Developing force measurement techniques for cell mechanics and adhesion.

Cellular force is essential in maintaining the normal function of a biological cell. The primary goal of this study is to develop experimental methods to quantitatively determine forces generated from cell contraction and cell-to-cell adhesion. A novel method has been developed to measure the cell contraction forces exerted within a cell-embedded collagen matrix. The technique provides a 3D cell-matrix model which allows estimation of the cell contraction forces over a certain period of time. It was found that embedded fibroblast cells are able to cause a shrinkage of their surrounding matrix due to cell contractility. Tailored equipment which has ultimate force and displacement resolutions of 10 nN and 100 nm respectively has been constructed to accurately determinate the elasticity of cell-embedded collagen matrix. In combination with a mathematical model, the cell contraction force can be calculated based on the geometric parameters of the collagen matrix before and after the shrinkage. Reagents of agonist (histamine) or antagonist (ML-7) have been used to stimulate or block the fibroblast contraction force. They both show the effect of altering the stiffness of an extracellular matrix which is critical in the determination of cell contraction forces. More importantly, the analysis of the measured data based on a non-linear mechanical model have also confirmed that the elasticity of the extracellular matrix will influence the fibroblast contraction force. Cell-to-cell adhesion is an intricate interplay of mechanical, chemical and electrical signals between cells. A novel method based on Atomic Force Microscopy Single Cell Force Spectroscopy (AFM-SCFS) has been applied to examine cell-to-cell adhesion of human kidney proximal tubule HK2 cells. Ketamine was used to evoke early changes in expression of proteins (E-cadherin, N-cadherin and β-catenin) central to adherens junctions that lead to the loss of cell-to-cell adhesion force. The results provide strong evidence that the illicit substance Ketamine has major impacts on renal function and the loss of intracellular adherent energy. Overall, both cell contraction and cell-to-cell adhesion experiments demonstrated that the changes of biological states evoke protein interactions which would ultimately lead to the biomechanical alterations. Therefore, characterising the changes of mechanobiological properties can provide new insight into the investigation of physiological and pathological issues

Collagen matrix stiffness influences on fibroblast contraction force

Cell-embedded hydrogel has been widely used as engineered tissue equivalents in biomedical applications. In this study, contraction force in human aortic adventitial fibroblasts seeded within a 3D collagen matrix was quantified by a novel force sensing technique. We demonstrate that contraction forces in cells treated with histamine are regulated by the gel stiffness in a linear manner. These findings provide novel insights for the design of collagen-based biomaterials for tissue engineering and clinical applications

Dicarbonyl stress and beta-cell dysfunction

Methylglyoxal may be involved in the early stages of decline in glucose tolerance and decline in pancreatic beta-cell function leading to type 2 diabetes. Methylglyoxal and methylglyoxal-modified proteins increase during short-term increases in glucose concentration, infusion of exogenous methylglyoxal in rats and mice impaired glucose tolerance and glyoxalase 1 (Glo1) prevented beta-cell toxicity. We investigated the role of methylglyoxal and protein glycation on beta-cell function and the development of diabetes, with focus on the interactions of cells with the extracellular matrix. Impairments in adhesion of MIN6 insulinoma cells to methylglyoxal-glycated collagen IV were assessed in vitro using atomic force microscopy force spectroscopy and methylglyoxal glycation adducts within the pancreas visualised by immunostaining. Minimal glycation of collagen IV at integrin binding sites impaired binding of MIN6 cells in vitro and resulted in a 91% decrease in the energy necessary to detach the cells from collagen. Evidence from high fat diet fed mice showed that the methylglyoxal concentration of the pancreas was increased in the insulin resistant, pre-diabetic state with methylglyoxal-derived glycation adducts within the pancreas predominantly localised to the extracellular matrix in vivo. We conclude that glycation of extracellular matrix collagen IV likely impairs adhesion of beta-cells to the extracellular matrix in pre-diabetes in vivo and may thereby contribute to beta-cell glucotoxicity and dysfunction with progression to type 2 diabetes mellitus

'Special K' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine

Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention

Vascular Adhesion Protein-1 Determines the Cellular Properties of Endometrial Pericytes

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule and a primary amine oxidase involved in immune cell trafficking. Leukocyte extravasation into tissues is mediated by adhesion molecules expressed on endothelial cells and pericytes. Pericytes play a major role in the angiogenesis and vascularization of cycling endometrium. However, the functional properties of pericytes in the human endometrium are not known. Here we show that pericytes surrounding the spiral arterioles in midluteal human endometrium constitutively express VAP-1. We first characterize these pericytes and demonstrate that knockdown of VAP-1 perturbed their biophysical properties and compromised their contractile, migratory, adhesive and clonogenic capacities. Furthermore, we show that loss of VAP-1 disrupts pericyte-uterine natural killer cell interactions in vitro. Taken together, the data not only reveal that endometrial pericytes represent a cell population with distinct biophysical and functional properties but also suggest a pivotal role for VAP-1 in regulating the recruitment of innate immune cells in human endometrium. We posit that VAP-1 could serve as a potential biomarker for pregnancy pathologies caused by a compromised perivascular environment prior to conception

Collagen matrix stiffness influencing on fibroblast contraction force

Fibroblast-seeded collagen gel which constructs a 3D bio-artificial extracellular matrix (ECM) has been widely used in wound healing, tissue engineering and regenerative medicine. Previous studies using fibroblast-populated collagen based assay to measure cell contractility were limited to examine the radius changes of gel and hence accurate determination of cell contraction force was difficult to achieve[1],[2]. Up to now, little has been known about how the stiffness of collagen matrix influences on cell contraction force. We have recently developed a novel nano-indentation device to measure the gel elasticity and in combination with a mathematical model, the cell contraction force can be determined accurately based on the measured gel thickness and area[3]. In this study, we apply the new technique to investigate the effect of gel stiffness on cell contraction force in a quantitative manner

A novel collagen gel-based measurement technique for quantitation of cell contraction force

Cell contraction force plays an important role in wound healing, inflammation, angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 µM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 µM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen, while the Young's modulus of the gel decreases due to the gel degradation

Ketamine reduces cell adhesion.

<p>AFM-single-cell force spectroscopy was used to measure the detachment energy (fJoules) and maximum unbinding force (nN) required to uncouple two HK2 cells. The energy required to separate the cells; grey area in panel A (control cells), panel B (0.1 mg/mL Ketamine treated cells), panel C (0.5 mg/mL Ketamine treated cells), and panel D (1 mg/mL Ketamine treated cells), and maximum force of detachment (red circle) was measured. The former is known as the “detachment energy” (panel E) and the later is “maximum unbinding force” (panel F). Ketamine decreased the maximum unbinding force and the work of adhesion in a dose dependent manner compared to control. Data is expressed as mean ± SEM. of multiple cells from 4 separate experiments, where key significances are shown, **** <i>P</i><0.0001.</p