The apelin receptor influences biomechanical and morphological properties of endothelial cells.

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions.

3D Micropillars Guide the Mechanobiology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

3D micropillars generated by photolithography are used as a platform to probe by atomic force microscopy the mechanodynamics of human induced pluripotent stem cell-derived cardiomyocytes. 3D micropillars guide subcellular cytoskeletal modifications of cardiomyocytes and lead to biochemical changes altering beating rate, stiffness, and calcium dynamics of the cells.

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

BACKGROUND: HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. STUDY DESIGN AND METHODS: The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. RESULTS: HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. CONCLUSION: Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

A novel contact model for AFM indentation experiments on soft spherical cell-like particles.

The use of the simple Hertz model for the analysis of Atomic Force Microscopy (AFM) force-distance curves measured on soft spherical cell-like particles leads to significant underestimations of the objects Young's modulus E. To correct this error, a mixed double contact model (based on the simple Hertz model and the Johnson-Kendall-Roberts (JKR) model) was derived. The model considers two independent particle deformation sites: (i) the upper part of the particle is deformed by the AFM indenter, (ii) the bottom part is deformed by the substrate, which is usually unnoticed. It becomes apparent that for soft particles even small forces between substrate and particle can influence the resulting force-distance curves. For instance we show, that a gravity-induced compression on the particle bottom side can have significant influence on the measurements. To highlight these observations, the deviation of the particle Young's modulus E between the simple Hertz model and our model is calculated. This error strongly depends on the ratio of the three involved radii: (i) the radius of the AFM indenter, (ii) the radius of the particle and (iii) the radius of the substrate as well as on the acting gravity force. Overall, the analysis suggests that for nanoscopic indenters the deviation is negligible, whereas the use of microscopic indenters results in significant errors that can be corrected via the presented model. This is important especially for very soft particles, since larger indenters can achieve higher signal to noise ratios. Furthermore, the applicability of the model was confirmed by indentation experiments on hydrogel microbeads. The mixed double contact model is applicable to a large range of indenter geometries and can be adapted for other contact models.

Stiffness of left ventricular cardiac fibroblasts is associated with ventricular dilation in patients with recent-onset nonischemic and nonvalvular cardiomyopathy.

BACKGROUND: Ventricular dilation is known as a pivotal predictor in recent-onset cardiomyopathy (ROCM), but its pathophysiology is not fully understood. In the present study we investigated whether single-cell stiffness of right and left ventricular-derived fibroblasts has an effect on cardiac phenotype in patients with ROCM. METHODS AND RESULTS: Patients with endomyocardial biopsy-proven ROCM were included (n=10). Primary cardiac fibroblasts (CFBs) were cultured from left and right ventricular endomyocardial biopsies and their single-cell stiffness was analyzed by quantification of Young's modulus using colloidal probe atomic force microscopy. Cardiac fibrosis was analyzed by Masson's trichrome staining. CFBs from the left ventricle showed significantly decreased stiffness when compared with CFBs from the right ventricle, indexed by decreased stiffness (Young's modulus 3,374±389 vs. 4,837±690 Pa; P<0.05). Young's modulus of CFBs derived from the left ventricle correlated negatively with the left ventricular end-diastolic dimension derived from 2-dimensional echocardiography (R(2)=0.77; P<0.01). Neither left nor right ventricular fibrosis correlated with the respective ventricular dimensions. CONCLUSIONS: Our data suggest that a decrease in single-cell stiffness of left ventricular fibroblasts could trigger left ventricular dilation in patients with ROCM. This implies a new potential mechanism for the ventricular dilation with this disease.