AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes.

Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11-19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

The impact of hyperglycemia on adhesion between endothelial and cancer cells revealed by single-cell force spectroscopy.

The impact of hyperglycemia on adhesion between lung carcinoma cells (A549) and pulmonary human aorta endothelial cells (PHAEC) was studied using the single-cell force spectroscopy. Cancer cells were immobilized on a tipless Atomic Force Microscopy (AFM) cantilever and a single layer of endothelial cells was prepared on a glass slide. The measured force-distance curves provided information about the detachment force and about the frequency of specific ligand-receptor rupture events. Measurements were performed for different times of short term (up to 2 h) and prolonged hyperglycemia (3 h - 24 h). Single-cell force results were correlated with the expression of cell adhesion molecules (intercellular adhesion molecule, P-selectin) and with the length and density of the PHAECs glycocalyx layer, which were measured by AFM nanoindentation. For short-term hyperglycemia, we observed a statistically significant increase of the adhesion parameters that was accompanied by an increase of the glycocalyx length and expression of P-selectin. Removal of hyaluronic acid from PHAECs glycocalyx significantly decreased the adhesion parameters, which indicates that hyaluronic acid has a strong impact on adhesion in A549/PHAEC system in short term of hyperglycemia. For prolonged hyperglycemia, the most significant increase of adhesion parameters was observed for 24 hours and this phenomenon correlated with the expression of adhesion molecules and a decrease of the glycocalyx length. Taking together, presented data indicate that both mechanical and structural properties of the endothelial glycocalyx strongly modulate the adhesion in the A549/PHAEC system.

Biocompatible and fluorescent superparamagnetic iron oxide nanoparticles with superior magnetic properties coated with charged polysaccharide derivatives.

Syntheses and characterizations of biocompatible superparamagnetic iron oxide nanoparticles with embedded curcumin and coated with ultrathin layer of hyaluronic acid-curcumin (HA-Cur) conjugate have been reported. Zeta potential measurements confirmed effective coating of native iron oxide nanoparticles stabilized by cationic derivative of chitosan (SPION-CCh) with the synthesized HA-Cur conjugate. Both SPIONs with embedded curcumin and the ones coated with HA-Cur (SPION-CCh/HA-Cur) revealed desired magnetic characteristics while fluorescent properties were much better for the coated nanoparticles. SPION-CCh/HA-Cur nanoparticles were shown to be very promising candidates for T2 MRI contrast agents as they can easily penetrate cell membrane and their relaxivity is exceptionally high (ca. 470mM-1s-1). They may be also tracked using confocal fluorescence microscopy due to the presence of fluorescent curcumin in the coating. In vitro studies indicated that the obtained SPIONs-CCh/HA-Cur were non-toxic for EA.hy926 endothelial cells.

Morphological and nanomechanical changes in mechanosensitive endothelial cells induced by colloidal AFM probes.

Mechanotransduction is one of the main properties of endothelial cells (ECs) phenotype. Hemodynamic forces like flow-generated endothelial shear stress play a fundamental role in ECs cytoskeletal remodeling and activate signaling cascades in ECs. AFM methods are widely used to characterize morphology as well as mechanical properties of cells. In both cases AFM probes directly interact with cell surface exerting mechanical forces on the cellular membrane, which in turn may stimulate mechanosensitive receptors present in EC. This article presents examples of how the colloidal AFM probes influence ECs during multiple scans. The results revealed that multiple scans of the ECs significantly influenced the morphology and elasticity of cells. Moreover, changes in the cell shape and mechanical properties were dependent on the scan direction (across or along the main axis of the cell). Multiple scans with a colloidal probe leaded to reorientation of the cell main axis and this effect was similar to the action of the shear stress induced by flow conditions. Furthermore, the correlation between the tip-induced modification of the cell properties and the remodeling of the cell's glycocalyx was observed. SCANNING 38:654-664, 2016. © 2016 Wiley Periodicals, Inc.

Nano-mechanical model of endothelial dysfunction for AFM-based diagnostics at the cellular level.

A review of recent experimental investigations on the nanomechanical response of individual endothelial cells to inflammation caused by environmental agents and selected chemical compounds is presented. We focus on the results obtained by means of the force spectroscopy using the tip of an atomic force microscope as an imaging and nanoindentation spectroscopic probe. The findings presented in this review allow validating the nanoindentation method as a tool for quantitative cell elasticity probing and thereby allow proposing a nanomechanical model of endothelial dysfunction that could be practically used for drug efficacy and toxicity profiling in the endothelium at the subcellular level.