Endothelial glycocalyx detection and characterization by means of atomic force spectroscopy: Comparison of various data analysis approaches.

In recent years, atomic force spectroscopy (AFS) has been used to detect and characterize the endothelial glycocalyx (eGlx) in in vitro and ex vivo experiments. Several analysis methods were proposed, which differ not only in the numerical implementations, but also in physical models of glycocalyx description. Therefore, it is difficult to directly relate the experiments performed by different groups. In this work, we compared different models used for quantitative analysis of atomic force spectroscopy datasets recorded for eGlx. To capture glycocalyx at various structural conditions, we used basic enzymatic protocols for glycocalyx removal and restoration in human aortal endothelial cells (HAEC). Nanoindentation experiments for this model system were performed for (i) untreated cells, (ii) for cells after heparinase incubation, which enzymatically removes glycocalyx, (iii) for cells with successive heparin treatment, which partially restores the glycocalyx layer. Analysis of nanoindentation data was performed using different models: (a) a single-layer contact mechanics, (b) a double-layer model contact mechanics, (c) a polymer "brush" two-layer model based on the Alexander - de Gennes theory and (d) a simple single-layer "mechanical spring" model. Although different physical parameters are evaluated in methods (a-d), we show that all approaches revealed similar qualitative changes of the glycocalyx layer, which reflected the processes of glycocalyx degradation and its partial restoration. This paper may facilitate a direct comparison of past and future glycocalyx oriented AFS experiments that are analysed with different approaches.

Metformin attenuates adhesion between cancer and endothelial cells in chronic hyperglycemia by recovery of the endothelial glycocalyx barrier.

BACKGROUND: Epidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized. METHODS: Methods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression. RESULTS: SCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1. CONCLUSION: Our results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells. GENERAL SIGNIFICANCE: Metformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.