Uri Zaretsky

Static mechanical stretching accelerates lipid production in 3T3-L1 adipocytes by activating the MEK signaling pathway.

Understanding mechanotransduction in adipocytes is important for research of obesity and related diseases. We cultured 3T3-L1 preadipocytes on elastic substrata and applied static tensile strains of 12% to the substrata while inducing differentiation. Using an image processing method, we monitored lipid production for a period of 3-4 wk. The ratio of %-lipid area per field of view (FOV) in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05), reaching ∼1.8 on average starting from experimental day ∼10. The superior coverage of the FOV by lipids in the stretched cultures was due to significantly greater sizes of lipid droplets (LDs) with respect to nonstretched cultures, starting from experimental day ∼10 (P < 0.05), and due to significantly more LDs per cell between days ∼10 and ∼17 (P < 0.05). The statically stretched cells also differentiated significantly faster than the nonstretched cells within the first ∼10 days (P < 0.05). Adding peroxisome proliferator-activated receptor-γ (PPARγ) antagonist did not change these trends, as the %-lipid area per FOV in the stretched cultures that received this treatment was still significantly greater than in the nonstretched cultures without the PPARγ antagonist (14.44 ± 1.96% vs. 10.21 ± 3%; P < 0.05). Hence, the accelerated adipogenesis in the stretched cultures was not mediated through PPARγ. Nonetheless, inhibiting the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched cultures (13.53 ± 5.63%), bringing it to the baseline level of the nonstretched cultures without the MEK inhibitor (10.21 ± 3.07%). Our results hence demonstrate that differentiation of adipocytes can be enhanced by sustained stretching, which activates the MEK signaling pathway.

Cellular Alterations in Cultured Endothelial Cells Exposed to Therapeutic Ultrasound Irradiation

Restoration of blood supply to tissue with impaired perfusion depends on spontaneous or mediated angiogenesis, which among other mechanisms includes stimulation, migration, and proliferation of endothelial cells (ECs). Therapeutic ultrasound (US) irradiation is known as an inducer of cellular modifications and is used to accelerate wound healing. An in vitro setup was developed in order to allow for a comprehensive investigation of cellular alterations induced in cultured ECs after exposure to different modes of therapeutic US irradiation. Viability assays revealed a higher rate of proliferation in the sonicated groups, although cell death was not observed. Visualization of actin stress fibers demonstrated partial disassembly of the fibers immediately after US sonication, with a maximum after about 2 h. However, 24 h following sonication the fibers regain normal appearance. A similar behavior was observed with the microtubules and focal adhesion complexes. Utilizing a wound healing assay revealed that migration rate of ECs is enhanced by US irradiation. These findings hint that therapeutic US sonication of ECs results in temporarily cellular alterations, which may induce tissue remodeling via stimulation of EC proliferation and migration.

Integrated approach for in vivo evaluation of respiratory muscles mechanics

The respiratory muscles constitute the respiratory pump, which determines the efficacy of ventilation. Any functional disorder in their performance may cause insufficient ventilation. This study was designed to quantitatively explore the relative contribution of major groups of respiratory muscles to global lung ventilation throughout a range of maneuvers in healthy subjects. A computerized experimental system was developed for simultaneous noninvasive measurement of inspired/expired airflow, mouth pressure and up to 8 channels of EMG surface signals from major respiratory muscles which are located near the skin (e.g., sternomastoid, external intercostal, rectus abdominis and external oblique) during various respiratory maneuvers. Lung volumes values were calculated by integration of airflow data. Hills muscle model was utilized to calculate the forces generated by the muscles from the acquired EMG data. Analysis of EMG measurements and respiratory muscles forces revealed the following characteristics: (a) muscle activity increased with increased breathing effort, (b) inspiratory muscles contributed to inspiration even at relatively low flow rates, while expiratory muscles are recruited at higher flow rates, (c) the forces generated by the muscle depended on the muscle properties as well as on their EMG performance and (d) the pattern of the muscles force curves varied between subjects, but were generally consistent for the same subject regardless of breathing effort.

Fluid-Flow Induced Wall Shear Stress and Epithelial Ovarian Cancer Peritoneal Spreading

Epithelial ovarian cancer (EOC) is usually discovered after extensive metastasis have developed in the peritoneal cavity. The ovarian surface is exposed to peritoneal fluid pressures and shear forces due to the continuous peristaltic motions of the gastro-intestinal system, creating a mechanical micro-environment for the cells. An in vitro experimental model was developed to expose EOC cells to steady fluid flow induced wall shear stresses (WSS). The EOC cells were cultured from OVCAR-3 cell line on denuded amniotic membranes in special wells. Wall shear stresses of 0.5, 1.0 and 1.5 dyne/cm2 were applied on the surface of the cells under conditions that mimic the physiological environment, followed by fluorescent stains of actin and β-tubulin fibers. The cytoskeleton response to WSS included cell elongation, stress fibers formation and generation of microtubules. More cytoskeletal components were produced by the cells and arranged in a denser and more organized structure within the cytoplasm. This suggests that WSS may have a significant role in the mechanical regulation of EOC peritoneal spreading.

Wall shear stress effects on endothelial-endothelial and endothelial-smooth muscle cell interactions in tissue engineered models of the vascular wall.

Vascular functions are affected by wall shear stresses (WSS) applied on the endothelial cells (EC), as well as by the interactions of the EC with the adjacent smooth muscle cells (SMC). The present study was designed to investigate the effects of WSS on the endothelial interactions with its surroundings. For this purpose we developed and constructed two co-culture models of EC and SMC, and compared their response to that of a single monolayer of cultured EC. In one co-culture model the EC were cultured on the SMC, whereas in the other model the EC and SMC were cultured on the opposite sides of a membrane. We studied EC-matrix interactions through focal adhesion kinase morphology, EC-EC interactions through VE-Cadherin expression and morphology, and EC-SMC interactions through the expression of Cx43 and Cx37. In the absence of WSS the SMC presence reduced EC-EC connectivity but produced EC-SMC connections using both connexins. The exposure to WSS produced discontinuity in the EC-EC connections, with a weaker effect in the co-culture models. In the EC monolayer, WSS exposure (12 and 4 dyne/cm2 for 30 min) increased the EC-EC interaction using both connexins. WSS exposure of 12 dyne/cm2 did not affect the EC-SMC interactions, whereas WSS of 4 dyne/cm2 elevated the amount of Cx43 and reduced the amount of Cx37, with a different magnitude between the models. The reduced endothelium connectivity suggests that the presence of SMC reduces the sealing properties of the endothelium, showing a more inflammatory phenotype while the distance between the two cell types reduced their interactions. These results demonstrate that EC-SMC interactions affect EC phenotype and change the EC response to WSS. Furthermore, the interactions formed between the EC and SMC demonstrate that the 1-side model can simulate better the arterioles, while the 2-side model provides better simulation of larger arteries.

General tube law for collapsible thin and thick-wall tubes

Modeling the complex deformations of cylindrical tubes under external pressure is of interest in engineering and physiological applications. The highly non-linear post-buckling behavior of cross-section of the tube during collapse attracted researchers for years. Major efforts were concentrated on studying the behavior of thin-wall tubes. Unfortunately, the knowledge on post-buckling of thick-wall tubes is still incomplete, although many experimental and several theoretical studies have been performed. In this study we systematically studied the effect of the wall thickness on post-buckling behavior of the tube. For this purpose, we utilized a computational model for evaluation of the real geometry of the deformed cross-sectional area due to negative transmural (internal minus external) pressure. We also developed an experimental method to validate the computational results. Based on the computed cross-sections of tubes with different wall thicknesses, we developed a general tube law that accounts for thin or thick wall tubes and fits the numerical data of computed cross-sectional areas versus transmural pressures.

A technique for global assessment of respiratory muscle performance at different lung volumes.

A system for noninvasive assessment of an all-inclusive function of respiratory muscles at different lung volumes is presented. The apparatus was based on the interrupter technique and facilitated simultaneous measurements of mouth pressure and airflow rate during dynamic or quasistatic manoeuvres. In this study, mouth pressure values were continuously acquired during and after interruption of a forced inspiratory or expiratory manoeuvre for as long as the subject could sustain an elevated mouth pressure against the obstructed opening. These measurements provided information on both muscle strength and power. A total of 420 forced maximal inspiratory and expiratory manoeuvres performed by six healthy subjects were monitored at different lung volumes. The pattern of maximal pressure-time curves was consistent for the same subject regardless of lung volume. Similar values of maximal mouth pressure can be generated by healthy subjects by using either a flange-style mouthpiece or facial mask. For both methods mouth pressure shows a significant (p < 0.05) second order dependency on lung volume for both inspiration and expiration. The standard deviation of measurements from a single subject about a second order curve is of the order of 5-15%. The findings of interchangeability between methods of measurement may be useful in allegedly non-compliant patients.

Hydrodynamic evaluation of intravenous infusion systems

STUDY OBJECTIVE We investigated the hydrodynamic characteristics of IV infusion sets for rapid fluid resuscitation. A simple technique has been devised for quantitative evaluation of the hydrodynamic characteristics of IV sets, including their components, for a range of infusion pressures. SETTING AND METHODS Previous investigations have measured the overall flow rate of infusion sets with and without IV catheters. This study presents a quantitative technique for measuring the resistance to flow of the IV delivery set as a whole as well as its components. An infusion set was measured with 14- and 18-gauge IV catheters while delivering fluid at infusion pressures between 50 (gravity) and 400 mm Hg. MEASUREMENTS AND MAIN RESULTS At gravity-driven infusion, the drip chamber imposes a resistance to flow of the same order as that of the catheter. At pressurized infusion with small-bore catheters, the catheter consumes the majority of the overall pressure drop. At pressurized infusion with a large-bore catheter or tubing, the standard drip chamber becomes the limiting component and imposes the largest resistance to flow. CONCLUSION At gravity-delivered pressures (50 and 100 mm Hg), the only effective way of increasing flow rate (more than twofold) is to use a low-resistance drip chamber or to use two infusion sites. At pressurized delivery pressures (more than 200 mm Hg), increasing catheter size from 18 to 14 gauge would be more effective than doubling the number of infusion sets. Also, a more efficient drip chamber adds an important advantage. Finally, increasing the tubing diameter adds only minimal benefit.

Custom-designed wells and flow chamber for exposing air-liquid interface cultures to wall shear stress.

The effects of mechanical stimuli such as wall shear stresses (WSS) on cellular processes have been studied in vitro in numerous cell types. In order to study WSS effects on cells cultured under air–liquid interface (ALI) conditions, we developed a custom-designed well that can be disassembled into sub-units that allow installation of the cultured cells in a flow chamber, and then, re-assembled for further incubation or biological tests. Human nasal epithelial cells were cultured in the new wells under ALI conditions, and some of their biological characteristics were compared with those cultured in commercial Millicells. The cultured cells from both types of wells secreted the same amount of mucin and had similar cytoskeletal structures. Preliminary WSS experiments demonstrated the advantage of the new wells and provided initial indications that WSS affects the performance of ALI cultured respiratory epithelial cells.

In vitro model of intravenous fluid administration: analysis of vein resistance to rapid fluid delivery

Rapid fluid administration is the cornerstone of successful trauma resuscitation of patients in a state of shock. Intravenous (IV) fluid delivery is a physical intrusion into a vein which results in a complex interaction between the rigid catheter and the compliant vein. We present an experimental model of IV infusion into a vein-like compliant tube that (a) demonstrated the interdependence between fluid administration and blood flow in a compliant tube and (b) allowed investigation of the contribution of the central venous system (between the infusion site and the heart) to the total resistance to infusion flow rate. The results show that in cases with very high resistance in the central venous system a significant increase of infusion flow rate cannot be achieved just by increasing the infusion pressure. Similarly, in cases of small veins when only small catheters can be used, infusate flow rate may be increased only by using two independent infusion ports. In cases with increased tissue pressure due to edema, gravity-driven infusion may not produce sufficient perfusion of the vascular compartments. It was also shown that the vein valves do not always close, and that peripheral blood flow may continue together with the infusate fluid (e.g., when there is a small downstream resistance and infusion with a small catheter).