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Biomedical subjects

John A Frangos

Publications and source records attributed to John A Frangos.

14 recordsLinked to original sources
PubMed2004

Stretch activation of GTP-binding proteins in C2C12 myoblasts.

Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.

Animals↗
PubMed2004

Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration.

Hand-arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynaud's phenomenon. Chronic exposure to hand-held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low-volume fluid oscillation. Rapid low-volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal-regulated kinase (ERK1/2) phosphorylation and endothelin-1 (ET-1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET-1 release compared to steady flow, and endothelin-1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium.

Blotting, Western↗
PubMed2004

Computerized methods for X-ray-based small bone densitometry.

Animal models have been widely used to correlate in vivo changes in bone mineral density (BMD) with changes in disease state of bone. In small animal models, e.g. the hindlimb suspension model of bone loss, a non-invasive assessment of BMD is required. X-ray radiography has been surpassed in some cases by quantitative computed tomography (QCT) and dual X-ray absorptiometry (DEXA) quantitation. However, there are drawbacks in using the computerized methods, especially for small animals. In this paper, we present image-processing algorithms to quantitatively determine bone area and mineral density in digitized radiographs. Image calibration is based on a calibration step wedge, and the algorithm automatically detects the steps and computes the calibration data. In addition, we demonstrate how the algorithm can accurately determine the cortical outline of the bone and provide reliable data and statistics for small animal studies. A downloadable implementation example for the popular NIH Image package is provided.

Absorptiometry, Photon↗
PubMed2004

Cell membrane fluidity changes and membrane undulations observed using a laser scattering technique.

Local transversal micromotions of cell membranes have been reported. These may provide the physical basis for changes in membrane fluidity. In this study, tissue scattering properties are used to measure the magnitude of transversal micromotions under varying stimuli. Laser light is directed at the cell surface at a low incident angle of 12 degrees so that reflected or refracted light does not enter the microscope objective. Scattered light showed strong low-frequency intensity fluctuations with random-walk behavior. Fluctuations were quantified by the median coefficient of variation over the entire observed cell area. Incubation of the cells with protein crosslinkers (paraformaldehyde) and metabolic suppressors (sodium azide) suppressed these fluctuations by 62 and 44%, respectively. The application of hypoosmotic media caused an increase of fluctuation magnitude by 23% (p < 0.005). Agents that increase membrane fluidity (2% ethanol and xenon) increased fluctuation magnitude by 15 and 31%, respectively (p < 0.05). Cessation of the ethanol and xenon exposure led to partial recovery of the fluctuation magnitude, which was nonsignificant for ethanol. This study shows a strong link between membrane fluidity and transversal membrane undulations and provides an important step in the understanding of the mechanosensing function of the cell membrane.

Cell Line, Tumor↗
PubMed2004

Oxygen delivery and consumption in the microcirculation after extreme hemodilution with perfluorocarbons.

The oxygen transport capacity of fluorocarbons was investigated in the hamster chamber window model microcirculation to determine the rate at which oxygen is delivered to the tissue in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Hydroxyethlyl starch (HES 200; 200 kDa molecular mass) was used as a plasma expander for two isovolemic hemodilutions performed with 10% HES 200 until a Hct of 65%. A third step reduced the Hct to 75% of baseline and was performed with either HES 200 or a 60% perfluorocarbon (PFC) emulsion. Comparisons of HES 200-only-hemodiluted animals versus 4.2 g/kg PFC emulsion-hemodiluted animals were made at 21% and 100% normobaric oxygen ventilation. It was found that systemic and microvascular oxygen delivery was 25% and 400% higher in the PFC animals compared with HES 200 animals, respectively, showing that PFCs deliver oxygen to the tissue when combined with hyperoxic ventilation in the present experiments, with no evidence of vasoconstriction or impaired microvascular function. Oxygen ventilation (100%) led to a positive base excess for the PFC group (5.5 +/- 2.5 mmol/l) versus a negative balance (-0.8 +/- 1.4 mmol/l) for the HES 200 group, suggesting that microvascular findings corresponded to systemic events.

Animals↗
PubMed2003

Reactive oxygen species inhibited by titanium oxide coatings.

Titanium is a successful biomaterial that possesses good biocompatibility. It is covered by a surface layer of titanium dioxide, and this oxide may play a critical role in inhibiting reactive oxygen species, such as peroxynitrite, produced during the inflammatory response. In the present study, titanium dioxide was coated onto silicone substrates by radio-frequency sputtering. Silicone coating with titanium dioxide enhanced the breakdown of peroxynitrite by 79%. At physiologic pH, the peroxynitrite donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) was used to nitrate 4-hydroxyphenylacetic acid (4-HPA) to form 4-hydroxy-3-nitrophenyl acetic acid (NHPA). Titanium dioxide-coated silicone inhibited the nitration of 4-HPA by 61% compared to aluminum oxide-coated silicone and 55% compared to uncoated silicone. J774A.1 mouse macrophages were plated on oxide-coated silicone and polystyrene and stimulated to produce superoxide and interleukin-6. Superoxide production was measured by the chemiluminescent reaction with 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA). Titanium dioxide-coated silicone exhibited a 55% decrease in superoxide compared to uncoated silicone and a 165% decrease in superoxide compared to uncoated polystyrene. Titanium dioxide-coated silicone inhibited IL-6 production by 77% compared to uncoated silicone. These results show that the anti-inflammatory properties of titanium dioxide can be transferred to the surfaces of silicone substrates.

Aluminum↗
PubMed2003

The use of dinoflagellate bioluminescence to characterize cell stimulation in bioreactors.

Bioluminescent dinoflagellates are flow-sensitive marine organisms that produce light emission almost instantaneously upon stimulation by fluid shear in a shear stress dose-dependent manner. In the present study we tested the hypothesis that monitoring bioluminescence by suspended dinoflagellates can be used as a tool to characterize cellular response to hydrodynamic forces in agitated bioreactors. Specific studies were performed to determine: (1) impeller configurations with minimum cell activation, (2) correlations of cellular response and an integrated shear factor, and (3) the effect of rapid acceleration in agitation. Results indicated that (1) at a volumetric mass transfer coefficient of 3 x 10(-4) s(-1), marine impeller configurations were less stimulatory than Rushton configurations, (2) bioluminescence response and a modified volumetric integrated shear factor had an excellent correlation, and (3) rapid acceleration in agitation was highly stimulatory, suggesting a profound effect of temporal gradients in shear in increasing cell stimulation. By using bioluminescence stimulation as an indicator of agitation-induced cell stimulation and/or damage in microcarrier cultures, the present study allows for the verification of hypotheses and development of novel mechanisms of cell damage in bioreactors.

Acceleration↗
PubMed2003

Rapid activation of Ras by fluid flow is mediated by Galpha(q) and Gbetagamma subunits of heterotrimeric G proteins in human endothelial cells.

OBJECTIVE: Temporal gradients in fluid shear stress have been shown to induce a proatherogenic phenotype in endothelial cells. The biomechanical mechanism(s) that enables the endothelium to respond to fluid shear stress requires rapid activation and signal transduction. The small G protein Ras has been identified as an early link between rapid mechanotransduction events and the effects of shear stress on downstream signal-transduction cascades. The aim of this study was to elucidate the upstream mechanotransduction signaling events mediating the rapid activation of Ras by fluid shear stress in human endothelial cells. METHODS AND RESULTS: Direct measurement of Ras-bound GTP and GDP showed that fluid-flow activation of Ras was rapid (10-fold within 5 seconds) and dose dependent on shear stress magnitude. Treatment with protein tyrosine kinase inhibitors or pertussis toxin did not significantly affect flow-induced Ras activation. However, activation was inhibited by transient transfection with antisense to Galpha(q) or the Gbetagamma scavenger beta-adrenergic receptor kinase carboxy terminus. Transfection with several Gbetagamma subunit isoforms revealed flow-induced Ras activation was most effectively enhanced by Gbeta1gamma2. CONCLUSIONS: These results suggest that the rapid, shear-induced activation of Ras is mediated by Galpha(q) through the activity of Gbetagamma subunits in human vascular endothelial cells.

Adaptation, Physiological↗
PubMed2003

Strain rate mechanotransduction in aligned human vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) exist in a dynamic mechanical environment and can sense and respond to mechanical stimuli in vivo. Stretch is known to stimulate intracellular biochemical events, but the influence of the rate at which stretch is applied has not been extensively investigated. Also, most studies of VSMC mechanotransduction use cell culture models not aligned in the direction of stretch. We aligned human VSMC in the direction of uniaxial stretch to examine the importance of strain rate and cell orientation. We demonstrate strain rate profoundly affects stretch-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Low strain rate induced dephosphorylation while physiologic and high rates increased phosphorylation. Dephosphorylation at low strain rate was dependent on cell orientation matching the strain field. Pretreatment with GDPbetaS indicated G proteins are required for ERK1/2 phosphorylation at physiologic strain rate. Apyrase addition to scavenge extracellular ATP inhibited ERK1/2 regulation at low and physiologic strain rates. These results indicate strain rate and cell orientation are important components of mechanotransduction.

Anisotropy↗
PubMed2002

Strain and strain rate activation of G proteins in human endothelial cells.

The endothelium is known to sense and respond to its physical environment, but the underlying mechanisms and early events of endothelial cell mechanotransduction are not well understood. The present study measured G protein activation by mechanical strain in human umbilical vein endothelial cells (HUVEC) directly by photoincorporation of a hydrolysis resistant, radiolabeled GTP analog. Ten percent uniaxial strain at a strain rate of 20% s(-1) over 1min activated a 38kDa Galpha subunit 167+/-17% relative to controls, while 2% cyclic strain failed to significantly activate the protein (117+/-19%). A single cycle of 10% strain at 20% s(-1) strain rate activated the Galpha subunit 152+/-25%, while activation at the same strain but lower strain rate (0.3% s(-1)) was not significantly different from controls (116+/-12%). Western blot analysis identified the 38kDa protein as Galpha(q/11). These results demonstrate the rapid activation of G proteins in HUVEC by cyclic uniaxial strain in a strain- and strain rate-dependent manner.

Cells, Cultured↗
PubMed2002

Phospholipid-bound molecular rotors: synthesis and characterization.

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotor's viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.

Cell Line↗
PubMed2002

Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein.

Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.

Animals↗
PubMed2002

A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors.

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa x s, and the mixtures ranged up to 4.5 mPa x s (21 degrees C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa x s (n = 5, P < 0.0001). The mathematical relationship between intensity (I) and viscosity (eta) was found to be eta = (kappaI)(nu). After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.

Blood Viscosity↗