ML exerted a cardioprotective effect, also independent of its inhibition of cellular ATRA uptake, the agent likely has another cardioprotective property, which may rely on the induction of TRX1 activity. In summary, this is the first study to show i) that local retinoids in the early MI zone may worsen disease outcome, ii) that inhibition of endothelial retinoid uptake using 5ML may constitute a novel treatment strategy, and iii) that targeting endothelial and myocardial retinoid uptake (e.g. via STRA6 inhibition) may constitute a novel treatment target in acute MI.

Early inhibition of endothelial retinoid uptake upon myocardial infarction restores cardiac function and prevents cell, tissue, and animal death.

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in  models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane  laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood-brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

Mario Mairhofer

Johannes Kepler University Linz

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells