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The toxic effects of acrylamide on cytoskeletal integrity and ion channel balance is well-established in many cell types, but there has been little examination regarding the effects of acrylamide on primary cardiomyocytes, despite the importance of such components in their function. Furthermore, acrylamide toxicity is generally examined using concentrations higher than those found in vivo under starch-rich diets. Accordingly, we sought to characterize the dose-dependent effects of acrylamide on various properties, including cell morphology, contraction patterns, and junctional connexin 43 staining, in primary cardiomyocytes. We show that several days exposure to 1-100 μM acrylamide resulted in altered morphology, irregular contraction patterns, and an increase in the amount of immunoreactive signal for connexin 43 at cell junctions. We conclude that dietary levels of acrylamide may alter cellular function with prolonged exposure, in primary cardiomyocytes.

Tetraspanin CD151 is highly expressed in endothelial cells and regulates pathologic angiogenesis. However, the mechanism by which CD151 promotes vascular morphogenesis and whether CD151 engages other vascular functions are unclear. Here we report that CD151 is required for maintaining endothelial capillary-like structures formed in vitro and the integrity of endothelial cell-cell and cell-matrix contacts in vivo. In addition, vascular permeability is markedly enhanced in the absence of CD151. As a global regulator of endothelial cell-cell and cell-matrix adhesions, CD151 is needed for the optimal functions of various cell adhesion proteins. The loss of CD151 elevates actin cytoskeletal traction by up-regulating RhoA signaling and diminishes actin cortical meshwork by down-regulating Rac1 activity. The inhibition of RhoA or activation of cAMP signaling stabilizes CD151-silenced or -null endothelial structure in vascular morphogenesis. Together, our data demonstrate that CD151 maintains vascular stability by promoting endothelial cell adhesions, especially cell-cell adhesion, and confining cytoskeletal tension.

Arrhythmogenic right ventricular cardiomyopathy is a primary heart muscle disorder characterized by the early occurrence of arrhythmias often out of proportion to the extent of structural remodeling and contractile derangement. Approximately 40% of patients with arrhythmogenic right ventricular cardiomyopathy have one or more mutations in genes encoding proteins in desmosomes, intercellular adhesion junctions which, in cardiac myocytes, reside within intercalated disks. Some desmosomal proteins fulfill roles both as structural proteins in cell–cell adhesion junctions and as signaling molecules in pathways mediated by Wnt ligands. Evidence is increasing that mutations in desmosomal proteins can perturb the normal balance of critical proteins in junctions and the cytosol which, in turn, could alter gene expression by circumventing normal Wnt signaling pathways. This review highlights recent advances in understanding the pathogenesis of arrhythmogenic right ventricular cardiomyopathy and presents evidence suggesting that the disease is caused by a combination of altered cellular biomechanical behavior and altered signaling.

Fluorescence laser tracking microrheometry (FLTM) is what we believe to be a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In an earlier article, we described the design, development, and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the work presented here, we demonstrate the suitability of FLTM to study local cellular rheology and obtain values for the storage and loss moduli G'(omega) and G''(omega) of fibroblasts consistent with past literature. We further establish that chemically induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provide a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogate and justify the relevance of bead endocytosis as a method of cellular internalization of 1-microm probes in FLTM. Second, we show that as sample temperature increases, FLTM findings are elevated toward higher frequencies. Third, we confirm that relevant bead sizes (1 and 2 microm) have no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (antiintegrin, antitransferrin, antidystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. Although these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(ω). This FLTM has nanometer spatial resolution over a frequency range extending from 1Hz to 50kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.

A 3D single-particle-tracking (SPT) system was developed based on two-photon excitation fluorescence microscopy that can track the motion of particles in three dimensions over a range of 100 mum and with a bandwidth up to 30 Hz. We have implemented two different techniques employing feedback control. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations that have been introduced into the optical system that break the axial symmetry of the point spread function and serve as an indicator of the particle's axial position. We verified the performance of the instrument by tracking particles in well-characterized models systems. We then studied the 3D viscoelastic mechanical response of 293 kidney cells using the techniques. Force was applied to the cells, by using a magnetic manipulator, onto the paramagnetic spheres attached to the cell via cellular integrin receptors. The deformation of the cytoskeleton was monitored by following the motion of nearby attached fluorescent polystyrene spheres. We showed that planar stress produces strain in all three dimensions, demonstrating that the 3D motion of the cell is required to fully model cellular mechanical responses.

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling.

Viscoelastic models of cells often treat cells as homogeneous objects. However, studies have demonstrated that cellular properties are local and can change dramatically on the basis of the location probed. Because membrane receptors are linked in various ways to the intracellular space, with some receptors linking to the cytoskeleton and others diffusing freely without apparent linkages, the cellular physical response to mechanical stresses is expected to depend on the receptor engaged. In this study, we tested the hypothesis that cellular mechanical stiffness as measured via cytoskeletally linked receptors is greater than stiffness measured via receptors that are not cytoskeletally linked. We used a magnetic micromanipulator to apply linear stresses to magnetic beads attached to living cells via selected receptors. One of the receptor classes probed, the dystroglycan receptors, is linked to the cytoskeleton, while the other, the transferrin receptors, is not. Fibronectin-coated beads were used to test cellular mechanical properties of the cytoskeleton without membrane dependence by allowing the beads to endocytose. For epithelial cells, transferrin-dependent stiffness and endocytosed bead-dependent stiffness were similar, while dystroglycan-dependent stiffness was significantly lower. For smooth muscle cells, dystroglycan-dependent stiffness was similar to the endocytosed bead-dependent stiffness, while the transferrin-dependent stiffness was lower. The conclusion of this study is that the measured cellular stiffness is critically influenced by specific receptor linkage and by cell type and raises the intriguing possibility of the existence of separate cytoskeletal networks with distinct mechanical properties that link different classes of receptors.

Cells face not only a complex biochemical environment but also a diverse biomechanical environment. How cells respond to variations in mechanical forces is critical in homeostasis and many diseases. The mechanisms by which mechanical forces lead to eventual biochemical and molecular responses remain undefined, and unraveling this mystery will undoubtedly provide new insight into strengthening bone, growing cartilage, improving cardiac contractility, and constructing tissues for artificial organs. In this article we review the physical bases underlying the mechanotransduction process, techniques used to apply controlled mechanical stresses on living cells and tissues to probe mechanotransduction, and some of the important lessons that we are learning from mechanical stimulation of cells with precisely controlled forces.

Background— Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. Methods and Results— Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127±5% of controls, P P P P P P P Conclusions— Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

A three-dimensional viscoelastic finite element model is developed for cell micromanipulation by magnetocytometry. The model provides a robust tool for analysis of detailed strain/stress fields induced in the cell monolayer produced by forcing one microbead attached atop a single cell or cell monolayer on a basal substrate. Both the membrane/cortex and the cytoskeleton are modeled as Maxwell viscoelastic materials, but the structural effect of the membrane/cortex was found to be negligible on the timescales corresponding to magnetocytometry. Numerical predictions are validated against experiments performed on NIH 3T3 fibroblasts and previous experimental work. The system proved to be linear with respect to cytoskeleton mechanical properties and bead forcing. Stress and strain patterns were highly localized, suggesting that the effects of magnetocytometry are confined to a region extending10 microm from the bead. Modulation of cell height has little effect on the results, provided the monolayer is5 micro m thick. NIH 3T3 fibroblasts exhibited a viscoelastic timescale of approximately 1 s and a shear modulus of approximately 1000 Pa.

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.

Aims The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins plakoglobin and plakophilin, which are linked to ARVC in patients. Methods and Results Cells expressing mutant plakoglobin or plakophilin showed no differences in cell-cell adhesion relative to controls, while knocking down these proteins weakened cell-cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell-cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared to controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3β inhibitor. Conclusions Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3β. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise.

Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients., We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg.

Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.

Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non–cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation (“stiffness”) was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required ≈10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor ( P

Background —Overloading the left ventricle in systole (pressure overload) is associated with a distinct morphological response compared with overload in diastole (volume overload). Methods and Results —We designed a novel computer-controlled experimental system that interfaces biaxially uniform strain with electrical pacing, so that cellular deformation can be imposed during a specified phase of the cardiac cycle. Cardiomyocytes were exposed to strain (4%) during either the first third (systolic phase) or last third (diastolic phase) of the cardiac cycle. Strain imposed during the systolic phase selectively activated p44/42 mitogen-activated protein kinase (MAPK) and MAPK/extracellular signal–regulated protein kinase kinase (MEK1/2, an activator of p44/42 MAPK) compared with strain imposed during the diastolic phase. In contrast, there was no difference in activation of p38 and c-Jun NH 2 -terminal kinases induced by strain imposed during the systolic phase (5.8- and 3.3-fold versus control, n=4) compared with the diastolic phase (5.5- and 3.1-fold). Induction of both brain natriuretic peptide (5.8-fold versus control, P P 3 H]leucine incorporation induced by strain imposed during the systolic phase (4.0-fold versus control) was greater than during the diastolic phase (2.7-fold, P Conclusions —Mechanical activation of p44/42 MAPK and MEK1/2, gene expression, and protein synthesis is regulated by the cardiac cycle, suggesting that mechanotransduction at the cellular level may underlie differences between pressure and volume overload of the heart.

Background —Increased biomechanical stresses in the fibrous cap of atherosclerotic plaques contribute to plaque rupture and, consequently, to thrombosis and myocardial infarction. Thin fibrous caps and large lipid pools are important determinants of increased plaque stresses. Although coronary calcification is associated with worse cardiovascular prognosis, the relationship between atheroma calcification and stresses is incompletely described. Methods and Results —To test the hypothesis that calcification impacts biomechanical stresses in human atherosclerotic lesions, we studied 20 human coronary lesions with techniques that have previously been shown to predict plaque rupture locations accurately. Ten ruptured and 10 stable lesions derived from post mortem coronary arteries were studied using large-strain finite element analysis. Maximum stress was not correlated with percentage of calcification, but it was positively correlated with the percentage of lipid ( P =0.024). When calcification was eliminated and replaced with fibrous plaque, stress changed insignificantly; the median increase in stress for all specimens was 0.1% (range, 0% to 8%; P =0.85). In contrast, stress decreased by a median of 26% (range, 1% to 78%; P =0.02) when lipid was replaced with fibrous plaque. Conclusions —Calcification does not increase fibrous cap stress in typical ruptured or stable human coronary atherosclerotic lesions. In contrast to lipid pools, which dramatically increase stresses, calcification does not seem to decrease the mechanical stability of the coronary atheroma.

There is a need to characterize biomechanical cell–cell interactions, but due to a lack of suitable experimental methods, relevant in vitro experimental data are often masked by cell–substrate interactions. This study describes a novel method to generate partially lifted substrate-free cell sheets that engage primarily in cell–cell interactions, yet are amenable to biological and chemical perturbations and, importantly, mechanical conditioning and characterization. A polydimethylsiloxane (PDMS) mold is used to isolate a patch of cells, and the patch is then enzymatically lifted. The cells outside the mold remain attached, creating a partially lifted cell sheet. This simple yet powerful tool enables the simultaneous examination of lifted and adherent cells. This tool was then deployed to test the hypothesis that the lifted cells would exhibit substantial reinforcement of key cytoskeletal and junctional components at cell–cell contacts, and that such reinforcement would be enhanced by mechanical conditioning. Results demonstrate that the mechanical strength and cohesion of the substrate-free cell sheets strongly depend on the integrity of the actomyosin cytoskeleton and the cell–cell junctional protein plakoglobin. Both actin and plakoglobin are significantly reinforced at junctions with mechanical conditioning. However, total cellular actin is significantly diminished on dissociation from a substrate and does not recover with mechanical conditioning. These results represent a first systematic examination of mechanical conditioning on cells with primarily intercellular interactions.

Contacting cells establish different classes of intricate structures at the cell-cell junctions. These structures are of increasing research interest as they regulate a broad variety of processes in development and disease. Further, in vitro studies are revealing that various cell-cell interaction proteins are involved not only in cell-cell processes but also in many additional aspects of physiology, such as migration and apoptosis. This chapter reviews the basic classification of cell-cell junctional structures and some of their representative proteins. Their roles in development and disease are briefly outlined, followed by a section on contemporary methods for probing cell-cell interactions and some recent developments. This chapter concludes with a few suggestions for potential research directions to further develop this promising area of study.

Connections between cell-cell junctions and cytoskeleton profoundly influence cell shape, motility and gene expression. However, cell-cell interactions are easily masked by cell-substrate interactions thus remains poorly characterized. A cell monolayer can be considered the simplest multicellular tissue structure, yet they fulfill critical roles in human physiology and tissue engineering. A systematic characterization of intercellular adhesion, cytoskeletal organization and signaling in a substrate-free cell monolayer is crucial for unlocking the potential for cell-sheet tissue engineering. To test our hypothesis that intercellular junctions and cytoskeleton components likely play significant roles in establishing cell sheet cohesion, we have developed a unique partial-lifting technique that allows generation and investigation of the biological and mechanical properties of substrate-freehuman keratinocytecell monolayer.Results demonstrated that through alterations of spatial organization and expression of their cytoskeleton and specialized intercellular adhesions, substrate-free cells create a monolayer tissue that has a much stronger mechanical strength than their substrate-adhering controls. To determine the contribution of intercellular junctions and cytoskeleton to cell sheet mechanics, we altered the junctional protein plakoglobin by siRNA interference, altered actin-myosin interactions by ROCK kinase inhibitor and actin disruption agent. Knockdown of plakoglobin significantly diminished its participation in intercellular junctions and resulted in weakened cell sheet strength. Similarly, actin disruption and actin-myosin contraction inhibition also diminished cell sheet strength. Next, we demonstrated that external mechanical stimuli further boosted the sheet's mechanical strength via increased junctional localizations and elevated expression levels, suggesting possible involvement of plakoglobin-related Wnt/β-catenin signaling and actin-related RhoA/ROCK signaling.Given the near-ubiquity of plakoglobin and actin in multicellular organisms, these findings could have significant implications for understanding the fundamental mechanisms that regulating cell adhesion and biomechanics, and improving the mechanical properties of tissue engineering constructs.

Rheological characterization of cells using passive particle tracking techniques can yield substantial information regarding local cellular material properties. However, limited work has been done to establish the changes in material properties of mechanically-responsive cells that experience external stimuli. In this study, cardiac fibroblasts plated on either fibronectin or collagen were treated with cytochalasin, mechanically stretched, or both, and their trajectories and complex moduli were extracted. Results demonstrate that both solid and fluid components were altered by such treatments in a receptor-dependent manner, and that, interestingly, cells treated with cytochalasin were still capable of stiffening in response to mechanical stimuli despite gross stress fiber disruption. These results suggest that the material properties of cells are dependent on a variety of environmental cues and can provide insight into physiological and disease processes.

Immortalized cells are commonly used as analogs for primary cells in many cell mechanics, tissue engineering, and biochemical assays. However, it is not well-established whether immortal cell lines can mimic the behavior of primary cells in more physiological (three-dimensional) environments. For this project, we investigate the mechanical properties of primary cardiac fibroblasts (CFs) and 3T3 transformed fibroblasts when cultured in cell bi-layers by comparing the cells’ viscoelastic properties.Copyright © 2012 by ASME

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

Background: Ex vivo T-cell depletion (TCD) by CD34+ selection has been used successfully for graft-versus-host disease prevention alleviating the need for additional pharmacologic immunosuppressants. TCD has been associated with higher rates of viral infections and cytomegalovirus (CMV) disease in particular compared to conventional (CONV) HCT. Modern methods of TCD achieve up to 5 log reduction of T-cells. At MSK, TCD HCT recipients (R) are routinely monitored for CMV, adenovirus (ADV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV6) in the blood by quantitative polymerase chain reaction (qPCR) assays. We report the incidence of viremia and end organ disease (EOD) by these viruses in TCD HCT recipients. Methods: Retrospective review of adults with acute leukemia (AL) and myelodysplastic syndrome (MDS) patients (pts) who received TCD HCT at MSK from June 2010 -December 2014. TCD was performed by the FDA approved CliniMACS CD34+ reagent system (Miltenyi Biotec, Gladbach, Germany). CMV was monitored at least weekly from day 14-100 and at least every 14 days until day 180. EBV was monitored through day 180. Monitoring for CMV and EBV was performed by the Clinical Microbiology Laboratory at MSK using Roche analyte specific reagents, and since March 2013, the Cobas Ampliprep/Cobas Taqman (CAP/CTM) CMV qPCR in plasma. Monitoring for ADV/HHV6 was performed by Viracor-IBT laboratories (Lenexa, KS). Preemptive treatment for CMV was started for ≥2 consecutive PCR >500copies/ml in whole blood or >300 IU/ml in plasma. Treatment of ADV/HHV6 viremia was at clinician's discretion. EBV post-transplant lymphoproliferative disorder (PTLD) was treated with rituximab as primary therapy. During the study, 11 pts were enrolled in CMV prevention trials of brincidofovir (BCV), and 5 pts received BCV for treatment of ADV. EOD was scored by standard criteria. Follow up was through June 2015. The cumulative incidence (CI) was estimated for viremia at 180 days and EOD at 365 days for CMV, ADV, HHV6, and EBV. Kaplan-Meier method and log-rank test were used to compare CI between groups. P Results: Of239 pts (median age 56.3 years, range 19.6-73.3), 161 (67%) pts had AL and 78 (33%) pts had MDS. Donors were matched-related 76 (32%), matched unrelated 116 (48%), or mismatched (related or unrelated) 47(20%); CMV R+ 148 (62%) or R- 91 (38%). CMV infection had the earliest onset post HCT at a median of 28 days (d) (IQR: 25-33), followed by HHV6 33d (25-52), ADV 71d (44-89), and EBV 85d (58-107). 188 (79%) pts had ≥1 dsDNA viral infection with 182 (76%) pts experiencing infection prior to d100. The CIs of viremia were: CMV 42% (68% in R+ vs 0% in R-, P Conclusions: 1) Viremias by dsDNA viruses occurred frequently and early post TCD HCT: 189 (79%) pts had at least 1 dsDNA viral infection, with onset of first infection before d100 in 183 (77%) pts. Around 58% of pts with CMV had coinfections with additional dsDNA virus(es). 2) Two thirds (67%) of CMV R+ and 41% of total pts received CMV antiviral treatment. 3) Overall, 18% of patients developed end organ disease by dsDNA viruses. 4) Viremias were managed effectively with intense monitoring and antiviral treatment, and rates of EOD were comparable to those of CONV HCT. Our data highlights the need for effective strategies to reduce the total burden of dsDNA viruses after TCD HCT. Figure 1. CIs of dsDNA viremia. Figure 1. CIs of dsDNA viremia. Figure 2. dsDNA viral co-infections Figure 2. dsDNA viral co-infections Figure 3. Coinfections among pts with CMV viremia Figure 3. Coinfections among pts with CMV viremia Disclosures Perales: Merck: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding.

Background: Streptococcus pneumoniae is a major cause of sepsis, pneumonia and meningitis leading to significant morbidity and mortality. Invasive pneumococcal disease (IPD) and mortality rates in cancer patients (pts) are higher than in the general population. Since the introduction of routine childhood immunization with the 7-valent pneumococcal conjugate vaccine (PREVNAR) in 2000, the overall rate of invasive pneumococcal disease (IPD) decreased in adults in the US. The impact of PREVNAR on rates of IPD in cancer pts is not well studied. Methods: Retrospective review of pts treated at Memorial Sloan-Kettering Cancer Center from 1991-2012. Unique patient visits per year were obtained from the hospital database. Prevalence was calculated as number of cases per 1000 patient-visits per year. Pts with positive culture(s) for S. pneumoniae were identified from microbiology records. Serotype data is available through 2001. IPD was defined as a positive S. pneumoniae culture from a sterile site such as blood, CSF, or pleural fluid culture. Pneumococcal pneumonia was defined as a positive S. pneumoniae culture from thelower respiratory tract or a positive upper respiratory culture with either radiographic evidence for pneumonia and/or ICD-9 code "pneumococcal pneumonia" or "pneumonia" associated with the positive culture for S. pneumoniae. Colonization was defined as positive culture for S. pneumoniae without IPD or pneumonia. We compared prevalence between "early" (prior to PREVNAR, 1992-2001) and "late" (2002-2012) periods, in those with diagnoses of solid tumors and hematologic malignancies, and between ages ≤21 and >21 years. Pneumococcal vaccination rates were calculated as the number of patients who received any pneumococcal vaccine divided by the number of patient-visits per year. Results: The mean number of patient-visits per year was 46,394 and 96,954, for the early and late period, respectively. Of 1,033 patients with ≥1 culture positive for S. pneumoniae during the study, 329 (32%) had IPD, 394 (38%) had pneumococcal pneumonia and 310 (30%) had colonization. S. pneumoniae type 6 accounted for 39 (22%) isolates where serotype data was available. The annual prevalence of IPD, pneumonia and colonization gradually declined over the course of the study (Figure 1). Compared to the early period, the prevalence of IPD, pneumonia and colonization were lower in the late period (Table 1). The highest prevalence of IPD was observed in pts ≤21 years of age (2.8 and 1.3 for early and late periods, respectively). The prevalence of IPD in pts with hematologic malignancies was 5-fold higher than pts with solid tumors in the early period (1.8 vs 0.39 respectively; P Conclusions: 1) Over a 20 year period we observed a decline in the prevalence of IPD, pneumococcal pneumonia and colonization in cancer patients at a tertiary cancer center in NYC. 2) The prevalence of IPD, pneumococcal pneumonia and colonization was significantly lower (P Table 1. Prevalence for IPD, Pneumonia and Colonization by Period Prevalence Period IPD PNA Colonization 1992-2001 0.36 0.48 0.48 2002-2012 0.16 0.16 0.08 Prevalence ratio (Early/Late) 2.25 3.0 6.0 95% CI 2.0-2.7 2.6-3.4 4.8-7.7 P value Figure 1. Annual prevalence of IPD, pneumonia and colonization Figure 1. Annual prevalence of IPD, pneumonia and colonization Disclosures Kaltsas: Pfizer: Research Funding.

Background: Hematopoietic cell transplant (HCT) patients are at risk for serious infections by Herpes simplex1/2 (HSV) and Varicella zoster(VZV). Current guidelines recommend prophylaxis (PPX) with acyclovir (ACV) during the peri-transplant period and continuing throughout the period of immunosuppression. Brincidofovir (BCV, CMX001) is a broad-spectrum nucleotide analog with in vitro activity against double stranded DNA (dsDNA) viruses including HSV and VZV. BCV is in Phase 3 development for the prevention of cytomegalovirus (CMV) infection post-HCT. At present, there is no clinical data to support the efficacy of BCV PPX for HSV/VZV post-HCT. At Memorial Sloan-Kettering Cancer Center (MSK) from 2010 to June 2015, approximately 70 alloHCT recipients were treated with BCV for various indications. We examined rates of breakthrough HSV and VZV infections during administration of BCV. Methods: Retrospective review of alloHCT patients (pts) treated for ≥14 days with BCV, on protocols or emergency investigational new drug application (EIND) at MSK. Administration of concomitant (CON) ACV for HSV PPX during BCV treatment was at clinician discretion. CMX001-350 was an expanded access study of BCV for treatment of subjects with dsDNA viruses and limited or no therapeutic options. CMX001-202 was a placebo-controlled study of BCV as preemption of adenovirus (ADV) post-HCT. CMX001-304 is a currently ongoing open label study of BCV for preemption of ADV disease. After June 2011, all pts received BCV 200mg total weekly dose [100mg twice weekly (BIW) or 200mg weekly (QW)] for adult/adolescent pts and 4mg/kg total weekly dose (2mg/kg BIW or 4mg/kg QW) for pediatric pts. Patients enrolled on CMX-304 through June 16, 2015 are included in this analysis. Based on CON ACV PPX during BCV treatment, 3 patient groups were identified: 1) No CON ACV PPX (group A); 2) Partial CON ACV PPX (group B); and 3) CON ACV PPX (group 3). Diagnosis of HSV or VZV infection was based on viral culture or PCR and clinical symptoms. Data was extracted from medical, pharmacy, and microbiology records. Results: Of 55 alloHCT pts who received BCV ≥14 days, 27 (49%) were treated on CMX001-350, 1 (2%) on CMX001-202, 12 (22%) on CMX001-304 and 15 (27%) under EIND. The median age was 53 years (range, 3-69). Thirty nine (71%) pts received T-cell depleted allografts, 9 (16%) cord blood, and 7 (13%) conventional allografts. BCV started a median of 85 days [Interquartile range (IQR), 59.5-209.5] post-HCT. The primary indication for BCV was: ACV resistant HSV in 2 (4%) pts, disseminated VZV in 1 (2%) pts, CMV in 28 (51%) pts, ADV in 21 (38%) pts and other dsDNA viruses (BK polyoma virus, Human herpesvirus 6, John Cunningham virus) in 3 (5%) pts. Thirty one (56%) pts received BCV 100mg BIW. Among 35 pts in group A, duration of BCV was a median of 39 days (IQR, 29.5-78). One pt in group A received concomitant anti-CMV antivirals for 12 days. Among 13 pts in group B, the median duration of BCV monotherapy was 23 days (IQR, 13-40). Two pts in group B had brief concomitant exposure to anti-CMV antivirals. Among 7 patients in group C, the median duration of BCV treatment was 71 days (IQR, 40-132) (Table 1). Two pts developed HSV infections during BCV treatment. For one pt, the primary indication for BCV was ACV resistant HSV. The pt initially responded to BCV with resolution of oral lesions. During his treatment with BCV, he had intermittent oral ulcers and oral cultures positive for HSV. The second pt developed breakthrough HSV infection during interruption of BCV for severe diarrhea related to graft versus-host disease. There were no breakthrough VZV infections. For one pt, the primary indication for BCV was disseminated VZV. The pt had complete resolution of VZV and no further VZV reactivation during BCV treatment. Conclusions: 1) During 2,806 patient-days of BCV monotherapy in highly immunosuppressed HCT pts, there were no VZV infections and only 2 breakthrough HSV infections. 2) BCV treatment interruptions and impaired gastrointestinal absorption likely explain the breakthrough HSV infections. 3) Our retrospective analysis in this heterogeneous cohort of sick alloHCT recipients supports that BCV administration at doses currently studied for CMV prevention is likely effective for prophylaxis of HSV/VZV in HCT. Disclosures Off Label Use: Brincidofovir (CMX001) for the efficacy of prophylaxis for Herpes simples 1/2 and Varicella zoster..

Cell–cell junctional proteins play important structural and functional roles in several physiological systems. Recent studies have illuminated key aspects in the relationship of junctional proteins with normal cell and tissue function as well as various pathologies. In this review article, the roles of cell–cell junctional proteins will be presented in four classes: adherens junctions, desmosomes, gap junctions, and tight junctions, and discussed primarily in the context of cardiovascular cell and tissue physiology and pathophysiology. The functions of the proteins are described from the perspective of mechanotransductive regulation of physiological and disease processes, with focus being laid on more biomechanical aspects, such as cell adhesion, migration, and mechanosignaling.

Heart failure remains a major cause of global morbidity and mortality. Since the benefits of heart transplantation are constrained by donor scarcity, and the ability of the heart to regenerate following infarction is limited, cell-based therapies have emerged as alternative treatments for the repair of damaged heart tissue. One method, myocardial cell sheet tissue engineering, detaches cultured cells from substrates without disrupting intercellular junctions critical for myocardial function. However, the challenges of generating mechanically strong and synchronously contractile cardiac sheets remain.In this study we characterized substrate-free cardiac cell sheets generated using a partial-lift method, whereby a portion of a cell monolayer is detached from the substrate. These cell sheets are dominated by cell-cell interactions and decoupled from cell-substrate interactions, but remain amenable to biological and chemical perturbations, and more importantly, mechanical conditioning and characterization. We show that lifted cardiac sheets exhibit significant changes in the distribution of cytoskeletal and junctional proteins, and that junctional expression of these proteins is enhanced by mechanical conditioning. Results further demonstrate that the mechanical strength and cohesion of these cell sheets depend on cytoskeletal and cell-cell junctional protein integrity. We also examined the microrheological characteristics, contraction frequency and calcium signaling of the cardiac cell sheets, which are critical for their potential clinical applications. Our results showed that mechanical conditioning the cell sheets alters several of these properties, with potential benefits to their function.This work represents a first systematic examination of mechanical conditioning on cardiac cells with primarily intercellular interactions. This method offers an unprecedented way to study cell junctions when substrate interactions are no longer dominant. The information gained from this study will help advance our understanding of cell-cell interactions and improve cardiac cell sheet biomechanical properties for tissue regeneration, and particularly heart repair.

The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low.To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples.We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum.Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

Cardiac architecture is inherently three- dimensional, yet most characterizations rely on two- dimensional histological slices or dissociated cells, which remove the native geometry of the heart. We previously developed a method for labeling intact heart sections without dissociation and imaging large volumes while preserving their three-dimensional structure. We further refine this method to permit quantitative analysis of im- aged sections. After data acquisition, these sections are assembled using image-processing tools, and qualitative and quantitative information is extracted. By examining the reconstructed cardiac blocks, one can observe end-to- end adjacent cardiac myocytes cardiac strands changing cross-sectional geometries, merging and separating from other strands. Quantitatively, representative cross- sectional areas typically used for determining hypertrophy omit the three-dimensional component; we show that tak- ing orientation into account can significantly alter the analysis. Using fast-Fourier transform analysis, we analyze the gross organization of cardiac strands in three dimen- sions. By characterizing cardiac structure in three dimen- sions, we are able to determine that the crystallin muta- tion leads to hypertrophy with cross-sectional area increases, but not necessarily via changes in fiber orienta- tion distribution. © 2009 Society of Photo-Optical Instrumentation Engi

3-D tissue cytometry has been successfully developed to optimize biological specimen throughput allowing the characterization of cell-cell, cell-tissue interaction to be quantified in 3-D tissue by combining high speed TPM, automated x-y specimen stage, and precision specimen sectioning mechanism. 3-D tissue cytometry is applied in two biomedical applications.

The contribution of structural remodeling to long-term adult brain plasticity is unclear. Here, we investigate features of GABAergic interneuron dendrite dynamics and extract clues regarding its potential role in cortical function and circuit plasticity. We show that remodeling interneurons are contained within a “dynamic zone” corresponding to a superficial strip of layers 2/3, and remodeling dendrites respect the lower border of this zone. Remodeling occurs primarily at the periphery of dendritic fields with addition and retraction of new branch tips. We further show that dendrite remodeling is not intrinsic to a specific interneuron class. These data suggest that interneuron remodeling is not a feature predetermined by genetic lineage, but rather, it is imposed by cortical laminar circuitry. Our findings are consistent with dynamic GABAergic modulation of feedforward and recurrent connections in response to top-down feedback and suggest a structural component to functional plasticity of supragranular neocortical laminae.

Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.

The response of cells to mechanical stresses is a field of growing inquiry. It is well known that both the morphologic and molecular expression of cells depend, in part, on the local mechanical environment, especially for cells such as endothelial cells that experience shear stress, stretch, and pressures. To systematically study the large variety of responses of cells to physical forces (e.g., signaling, adhesion, or stiffness changes), a number of techniques have been developed and used. Here we present methods for three types of cell mechanical studies, from the multicellular to the subcellular scales, and describe the basic principle and main use of each technique along with some design and setup considerations.

One of the challenges in labeling tissues for fluorescence microscopy is minimizing sample processing while maintaining or improving the information generated by the fluorescent label. Generally, tissues are extracted, fixed, and embedded in mounting media (such as paraffin), sectioned, and then postprocessed by removing the paraffin, blocking, labeling, and washing. Despite all of these steps, the consistency of labeling quality can vary as a result of several factors, including heterogeneity in labeling efficiency from slide to slide, the necessity of postprocessing to obtain information on sequential sections of tissue, interference from the mounting media, and loss of native three-dimensional structural information, especially in thicker sections. A method for embedding and processing tissues that have been labeled by intravital staining is described in this study. Intravital staining is the process in which live-cell dyes and other labels are injected into the bloodstream before fixation of the tissues. Tissues processed this way can be imaged upon sectioning without further staining and retain their native, three-dimensional information, thereby improving the information retained by the labels and speeding up sample processing.