Gerald A. Meininger

Cancer cell adhesion to the endothelium is a crucial process in hematogenous metastasis, but how the integrity of the endothelial barrier and endothelial cell (EC) mechanical properties influence the adhesion between metastatic cancer cells and the endothelium remain unclear. In the present study, we have measured the adhesion between single cancer cells and two types of ECs at various growth states and their mechanical properties (elasticity) using atomic force microscopy single cell force spectroscopy. We demonstrated that the EC stiffness increased and adhesion with cancer cells decreased, as ECs grew from a single cell to a confluent state and developed cell-cell contacts, but this was reversed when confluent cells returned to a single state in a scratch assay. Our results suggest that the integrity of the endothelial barrier is an important factor in reducing the ability of the metastatic tumor cells to adhere to the vascular endothelium, extravasate and lodge in the vasculature of a distant organ where secondary metastatic tumors would develop.

Editorial: Traditional Chinese Medicine: Organ Vascular Injury - Volume II

Not applicable - article is an editorial/commentary

Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca²⁺ ([Ca²⁺]_i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-α₅ antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an α₅ integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-α₅ antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca²⁺]_i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca²⁺]_i, which was measured using the Ca²⁺ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca²⁺]_i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca²⁺]_i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.

Bone is a common site of metastasis for breast cancer and the mechanisms of metastasis are not fully elucidated. The purpose of our study was to characterize temporal and molecular dynamics of adhesive interactions between human breast cancer cells (HBCC) and human bone marrow endothelium (HBME) with piconewton resolution using atomic force microscopy (AFM). In adhesion experiments, a single breast cancer cell, MDA-MB-231 (MB231) or MDA-MB-435 (MB435) was attached to the AFM cantilever and brought into contact with a confluent HBME monolayer for different time periods (0.5 to 300 sec). The forces required to rupture individual molecular interactions and completely separate interacting cells were analyzed as measures of cell-cell adhesion. Adhesive interactions between HBME and either MB231 or MB435 cells increased progressively as cell-cell contact time was prolonged from 0.5 to 300 sec due to the time-dependent increase in the number and frequency of individual adhesive events, as well as to the involvement of stronger ligand-receptor interactions over time. Studies of the individual molecule involvement revealed that Thomsen-Friedenreich antigen (TF-Ag), galectin-3, integrin-β1, and integrin-α3 are all contributing to HBCC/HBME adhesion to various degrees in a temporally defined fashion. In conclusion, cell-cell contact time enhances adhesion of HBCC to HBME and the adhesion is mediated, in part, by TF-Ag, galectin-3, integrin-α3, and integrin-β1.

Adhesive interactions between living cells or ligand-receptor interactions can be studied at the molecular level using atomic force microscopy (AFM). Adhesion force measurements are performed with functionalized AFM probes. In order to measure single ligand-receptor interactions, a cantilever with a pyramidal tip is functionalized with a bio-recognized ligand (e.g., extracellular matrix protein). The ligand-functionalized probe is then brought into contact with a cell in culture to investigate adhesion between the respective probe-bound ligand and endogenously expressed cell surface receptors (e.g., integrins or other adhesion receptor). For experiments designed to examine cell-cell adhesions, a single cell is attached to a tipless cantilever which is then brought into contact with other cultured cells. Force curves are recorded to determine the forces necessary to rupture discrete adhesions between the probe-bound ligand and receptor, or to determine total adhesion force at cell-cell contacts. Here, we describe the procedures for measuring adhesions between (a) fibronectin and α5β1 integrin, and (b) breast cancer cells and bone marrow endothelial cells.

Atomic force microscopy (AFM) is an attractive technique for studying biomechanical and morphological changes in live cells. Using real-time AFM monitoring of cellular mechanical properties, spontaneous oscillations in cell stiffness and cell adhesion to the extracellular matrix (ECM) have been found. However, the lack of automated analytical approaches to systematically extract oscillatory signals, and noise filtering from a large set of AFM data, is a significant obstacle when quantifying and interpreting the dynamic characteristics of live cells. Here we demonstrate a method that extends the usage of AFM to quantitatively investigate live cell dynamics. Approaches such as singular spectrum analysis (SSA), and fast Fourier transform (FFT) were introduced to analyze a real-time recording of cell stiffness and the unbinding force between the ECM protein-decorated AFM probe and vascular smooth muscle cells (VSMCs). The time series cell adhesion and stiffness data were first filtered with SSA and the principal oscillatory components were isolated from the noise floor with the computed eigenvalue from the lagged-covariance matrix. Following the SSA, the oscillatory parameters were detected by FFT from the noise-reduced time series data sets and the sinusoidal oscillatory components were constructed with the parameters obtained by FFT.

Key points: N‐cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N‐cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10⁻⁵ m) and ACh (10⁻⁵ m). N‐cadherin‐coated beads were able to induce clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10⁻⁵ m) or sodium nitroprusside (10⁻⁵ m) induced a significant increase or decrease in the N‐cadherin‐EGFP clustering, respectively. Application of pulling force (∼1 nN) to the N‐cadherin‐coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. N‐cadherin is the major cell–cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N‐cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N‐cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90 mmHg, both the density and the average size of N‐cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10^–5 m) induced a significant increase of N‐cadherin AJ density at 50 mmHg, whereas vasodilatation induced by ACh (10^–5 m) was accompanied by a significant decrease in density and size of N‐cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano‐responsive properties of N‐cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N‐cadherin‐coated microbead (5 μm) induced a progressive clustering of N‐cadherin‐enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1 nN) to the N‐cadherin‐coated‐beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10^–5 m) or sodium nitroprusside (10^–5 m) induced a significant increase or decrease of the N‐cadherin‐EGFP clustering, respectively. These observations provide compelling evidence that N‐cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N‐cadherin AJs in vasomotor regulation.

Small arteries and their component cellular and non‐cellular elements are continually subjected to, and interact with, mechanical forces. Such interactions are key in both short‐ and long‐term adaptation of the structure and function of the microcirculation to its local environment and metabolic requirements. Following this brief introduction is a series of papers presented as a symposium (Small Artery Mechanobiology: Roles of Cellular and Non‐Cellular Elements) at the World Congress for Microcirculation, Kyoto 2015.

Vascular stiffening, a risk factor for cardiovascular disease, is accelerated, particularly in women with obesity and type 2 diabetes. Preclinical evidence suggests that dipeptidylpeptidase-4 (DPP-4) inhibitors may have cardiovascular benefits independent of glycemic lowering effects. Recent studies show that consumption of a western diet (WD) high in fat and simple sugars induces aortic stiffening in female C57BL/6J mice in advance of increasing blood pressure. The aims of this study were to determine whether administration of the DPP-4 inhibitor, linagliptin (LGT), prevents the development of aortic and endothelial stiffness induced by a WD in female mice. C56Bl6/J female mice were fed a WD for 4 months. Aortic stiffness and ex vivo endothelial stiffness were evaluated by Doppler pulse wave velocity (PWV) and atomic force microscopy (AFM), respectively. In addition, we examined aortic vasomotor responses and remodeling markers via immunohistochemistry. Results were analyzed via 2-way ANOVA, p < 0.05 was considered as statistically significant. Compared to mice fed a control diet (CD), WD-fed mice exhibited a 24 % increase in aortic PWV, a five-fold increase in aortic endothelial stiffness, and impaired endothelium-dependent vasodilation. In aorta, these findings were accompanied by medial wall thickening, adventitial fibrosis, increased fibroblast growth factor 23 (FGF-23), decreased Klotho, enhanced oxidative stress, and endothelial cell ultrastructural changes, all of which were prevented with administration of LGT. The present findings support the notion that DPP-4 plays a role in development of WD-induced aortic stiffening, vascular oxidative stress, endothelial dysfunction, and vascular remodeling. Whether, DPP-4 inhibition could be a therapeutic tool used to prevent the development of aortic stiffening and the associated cardiovascular complications in obese and diabetic females remains to be elucidated.