Stiffness memory of EA.hy926 endothelial cells in response to chronic hyperglycemia.

BACKGROUND: Glycemic memory of endothelial cells is an effect of long-lasting hyperglycemia and is a cause of various diabetics complications, that arises despite of the treatment targeted towards returning low glucose level in blood system. On the other hand, endothelial dysfunction, which is believed to be a main cause of cardiovascular complications, is exhibited in the changes of mechanical properties of cells. Although formation of the glycemic memory was widely investigated, its impact on the mechanical properties of endothelial cells has not been studied yet. METHODS: In this study, nanoindentaion with a tip of an atomic force microscope was used to probe the long-term changes (through 26 passages, c.a. 80 days) in mechanical properties of EA.hy926 endothelial cells cultured in hyperglycemic conditions. As a complementary method, alterations in the structure of actin cytoskeleton were visualized by fluorescent staining of F-actin. RESULTS: We observed a gradual stiffening of the cells up to 20th passage for cells cultured in high glucose (25 mM). Fluorescence imaging has revealed that this behavior resulted from systematic remodeling of the actin cytoskeleton. In further passages, a drop in stiffness had occurred. The most interesting finding was recorded for cells transferred after 14 passages from high glucose to normal glucose conditions (5mM). After the transfer, the initial drop in stiffness was followed by a return of the cell stiffness to the value previously observed for cells cultured constantly in high glucose CONCLUSIONS: Our results indicate that glycemic memory causes irreversible changes in stiffness of endothelial cells. The formation of the observed "stiffness memory" could be important in the context of vascular complications which develop despite the normalization of the glucose level.

Elasticity changes anti-correlate with NO production for human endothelial cells stimulated with TNF-α.

Tumor necrosis factor alpha (TNF-α) is a critical cytokine that is involved in systemic inflammatory response and contributes to the activation of the pro-inflammatory phenotype of the endothelium. In the present study, effects of TNF-α on morphology and elasticity of endothelium in relation to the production of NO and actin fiber reorganization were analyzed in human dermal microvascular endothelial cells. The cells were incubated in MCDB medium solution and stimulated with [Formula: see text] of TNF-α. Atomic force microscopy measurements have enabled characterization of cell morphology and elastic properties in physiological conditions. The spectrophotometric Griess method was applied to estimate nitric oxide (NO) production of the cells. We demonstrated that TNF-α-induced changes in elasticity of endothelium anti-correlate with NO production and are associated with the reorganization of actin cytoskeleton.

Stiffness changes of tumor HEp2 cells correlates with the inhibition and release of TRAIL-induced apoptosis pathways.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a promising apoptotic agent that can selectively act on tumor cells. However, some cancer cells are resistant to TRAIL mediated apoptosis. In specific type of cells, sensitization by chemotherapeutic drugs may overcome the resistance to TRAIL induced apoptosis. In this work, atomic force microscopy (AFM) nanoindentation spectroscopy combined with fluorescence methods were used to investigate the biomechanical aspects of the resistance and unblocking of apoptosis in larynx carcinoma HEp2 cells treated with TRAIL. It is shown that there is a direct correlation between the increase in mechanical cell stiffness and the inhibition of apoptosis induced by TRAIL in HEp2 cells. Conversely, unblocking of apoptosis by sensitization of HEp2 cells with a chemotherapeutic drug Actinomycin D is related to the depolymerization of F-actin and to the decrease in the cell stiffness. Both effects, that is, changes in the mechanical stiffness of the cell and the inhibition of apoptotic pathway, are closely related to the Bcl-2 activity. Most probably, the depolymerization of F-actin results from downregulation of Rho protein, which in turn is accompanied by a lower activity of Bcl-2 and in consequence releases the intrinsic apoptotic channel. The presented results reveal a promising application of nanoindentation spectroscopy with an AFM tip as a novel tool for monitoring the processes of apoptosis inhibition.

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.

Glutaraldehyde fixation preserves the trend of elasticity alterations for endothelial cells exposed to TNF-α.

Among the users of atomic force microscopy based techniques, there is an ongoing discussion, whether cell elasticity measurements performed on fixed cells could be used for determination of the relative elasticity changes of the native (unfixed) cells subjected to physiologically active external agents. In this article, we present a case, for which the legitimacy of cell fixation for elasticity measurements is justified. We provide an evidence that the alterations of cell elasticity triggered by tumor necrosis factor alpha (TNF-α) in EA.hy926 endothelial cells are preserved after glutaraldehyde (GA) fixation. The value of post-fixation elasticity parameter is a product of the elasticity parameter obtained for living cells and a constant value, dependent on the GA concentration. The modification of the initial value of elasticity parameter caused by remodeling of the cortical actin cytoskeleton is reflected in the elasticity measurements performed on fixed cells. Thus, such fixation procedure may be particularly helpful for experiments, where the influence of an external agent on the cell cortex should be assessed and AFM measurements of living cells are problematic or better statistics is needed.