Localized Tensional Forces on PECAM-1 Elicit a Global Mechanotransduction Response via the Integrin-RhoA Pathway

SummaryBackgroundMechanical forces regulate cell behavior and function during development, differentiation, and tissue morphogenesis. In the vascular system, forces produced by blood flow are critical determinants not only of morphogenesis and function, but also of pathological states such as atherosclerosis. Endothelial cells (ECs) have numerous mechanotransducers, including platelet endothelial cell adhesion molecule-1 (PECAM-1) at cell-cell junctions and integrins at cell-matrix adhesions. However, the processes by which forces are transduced to biochemical signals and subsequently translated into downstream effects are poorly understood.ResultsHere, we examine mechanochemical signaling in response to direct force application on PECAM-1. We demonstrate that localized tensional forces on PECAM-1 result in, surprisingly, global signaling responses. Specifically, force-dependent activation of phosphatidylinositol 3-kinase (PI3K) downstream of PECAM-1 promotes cell-wide activation of integrins and the small GTPase RhoA. These signaling events facilitate changes in cytoskeletal architecture, including growth of focal adhesions and adaptive cytoskeletal stiffening.ConclusionsTaken together, our work provides the first evidence of a global signaling event in response to a localized mechanical stress. In addition, these data provide a possible mechanism for the differential stiffness of vessels exposed to distinct hemodynamic force patterns in vivo

Visualization of individual carbon nanotubes with fluorescence microscopy using conventional fluorophores

We demonstrate that individual carbon nanotubes (CNTs) can be visualized with fluorescence microscopy through noncovalent labeling with conventional fluorophores. Reversal of contrast in fluorescence imaging of the CNTs was observed when performing labeling procedure in a nonpolar solvent. Our results are consistent with a CNT-fluorophore affinity mediated by hydrophobic interaction. The reverse-contrast images also provide clear indication of nanotube location. © 2003 American Institute of Physics

A pediatric airway atlas and its application in subglottic stenosis

Young children with upper airway problems are at risk for hypoxia, respiratory insufficiency and long term morbidity. Computational models and quantitative analysis would reveal airway growth patterns and benefit clinical care. To capture expected growth patterns we propose a method to build a pediatric airway atlas as a function of age. The atlas is based on a simplified airway model in combination with kernel regression. We show experimental results on children with subglottic stenosis to demonstrate that our method is able to track and measure the stenosis in pediatric airways

The Virtual Pediatric Airways Workbench

The Virtual Pediatric Airways Workbench (VPAW) is a patient-centered surgical planning software system targeted to pediatric patients with airway obstruction. VPAW provides an intuitive surgical planning interface for clinicians and supports quantitative analysis regarding prospective surgeries to aid clinicians deciding on potential surgical intervention. VPAW enables a full surgical planning pipeline, including importing DICOM images, segmenting the airway, interactive 3D editing of airway geometries to express potential surgical treatment planning options, and creating input files for offline geometric analysis and computational fluid dynamics simulations for evaluation of surgical outcomes. In this paper, we describe the VPAW system and its use in one case study with a clinician to successfully describe an intended surgery outcome

Size-Uniform 200 nm Particles: Fabrication and Application to Magnetofection

We report on the fabrication of arrays of mono- and multimetallic particles via metal evaporation onto lithographically patterned posts, as well as the magnetic force calibration and successful magnetofection of iron particles grown via this method. This work represents the first instance in which metal evaporation onto post structures was used for the formation of released, shape-defined metal particles. Also, our work represents the first use of lithographically defined particles as agents of magnetofection. Using these techniques it is possible to create particles with complex shapes and lateral dimensions as small as 40 nm. Our demonstrated compositionally flexible particles are highly size-uniform due to their photolithographically defined growth substrates, with particle dimensions along two axes fixed at 200 nm; the third axis dimension can be varied from 20 nm to 300 nm during the deposition procedure. Atomic percent of metals incorporated into the particle volume is highly tunable and particles have been synthesized with as many as four different metals. We performed magnetic force calibrations on a single particle size for iron particles using an axially magnetized NeFeB permanent magnet and comparisons are made with commercially available magnetic beads. In order to evalutate their usefulness as magnetofection agents, an antisense oligonucleotide (ODN) designed to correct the aberrant splicing of enhanced green fluorescent protein mRNA, was successfully transfected into a modified HeLa cell line. Magnetically enhanced gene delivery was accomplished in vitro using antisense ODN-laden iron particles followed by application of a field gradient. Magnetically enhanced transfection resulted in a 76% and 139% increase in fluorescence intensity when compared to Lipofectamine and antisense ODN-loaded particles delivered without magnetic treatment, respectively. To our knowledge, these experiments constitute the first use of lithographically defined particles as successful agents for magnetically enhanced transfection of an antisense oligonucleotide

Pediatric sleep-related breathing disorders: advances in imaging and computational modeling

We understand now that sleep of sufficient length and quality is required for good health. This is particularly true for infants and children, who have the added physiologic task of growth and development, as compared to their adult counterparts. Sleep-related breathing disorders (SRBDs) are common in childhood and if unrecognized and not treated can result in significant morbidity. For example, children with obstructive sleep apnea (OSA) can exhibit behavioral, mood, and learning difficulties. If left untreated, alterations in the function of the autonomic nervous system and a chronic inflammatory state result, contributing to the risk of heart disease, stroke, glucose intolerance, and hypertension in adulthood

Mechanical Stiffness Grades Metastatic Potential in Patient Tumor Cells and in Cancer Cell Lines

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here we apply a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacological inhibition of myosin II increases invasiveness, while increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By demonstrating that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer

Volumetric nasal cavity analysis in children with unilateral and bilateral cleft lip and palate

OBJECTIVES/HYPOTHESIS: Children with cleft lip and palate (CLP) often suffer from nasal obstruction that may be related to effects on nasal volume. The objective of this study was to compare side:side volume ratios and nasal volume in patients with unilateral (UCLP) and bilateral (BCLP) clefts with age-matched controls. STUDY DESIGN: Retrospective case-control study using three-dimensional (3D) nasal airway reconstructions. METHODS: We analyzed 20 subjects (age range = 7-12 years) with UCLP and BCLP from a regional craniofacial center who underwent cone beam computed tomography (CT) prior to alveolar grafting. Ten multislice CT images from age-matched controls were also analyzed. Mimics software (Materialise, Plymouth, MI) was used to create 3D reconstructions of the main nasal cavity and compute total and side-specific nasal volumes. Subjects imaged during active nasal cycling phases were excluded. RESULTS: There was no statistically significant difference in affected:unaffected side volume ratios in UCLP (P = .48) or left:right ratios in BCLP (P = .25) when compared to left:right ratios in controls. Mean overall nasal volumes were 9,932 ± 1,807, 7,097 ± 2,596, and 6,715 ± 2,115 mm(3) for control, UCLP, and BCLP patients, respectively, with statistically significant volume decreases for both UCLP and BCLP subjects from controls (P < .05). CONCLUSIONS: This is the first study to analyze total nasal volumes in BCLP patients. Overall nasal volume is compromised in UCLP and BCLP by approximately 30%. Additionally, our finding of no major difference in side:side ratios in UCLP and BCLP compared to controls conflicts with pre-existing literature, likely due to exclusion of actively cycling scans and our measurement of the functional nasal cavity

Quantitative assessment of the upper airway in infants and children with subglottic stenosis

OBJECTIVES/HYPOTHESIS: Determine whether quantitative geometric measures and a computational fluid dynamic (CFD) model derived from medical imaging of children with subglottic stenosis (SGS) can be effective diagnostic and treatment planning tools. STUDY DESIGN: Retrospective chart and imaging review in a tertiary care hospital. METHODS: Computed tomography scans (n = 17) of children with SGS were analyzed by geometric and CFD methods. Polysomnograms (n = 15) were also analyzed. Radiographic data were age/weight flow normalized and were compared to an atlas created from radiographically normal airways. Five geometric, seven CFD, and five polysomnography measures were analyzed. Statistical analysis utilized a two-sample t test with Bonferroni correction and area under the curve analysis. RESULTS: Two geometric indices (the ratio of the subglottic to midtracheal airway, the percent relative reduction of the subglottic airway) and one CFD measure (the percent relative reduction of the hydraulic diameter of the subglottic airway) were significant for determining which children with SGS received surgical intervention. Optimal cutoffs for these values were determined. Polysomnography, the respiratory effort-related arousals index, was significant only prior to Bonferroni correction for determining which children received surgical intervention. CONCLUSIONS: Geometric and CFD variables were sensitive at determining which patients with SGS received surgical intervention. Discrete quantitative assessment of the pediatric airway was performed, yielding preliminary data regarding possible objective thresholds for surgical versus nonsurgical treatment of disease. This study is limited by its small, retrospective, single-institution nature. Further studies to validate these findings and possibly optimize treatment threshold recommendations are warranted

Mechanical Properties and Gene Expression of Chondrocytes on Micropatterned Substrates Following Dedifferentiation in Monolayer

Chondrocytes in articular cartilage normally exhibit high expression of collagen II and aggrecan but rapidly dedifferentiate to a fibroblastic phenotype if passaged in culture. Previous studies have suggested that the loss of chondrocyte phenotype is associated with changes in the structure of the F-actin cytoskeleton, which also controls cell mechanical properties. In this study, we examined how dedifferentiation in monolayer influences the mechanical properties of chondrocytes isolated from different zones of articular cartilage. Atomic force microscopy was used to measure the mechanical properties of superficial and middle/deep zone chondrocytes as they underwent serial passaging and subsequent growth on fibronectin-coated, micropatterned self-assembled monolayers (MSAMs) that restored a rounded cell shape in 2D culture. Chondrocytes exhibited significant increases in elastic and viscoelastic moduli with dedifferentiation in culture. These changes were only partially ameliorated by the restoration of a rounded shape on micropatterned surfaces. Furthermore, intrinsic zonal differences in cell mechanical properties were rapidly lost with passage. These findings indicate that cell mechanical properties may provide additional measures of phenotypic expression of chondrocytes as they undergo dedifferentiation and possibly redifferentiation in culture