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In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesteroldepleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD-and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.

Abstract-Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3␤ in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3␤ (Tg-GSK-3␤-DN) and tetracycline-regulatable wild-type GSK-3␤. GSK-3␤-DN significantly reduced the kinase activity of endogenous GSK-3␤, inhibited phosphorylation of eukaryotic translation initiation factor 2B, and induced accumulation of ␤-catenin and myeloid cell leukemia-1, confirming that GSK-3␤-DN acts as a dominant negative in vivo. Tg-GSK-3␤-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the ␣-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater E max after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3␤ induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3␤-DN and nontransgenic mice, Tg-GSK-3␤-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3␤ transgene in tetracycline-regulatable wild-type GSK-3␤ mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3␤-DN in cardiac myocytes inhibited tumor necrosis factor-␣-induced apoptosis, and the antiapoptotic effect of GSK-3␤-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3␤ induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3␤ inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3␤ during heart failure could be compensatory. Key Words: GSK-3␤ Ⅲ heart failure Ⅲ cardiac hypertrophy Ⅲ apoptosis G SK-3␤ is a ubiquitously expressed serine/threonine kinase that has versatile biological functions in cells, including regulation of metabolism, cell growth/death, and protein translation and transcription. 1,2 Unlike most protein kinases, GSK-3␤ remains active in the resting state and is inactivated when cells are stimulated by mitogens, by other protein kinases, such as Akt, or by the Wnt pathway. In cardiac myocytes, GSK-3␤ phosphorylates ␤-catenin, 3 eukaryotic translation initiation factor (eIF)2B, 4 NFAT, 5 GATA4, 6 myocardin, 7 and other proteins, thereby negatively regulating protein synthesis and gene expression. GSK-3␤ downregulates SERCA2a 8 and enhances mitochondrial permeability transition, 9 thereby leading to an inability to normalize cytosolic Ca 2ϩ in diastole and reduced cell survival, respectively.GSK-3␤ is an important negative regulator of cardiac hypertrophy. 10 GSK-3␤ negatively regulates ␤-adrenergic and endothelin-induced cardiac hypertrophy in cultured ...

This paper studies the application of the discrete Fourier transform (DFT) to predict angular orientation distributions from images of fibers and cells. Angular distributions of fibers in composites define their material properties. In biological tissues, cell and fiber orientation distributions are important since they define their mechanical properties and function.We developed a filtering scheme for the DFT to predict angular distributions accurately. The errors involved in this DFT technique and their sources were quantified through Monte Carlo simulation of computer-generated images. The knowledge of these errors allows one to verify the suitability of the method for a particular application. We found that the DFT method is most accurate for slender fibers, and propose a means to minimize errors by optimizing parameters. This method was applied to predict orientation distribution of cells and actin fibers in bio-artificial tissue constructs.

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development. IntroductionM usculoskeletal injuries are a common cause of pain and disability, and result in significant healthcare costs.1 Many of these injuries require regeneration of fibrocartilage (tissue composed of fibrous and cartilaginous components) for effective healing.2-4 For example, meniscus healing is typically insufficient due to a lack of fibrocartilage regeneration.3 Similarly, tendon-to-bone healing and repair, as frequently required after rotator cuff injury, often fails due to a lack of fibrocartilage formation at the tendon-to-bone interface. 4 Little is known about natural fibrocartilage healing, and hence little can be done to improve it. We and others have hypothesized that rebuilding the fibrocartilaginous insertion site of the tendon or ligament into bone is critical for restoration of function and for prevention of re-injury. [4][5][6] Several studies provide evidence that the stress environment influences cell morphology and the fibrocartilage production. 7,8 Compressive loads in vivo have been shown to change tendon composition and structur...

Continuum constitutive laws are needed to ensure that bio-artificial tissue constructs replicate the mechanical response of the tissues they replace, and to understand how the constituents of these constructs contribute to their overall mechanical response. One model designed to achieve both of these aims is the Zahalak model, which was modified by Marquez and co-workers to incorporate inhomogeneous strain fields within very thin tissues. When applied to reinterpret previous measurements, the modified Zahalak model predicted higher values of the continuum stiffness of fibroblasts than earlier estimates. In this work, we further modify the Zahalak model to account for inhomogeneous strain fields in constructs whose cell orientations have a significant out-of-plane component. When applied to reinterpret results from the literature, the new model shows that estimates of continuum cell stiffness might need to be revised upward. As in this article's companion, we updated the average cell strain by defining a correction factor ("strain factor"), based upon the elastic response. Three different cell orientation distributions were studied. We derived an approximate scaling model for the strain factor, and validated it against exact and self-consistent (mean-field) solutions from the literature for dilute cell concentrations, and Monte Carlo simulations involving three-dimensional finite element analyses for high cell concentrations.

Engineered tissues represent a natural environment for studying cell physiology, mechanics, and function. Cellular interactions with the extracellular matrix proteins are important determinants of cell physiology and tissue mechanics. Dysregulation of these parameters can result in diseases such as cardiac fibrosis and atherosclerosis. In this report we present a novel system to produce hydrogel tissue constructs (HTCs) and to characterize their mechanical properties. HTCs are grown in custom chambers and a robotic system is used to indent them and measure the resulting forces. Force measurements are then used to estimate HTC pretension (cellular contractility). Pretension was reduced in a dose-dependent manner by cytochalasin D (CD) treatment; the highest concentration (2 μM) resulted in ~10-fold decrease. On the other hand, treatment with fetal bovine serum (20%) resulted in approximately threefold increase in pretension. Excellent repeatability and precision were observed in measurements from replicate HTCs. The coefficient of statistical variance of quantified pretension ranged from 7% to 15% (n = 4). Due to the small size (4×4×0.8 mm) of the HTCs, this system of profiling HTC mechanics can readily be used in high-throughput applications. In particular, it can be used for screening chemical libraries in search of drugs that can alter tissue mechanics.

The mechanics of bio-artificial tissue constructs result from active and passive contributions of cells and extracellular matrix (ECM). We delineated these for a fibroblast-populated matrix (FPM) consisting of chick embryo fibroblast cells in a type I collagen ECM through mechanical testing, mechanical modeling, and selective biochemical elimination of tissue components. From a series of relaxation tests, we found that contributions to overall tissue mechanics from both cells and ECM increase exponentially with the cell concentration. The force responses in these relaxation tests exhibited a logarithmic decay over the 3600 second test duration. The amplitudes of these responses were nearly linear with the amplitude of the applied stretch. The active component of cellular forces rose dramatically for FPMs containing higher cell concentrations.