R-3 Repository :: Browsing by Author "McIntire, Larry V."

Browsing by Author "McIntire, Larry V."

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2D timelapse and 3D fluorescence microscopy with applications to vascular tissue engineering
(1998) Stamatas, Georgios Nikolaou; McIntire, Larry V.
Part of the definition given to the new emerging science discipline of tissue engineering is the understanding of the structure-function relationships at the cellular level. In this context it is important for vascular tissue engineering to understand the mechanisms involved in the vascular cell responses to their mechanically active environment. This work has elucidated some aspects of the complicated puzzle of mechanotransduction in vascular smooth muscle cells (SMC) and endothelial cells (EC). Two dimensional timelapse fluorescence microscopy revealed rapid alkalinization occurring in cultured human aortic SMC exposed to well defined fluid flow profiles. The response was reversible and persisted for at least 20 min after flow initiation. The magnitude of the alkalinization (0.14 pH units) was enough to increase the nitric oxide synthase activity and account at least in part for the flow-induced increases in NO production by SMC. Use of specific inhibitors demonstrated the involvement of the Na$\sp+$/H$\sp+$ exchanger in the flow-induced response, whereas the Cl$\sp-$/HCO$\sb3\sp-$ exchanger was active even under stationary conditions. The involvement of calcium as a second messenger in the EC flow-induced mechanotransduction and the localization of possible signals within the cell was addressed by a three dimensional fluorescence microscopy technique. After 5 min of flow initiation there was a significant calcium increase in the nuclear region. The response was cytoskeleton independent. The same technique revealed early flow-induced changes in the three dimensional EC architecture. Nuclear and whole cell heights were reduced by about 1 $\mu$m with a corresponding increase in the cross-sectional area at lower optical sections. Using specific cytoskeleton disrupters we demonstrated that the whole cell height response was cytoskeleton independent and the nuclear height response was microtubule dependent. Thus, it has been shown that force imposed on the EC membrane is rapidly transmitted by microtubules to the endothelial nucleus. A mechanical equivalent model is presented to explain the cytoskeleton involvement in flow-induced structural changes based on tensegrity arguments. The early responses in the nuclear calcium and structure demonstrated in this study may be important for the shear-induced gene regulation.
A blood flow model for sickle cell anemia
(1974) Nicholas, Dianne Wheatley; McIntire, Larry V.
A mathematical model has been developed to simulate the time average blood flow throughout the circulatory system of the human body. It was assumed that whole blood obeyed Cassons equation and the flow properties were calculated by means of this relationship. The model is a complete computer simulation designed to calculate the steady flow rates, average pressure gradients, and mean hemoglobin flow rates over the entire vascular system. Pertinent shear stress and shear rate data were submitted to the model to determine the extent to which the above properties were affected by the disease, sickle cell anemia. Possible extension of the model for evaluation of proposed theraputics for sickle cell anemia are discussed.
Adhesion of murine RAW117 lymphoma cells to hepatic sinusoidal endothelial cells: Study of surface molecule interactions under flow and effect of cyclooxygenase and lipoxygenase inhibitors
(2000) Papadimitriou, Minas N. B.; McIntire, Larry V.
Adhesion of malignant tumor cells under flow conditions to the endothelial monolayer lining the interior of the blood vessels is an important step in the metastatic cascade. This project examined the adhesive interactions of murine RAW117 large-cell lymphoma cells to murine hepatic sinusoidal endothelial cells (HSE). Flow cytometric analysis demonstrated constitutive-expression of VCAM-1, ICAM-1, PECAM-1 and beta1 integrin subunit on the surface of both HSE and RAW117 cells. Additionally, alpha4 and beta 7 integrin subunits and MAdCAM-1 were present on the RAW117 cell surface. The dynamic adhesion assay used employs a parallel-plate flow chamber coupled with video microscopy and digital image processing. It is capable of distinguishing initial attachment from adhesion stabilization. Using monoclonal antibodies to block the surface molecules mentioned above, we determined that an interaction of integrin alpha4beta1 on RAW117 cells with liver endothelial VCAM-1 occurs during the early stages of the adhesion process and may be important in liver metastasis. The second part of this project focused on the potential role of the cyclooxygenase (COX) pathway in the adhesive interactions of the cells studied. COX-1 isoform was detected in unstimulated RAW117 cells. Blocking it with COX inhibitors such as indomethacin, aspirin, sulindac and piroxicam resulted in increase in adhesion under static conditions. However, when lipoxygenase (LOX) inhibitors (such as ETI, NDGA and Esculetin) were used either alone or in combination with COX inhibitors, there was a significant decrease in adhesion. This observation suggested an involvement of the LOX pathway in the adhesion process. COX-2 isoform was detected in unstimulated HSE and was upregulated after stimulation with TNF-alpha. Blocking it in unstimulated HSE resulted in an increase in adhesion of RAW117. However, in the case of TNF-alpha stimulated HSE, NS-398 (a COX-2 specific inhibitor) caused a significant decrease in adhesion, indicating a potential role for COX-2 in the adhesion of RAW117 to stimulated HSE.
Automated tracking of tumor invasion in three dimensional extracellular matrix analogs and a novel stochastic analysis of the cell trajectories
(2001) Demou, Zoe NM; McIntire, Larry V.; Zygourakis, Kyriacos
Tumor cell migration and invasion of body tissues are prerequisite mediators for lymphatic or hematogenous cancer dissemination. To date, there is insufficient understanding of what triggers the metastatic cascade, and of how the interplay among cell receptors, the cellular and acellular components of the extracellular matrix and proteolytic enzymes mediate cancer migration, invasion, proliferation and survival. In addition to the inherent complexity of each one of the aforementioned phenomena is the lack of an experimental technique capable of dissecting the mechanisms that mediate the dynamic invasive and migratory behavior at the cellular level and with respect to the properties of the cell environment. The goal of my thesis was to develop an automated system for cell tracking in three dimensions and use it to model the dynamics of cancer invasion and migration. Therefore the hardware and software were designed for a fully automated optical 3D cell tracking system that quantified long-term invasion and migration of cancer cells infiltrating 3D extracellular matrix analogs. The quantitative analysis of the cell trajectories employed a novel formulation of the continuous Markov model that evaluated the potential for invasive or lateral motion and cell stops. The infiltration of human HT1080 fibrosarcoma and human MDA-MB-231 adenocarcinoma cells, was monitored in plain or Matrigel-containing collagen type I gels. Parameters such as the speed subpopulations, the persistence of motion in certain directions, the turning frequency of the cells, the preferred directions of motion, and the invasion depth profiles over time quantified infiltration at the cellular level. Distinct migratory and invasive phenotypes significantly dependent on the gel composition were identified for the two cell types. The HT1080 cell line expressed a high motility phenotype and well-preserved lateral motion on the plain collagen gel surface. The basement membrane components transformed the HT1080 cells to robust invaders by significantly enhancing the matrix infiltration and the turning frequency. The low motility, slow invasion and low turning behavior of MDA-MB-231 cells indicated that their invasiveness may depend on matrix-degrading activity. To the best of my knowledge this is the first study employing a detailed set of quantitative descriptors to demonstrate that tumor invasion and migration are dynamic processes of individual cells that depend significantly on the cell type and the tumor microenvironment.
Characterization of platelet glycoprotein Ib-IX-V: von Willebrand factor interaction under shear conditions
(2004) Ramasubramanian, Anand; McIntire, Larry V.
Arterial thrombosis is one of the important pathophysiological mechanisms that lead to cardiovascular diseases. In this thesis, we have made an attempt to better characterize the kinetic and molecular mechanisms that underlie the critical first step in arterial thrombosis, namely, the interaction between platelet glycoprotein (GP) Ib and von Willebrand factor (VWF). In the first part of the work, we evaluated the kinetics of interaction between platelet GP Ib-IX-V complex and VWF under arterial flow conditions. The GP Ibalpha subunit of GP Ib complex binds to VWF through the Al domain of VWF. Impaired GP Ib-VWF interaction due to GP Ibalpha mutations can result in bleeding abnormalities including platelet-type von Willebrand disease (ptVWD). We measured the cellular on- and off-rate constants of CHO cells expressing wild-type or gain- or loss-of-function mutant GP Ibalpha interacting with VWF-Al-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on VWF-Al surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of VWF-Al, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency was 3-fold more and 3-fold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GP Ibalpha affect both the association and dissociation kinetics of the GP Ibalpha-VWF-Al bond. In the second part of the work, we compared the interaction of unusually large multimers of VWF (ULVWF) and that of the normal plasma multimers of VWF (P-VWF) platelets. ULVWF multimers are implicated in the pathology of a thrombotic disorder, thrombotic thrombocytopenic purpura (TTP) due to their increased affinity for platelets. We found that the ULVWF multimers are more effective than the normal P-VWF multimers in mediating (a) platelet aggregation in solution at high shear stress; (b) ristocetin-modulated platelet agglutination and (c) platelet adhesion to immobilized VWF under arterial shear conditions.
Cone-plate viscometric studies of neutrophil adhesion to the endothelial ligands E-selectin and ICAM-1
(2000) Hentzen, Eric; McIntire, Larry V.
Neutrophil adhesion to endothelium is prerequisite for extravasation and localization at inflammatory sites. In the vasculature this process occurs under conditions of hydrodynamic shear requiring that bonds formed between neutrophils and endothelium withstand forces imposed by flowing blood. In this work we investigated interactions of neutrophils with the endothelial ligands ICAM-1 and E-selectin. Detailed examination of the effects of hydrodynamic forces and molecular mechanisms of neutrophil adhesion to cell lines expressing these molecules was performed using cone-plate viscometry and two-color flow cytometry. The relative contributions of LFA-1 and Mac-1 to the dynamics and strength of neutrophil adhesion to ICAM-1 varied with hydrodynamic conditions, activation state, and ICAM-1 expression. Target cells expressing ∼1000 ICAM-1 sites/mum2 were captured with an efficiency of 0.15 at 100s-1. An eight-fold increase in ICAM-1 surface density doubled adhesion efficiency to ∼0.3 at low shear and increased the shear range over which adhesion occurred. The collisional contact duration required for successful capture was predicted to be ∼9ms for Ilow and ∼4ms for Ihigh. Shear alone was sufficient for LFA-1 mediated adhesion to ICAM-1 and fMLP stimulation boosted capture efficiency four-fold. Mac-1 was one-third as efficient in capture, but stabilized aggregates over several minutes of shear and at stresses exceeding 5 dyn/cm2. beta 2-integrin mediated adhesion to ICAM-1 appears to be a cooperative and sequential process of LFA-1-dependent capture followed by Mac-1-mediated stabilization. Studies with selectin transfectants indicated that tethering through E-selectin was more efficient than L-selectin in promoting beta2-integrin mediated adhesion. fMLP-stimulated neutrophils bound E-selectin at two-fold higher levels than L-selectin. Shear with E-selectin transfectants over a discrete range of shear rates (∼400 to 600s-1) induced adhesion of ∼30% of unstimulated neutrophils. This correlated with a 50% increase in Mac-1 expression within one minute. Treatment of neutrophils with a specific inhibitor of p38 MAP kinase decreased both of these processes to background levels, implicating a p38 pathway in transduction of intracellular signals induced by E-selectin binding. Together these studies provide evidence that neutrophil binding to endothelial ligands functions optimally under discrete hydrodynamic conditions, providing an intrinsic mechanism regulating the transition from transient tethering to stable but reversible adhesion.
Determination of chemical tracer partition coefficients
(1980) Kapoor, Sunil Kumar; Deans, H. A.; Davis, S. H.; McIntire, Larry V.
The purpose of this investigation was to develop an apparatus for determining the partition coefficients of the esters used in single well tracer tests. The single well tracer test has been used since 1968 to determine the residual oil saturations in oil reservoirs. Evaluation of the residual oil saturation requires knowledge of the partition coefficient for the chemical tracer used. In this thesis, an experimental apparatus has been developed to determine partition coefficients at reservoir conditions of pressure, temperature, tracer concentration and brine salinity. The apparatus has been designed for pressures up to 5 psi and temperatures up to 2°F. These will cover the range of most oil reservoirs. It consists of a closed loop in which the oil and the brine phases are contacted. Pure tracer is injected in known amounts, and the concentration of tracer in the brine phase is measured while the brine is being continuously circulated. After tracer concentration reaches an equilibrium value, a mass balance calculation gives the concentration of the tracer in the oil phase. Hence, the value of the partition coefficient can be calculated. Another injection of tracer is then made, and the process repeated. In this way the dependence of the partition coefficient on concentration is obtained at a particular temperature and pressure. Preliminary tests were run to determine the partition coefficients of ethyl acetate in synthetic brine (containing 1, ppm sodium chloride) and white oil. The partition coefficient was obtained at 78°F, 12°F and 16°F for tracer concentrations up to .8%. The apparatus developed could be used to determine the partition coefficients for live reservoir oil and samples and formation water.
Differential roles of adhesion receptors during neutrophil adhesion to endothelial cells under flow conditions
(1998) Gopalan, Priya Kadambi; Akin, John Edward.; McIntire, Larry V.
Neutrophil emigration from the vasculature is an essential part of the primary immune response to infections. It is a multi-step process of cell interaction with the endothelium (capture, rolling, arrest and transmigration) that involves several classes of receptors. Neutrophils are captured (tethered) and then roll on the endothelial surface. This initial interaction is thought to involve the selectin family of cell adhesion molecules. Cell arrest (firm adhesion) follows neutrophil activation. Activated CD18 integrins and ICAM-1, a member of the immunoglobulin gene superfamily, mediate arrest. Finally, the neutrophils transmigrate beneath the endothelial cells, a process also thought to be dependent on the CD18 integrins and ICAM-1. We used a parallel plate flow chamber to simulate laminar flow along vessel walls in the vasculature. We demonstrate that L-selectin can activate the adhesive function the CD18 integrins. In this work, we show for the first time that L-selectin can activate LFA-1. We also show that two of the members of the CD18 integrin family, LFA-1 and Mac-1, are both sufficient to mediate neutrophil arrest and transmigration across endothelial cells stimulated for 4 hr with IL-$1\beta .$ We provide evidence for the existence of a pathway independent of the CD18 integrins that can mediate arrest and transmigration. Neutrophils utilizing this pathway are able to transmigrate with a rate comparable to CD18-dependent transmigration. We provide some preliminary data that demonstrates that PECAM-1 ligation does not activate neutrophil CD18 integrin-mediated arrest, in contrast to published reports. Finally, we report the results of some initial experiments that suggest that L-selectin and PSGL-1 mediate the capture of neutrophils by endothelial monolayers stimulated for 4 hr with IL-$1\beta ,$ while E-selectin and sialic acids are involved in rolling, but not in capture. Our results give insight into the interplay between laminar shear flow and adhesion receptor function which enable neutrophil emigration during inflammatory conditions.
Diffusion thermoeffect in ternary liquid mixtures
(1980) Platt, Gerald Edwin; Rowley, Richard L.; McIntire, Larry V.; Leland, Thomas W.
The diffusion thermoeffect or Dufour effect, a heat flux induced by an isothermal chemical potential gradient, is usually characterized by the heat of transport. The heat of transport is defined as the ratio of the heat flux to the mass flux under isothermal conditions. Although heats of transport have been measured in binary liquid mixtures, no direct measurements have been made in ternary nonelectrolyte liquid mixtures. In this work, the two independent heats of transport for toluene-chlorobenzene-bromobenzene mixtures at 25°C were determined at various compositions using a withdrawable liquid gate cell. Values were obtained by fitting calculated to measured temperature differences via a weighted non-linear least squares fitting routine using heats of transport as adjustable parameters. Experimental temperature differences were measured at points symmetric about the initial diffusion interface. The diffusion thermoeffect can be described mathematically by the hydrodynamic equations into which fluxes, obtained using methods of nonequilibrium thermodynamics, have been substituted. The resulting set of coupled differential equations were solved using a perturbation scheme for the composition and temperature profiles as functions of time. Initial and boundary conditions used corresponded closely to the experimental configuration. In addition to heats of transport, the Onsager coefficients were determined. The effect of a third component on ternary solution thermal diffusion experiments was evaluated using Onsager reciprocity.
Dynamic mathematical modeling of mural thrombogenesis over type I collagen
(1996) Zaidi, Tabish Naseem; McIntire, Larry V.
We have developed a transient computational model of mural thrombogenesis, simulating whole blood flow in real time in a parallel plate flow chamber. Finite element computational methods were utilized to solve governing partial differential equations of mass conservation and fluid flow. The spatial and temporal variation of concentrations of four platelet agonists known to be synthesized by or released from activated platelets (ADP, vWF, thromboxane A$\sb2$ and thrombin) were computed. The embolizing shear, tensile stresses and torque acting on the aggregates due to fluid forces caused by blood flow were also quantitated. The maximum thrombin concentrations (occurring in stagnating fluid pockets proximal to the thrombi) at the end of two minute blood perfusion are more than 800 fold larger compared to those required for irreversible platelet aggregation in vitro (1-3 nM) under all blood flow conditions. While ADP and vWF might only be important at the initial stages of thrombogenesis and at lower wall shear rates, TxA$\sb2$ concentrations by themselves are not sufficient to cause platelet activation. All fluid stresses increase with wall shear rates and are independent of time. The torque on platelet thrombi increases with time and wall shear rate.
Effect of fluid rheology of hole cleaning in highly-deviated wells
(1995) Vinod, Palathinkara S.; McIntire, Larry V.
One of the technical challenges in deviated drilling is the transport of drill bit cuttings to the surface. The complexity arises due to the narrow settling clearance for the cuttings and the presence of a cuttings bed on the low side of the bore hole. Fluid rheology is the determining factor in the efficiency of this operation. Drilling fluid selection for possible field use is the focus of this dissertation. The problem has been treated with a two-pronged strategy: (i) macroscopic studies that involve numerical models for the prediction of effects of rheology on drilling fluid flow through deviated bore holes; and (ii) microscopic experimental studies that provide physical insights into the fluid forces and relevant rheological parameters in cuttings resuspension. The objective of this study is to propose guidelines for fluid selection and rheological characterization of drilling fluids for the industry. The numerical study demonstrates that power law index is a significant parameter in determining the local flow regime in the different regions of the annulus and hence accurate control of power law index is critical in optimizing bore hole flow. In laminar flow, the shear thinning nature of a fluid does not aid flow through the narrow regions. Turbulence in the wider regions of the annuli aids flow through the narrow regions. The wall shear stresses are dependent only on the pressure drop, gap width and the local flow regimes. Eccentricity is found to hinder flow through the narrow regions of the annuli and certain combinations of rheology and eccentricity can cause stationary 'plug like' zones inside the annuli. The experimental study combined with microscopic calculations identified lift force as the limiting force in particle mobilization and put in perspective the possible importance of normal stresses due to the viscoelastic behavior of the fluid. It is shown that viscous characterization of the fluid is inadequate to predict the particle mobilization velocities even for very simple situations. Characterization of the fluid viscoelastic properties can provide qualitative information on the importance of fluid rheology for particle mobilization. The parameters of interest identified are the magnitude and strain sensitivity of linear viscoelastic moduli.
Effect of novel antithrombotic agents on platelet aggregation under flow conditions
(1992) Natarajan, Maya; McIntire, Larry V.
Epi-fluorescence video microscopy and digital image processing were used to evaluate the effects of three novel anti-platelet agents namely GT-12/I.2HBr (GT-12/I), A4.2HBr and IC, a series of compounds containing the $-$3-carboxylic acid piperidine moiety as their functional group, on platelet aggregation. GT-12/I was evaluated at a concentration of 100$\mu$M so that it could be used as a reference compound against which the effects of A4.2HBr and IC could be compared. Results show that at this concentration GT-12/I inhibited platelet accumulation by 59.0 $\pm$ 3.8% whereas A4.2HBr, the more hydrophobic molecule, inhibited aggregation by 73.5 $\pm$ 1.9% at the same concentration. This confirms earlier predictions that the optimization of hydrophobicity within a compound maximizes its inhibitory potential. Stereochemistry, however, seems to dominate over all other effects as it was observed that IC, an enantiomer of GT-12/I, drastically reduced platelet aggregation by 84.8 $\pm$ 2.8%. (Abstract shortened with permission of author.)
Effect of shear stress on leukocyte adhesion to vascular endothelium
(1988) Lawrence, Michael Brandeau; McIntire, Larry V.
The effect of flow on the adhesion of neutrophils (PMNL) to vascular endothelium was investigated using a parallel plate flow chamber. Human umbilical vein endothelial cells (HUVEC) were cultured on a glass slide which was fitted onto the flow cell. Formyl-methionyl-leucyl-phenyl-alanine (FMLP), a chemotactic tripeptide, interleukin-1 (IL1), lipopolysaccharide (LPS), and thrombin were used to stimulate the PMNL and HUVEC in order to model the effect of inflammatory mediators on leukocyte adhesion under controlled flow conditions. PMNL adhesion to HUVEC monolayers was measured over a range of wall shear stresses estimated to be representative of flow conditions in the microcirculation. HUVEC monolayers were treated with interleukin-1 (IL1, 2U/ml, 4 hours) preceding the experiment. At 2.0 dynes/cm$\sp2$ wall shear stress, 371 $\pm$ 25.8 PMNL/mm$\sp2$ (mean $\pm$ SEM) adhered to IL1-treated HUVEC and 28 $\pm$ 2.9 PMNL/mm$\sp2$ adhered to control HUVEC after ten minutes of flow (p $<$ 0.01 on the adhesion ratio, n = 5). At 3.0 dynes/cm$\sp2$ wall shear stress, 10.2 $\pm$ 3.8 PMNL/mm$\sp2$ adhered to IL1-treated HUVEC and 6.8 $\pm$ 3.5 PMNL/mm$\sp2$ adhered to control HUVEC (n = 5). PMNL adherence to IL1-treated HUVEC decreased significantly at 3.0 dynes/cm$\sp2$ compared to adherence at 2.0 dynes/cm$\sp2$ (p $<$ 0.005). The CD18 family of leukocyte glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to the inflammatory response. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to IL1-treated HUVEC at 2.0 dynes/cm$\sp2$. TS1/18 inhibited migration of PMNL beneath IL1-treated HUVEC monolayers by 82 $\pm$ 6.8%. In flow experiments with CD18-deficient PMNL, no transendothelial migration was observed. The effect of FMLP (10$\sp{-8}$ M) on PMNL adhesion to untreated HUVEC was investigated at wall shear stresses ranging from 0.25 to 2.0 dynes/cm$\sp2$. FMLP stimulation did not significantly increase PMNL adherence at shear stresses above 0.5 dynes/cm$\sp2$. It was possible using a flow system to demonstrate that the initial attachment (or margination) of PMNL does not involve the same membrane associated adhesive proteins as does subsequent migration beneath HUVEC monolayers. Additionally, by controlling the level of shear force, it was possible to distinguish CD18/ICAM-1 mediated attachments to HUVEC from CD18-independent ones. The CD18/ICAM-1 dependent component appears to contribute to attachment at wall shear stresses below 1.0 dyne/cm$\sp2$, while the CD18/ICAM-1 independent component appears to form stronger or more numerous bonds that mediate adhesion at higher wall shear stresses. These observations indicate that local blood flow rates in the vasculature can play an important role in regulating the margination and attachment of leukocytes to the blood vessel wall.
Effect of shear stress on surface membrane potassium ion permeability of calf pulmonary artery endothelial cells (Potassium ion)
(1989) Alevriadou, Barbara Rita; McIntire, Larry V.
This study aims to determine the effect of shear stress on membrane potassium (K$\sp+$) permeability in vascular endothelial cells. Cultured monolayers of calf pulmonary artery endothelial cells, preloaded with $\rm \sp{86}Rb\sp+$, were subjected to different levels of fluid shear stress in the range 1-10 dynes/cm$\sp2$, in a parallel-plate geometry flow chamber, and the radioactivity of the effluent was monitored with time. Increase in laminar shear stress, from 1 dyne/cm$\sp2$ to a higher level, resulted in a rapid transient increase in the rate constant for $\rm \sp{86}Rb\sp+$ release. The difference of efflux rate coefficients, between the peak and the baseline at 1 dyne/cm$\sp2$, varied with shear stress in a "dose-dependent" manner. Changes in K$\sp+$ permeability may occur via activation of shear-stress-activated K$\sp+$ channels, Ca$\sp{2+}$-activated K$\sp+$ channels, or both. The Ca$\sp{2+}$-activated K$\sp+$ channels were shown to respond to bradykinin stimulation under flow conditions. The significance of such flow studies is that they provide more knowledge about the flow-associated changes in ionic channels and intracellular second messengers in endothelial cells.
Effects of antiplatelet agents on polymorphonuclear leukocytes exposed to mechanical trauma
(1981) Rothberg, Jeanne Mays; McIntire, Larry V.; Hellums, Jesse D.; Martin, R. Russell
The effects of shear stress on polymorphonuclear leukocytes (PMNs) were investigated to ascertain whether mechanical trauma induces lysosomal granule release or membrane lysis. In addition, the effects of antiplatelet agents on PMN leukocyte response to shear stress were studied. The antiplatelet agents, known to elevate intracellular and cyclic adenosine monophosphate (cAMP), included the following: prostaglandin Ej (PGEi), prostaglandin I2 (PGI2), theophylline, RA-8 (dipyridamole), and the dipyridamole analog, RA-233. PMN leukocyte suspensions were exposed to shear stresses of 1 or 3 dynes/cm2 at room temperature (23°C) for 1 minutes. Leukocyte responses were monitored by the following measurements. The electronic particle count was measured to indicate shear-induced aggregation and lysis. 8-Glucuronidase in the extracellular fluid was assayed to indicate azurophilic granule release. Finally, lactic dehydrogenase release was measured to indicate cell lysis due to PMN exposure to shear stress. Results of the shear-induced effects on PMNs without drug treatment show that cell loss, granule release, and cell lysis increase with increasing shear stress. Furthermoré, PMNs exposed to a shear stress of 1 dynes/cm2 release approximately equal percentages of 3-glucuronidase and lactic dehydrogenase. Percent cell loss approximates enzyme release indicating cell disruption at this shear stress. At a shear stress of 3 dynes/cm2, 3-glucuronidase release is greater than LDH release indicating that the PMNs are expelling granules into the extracellular fluid at this higher shear stress without concomitant cytoplasmic enzyme release. Pretreatment of PMNs with PGEi plus RA-233 or PGEi plus theophylline produces an approximately 25% reduction of cell loss and release of 3-glucuronidase and LDH release after exposure to a shear stress of 3 dynes/cm2. No significant effects of PGEi plus RA-233 are observed at 1 dynes/cm2. Pretreatment of PMNs with PGI2 plus RA-233 produces no reduction of PMN response to mechanical stimuli. PMNs incubated with dipyridamole and exposed to a shear stress of 3 dynes/cm2 show a significant reduction in cell loss, granule release, and lysis. The suppressive effect of dipyridamole appears to be additive with the effect of PGEi. Leukocytes pretreated with PGEi plus dipyridamole and exposed to shear stresses of 3 dynes/cm2 show a greater decrease of cell loss and enzyme release than pretreatment with dipyridamole alone or PGEi plus a phosphodiesterase inhibitor.
Effects of cyclic strain on the gene expression of human umbilical vein endothelial cells as determined through microarray technology
(2003) Frye, Stacie Renee; McIntire, Larry V.
This thesis work resulted in microarray expression data of thousands of genes in vascular endothelial cells subjected to cyclic strain; and this data revealed genes showing expression changes between strained and control cells. The knowledge of gene expression changes due to cyclic strain helps reveal the mechanisms of vascular pathogenesis, which has been linked to the elevated levels of strain seen at arterial bifurcations and curves. Previous microarray studies on endothelial cells exposed to shear stress and smooth muscle cells subjected to cyclic strain indicated a number of genes that are responsive to hemodynamic forces in vascular cells. However, the studies in this thesis are the first to my knowledge to use microarray analysis to study vascular endothelial cells subjected to cyclic strain. Using microarrays equipped to study over 4000 genes simultaneously, expression levels of human umbilical vein endothelial cells exposed to cyclic strain (10%, 1 Hz), static control, and motion control for six and twenty-four hours indicated genes with differential expression between at least two of the three experimental conditions. Two different analysis methods, k-fold with a 2-fold threshold of significance and statistical testing with calculation of an adjusted p-value for significance, resulted in a total of 30 and 11 differentially expressed genes, respectively. Polymerase chain reaction verified the results of five genes and found that each method resulted in valid results. The k-fold method of analysis applied to expression data from two progressively higher wash temperatures of the microarrays resulted in an increasing number of differentially expressed genes. This study was the first to illustrate the strain-responsiveness of many of these differentially expressed genes. A number of the differentially expressed genes in this study fit into the functional classes of oxidative stress protection, regulation of proliferation or leukocyte recruitment, all of which are important to maintaining vascular health. Future in depth analysis of these differentially expressed genes will elucidate their functional activities in the cellular response to hemodynamic forces.
Effects of dynamic mechanical stresses on mammalian cell metabolism
(1991) Rajagopalan, Sridhar; McIntire, Larry V.
The effects of dynamic mechanical stresses on the arachidonic acid (AA) metabolism of human platelets and on the cell growth properties of murine tumor cells were studied. The cells were stressed by suspending them in laminar simple shear flows. Washed human platelet suspensions were stressed at 75 dynes/cm$\sp2$ and 150 dynes/cm$\sp2$ for 5 to 10 min. at 25$\sp\circ$C. The stress stimulated AA metabolism above basal levels. The lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid was preferentially formed, but the cyclooxygenase metabolite thromboxane B$\sb2$ was not detected. Thrombin-stimulated platelets, however, formed cyclooxygenase metabolites in addition to the lipoxygenase metabolites. This result indicates that the physical stresses and chemical agonists (like thrombin) affect platelet AA metabolism differently. Stress effects on tumor cells were studied using high and low metastatic sub-lines of the B16 melanoma and the RAW117 large cell lymphoma. The cells were stressed at 0, 450 and 900 dynes/cm$\sp2$ at 37$\sp\circ$C for 5 min. Damage caused by the stresses was determined by measuring the lysis in the shear field and measuring the relative reduction in cell numbers in stressed cultures compared to unstressed controls. Also, cell growth and lysis in stressed and unstressed cultures were monitored over time. The results could by modeled assuming that a subpopulation of stressed cells was lethally damaged and ultimately died while a second group of stressed cells were only sublethally damaged and grew normally after a brief lag-time. The data indicate that there was not much difference in the damage levels between high and low metastatic sub-lines and that a considerable fraction of stressed cells were not lethally damaged even at bulk shear stresses as high as 900 dynes/cm$\sp2$ for 5 min. Although the stresses applied were higher than normally seen in vivo, the results are in contrast to the accepted view that mechanical stresses developed in the circulation directly cause the destruction of most of the tumor cells during blood-borne metastasis and that highly metastatic cells can resist these stresses more effectively than poorly metastatic cells.
Effects of mechanical loading on osteoblast function using a three dimensional celijpolymer model
(1998) Jen, Anna Hsiao-Chieh; McIntire, Larry V.; Mikos, Antonios G.; Rudolph, Frederick B.; Gustin, Michael C.; Farach-Carson, Cindy
Mechanisms which trigger bone modeling/remodeling in response to changes in the mechanical environment are still unclear. In a three part study, effects of loading on osteoblast function were investigated using a three dimensional (3-D) ceWpolymer model. The 3-D model has advantages of cell culture while maintaining the natural matrix architecture of bone. Such celVpolymer constructs have been shown to form bone in vitro. Osteoblasts in 3-D ceWpolymer constructs were cyclically loaded (5% ). After five days, compressed constructs decreased in alkaline phosphatase activity, a marker of osteoblast maturation. After three weeks, loaded constructs showed lower alkaline phosphatase activity but higher RNA level of L-type calcium channels, involved in calcium signaling cascades. No difference was detected after twelve weeks. Results suggest osteoblasts sensed loading and altered functional activities in response. Use of the 3-D model to study other osteoblast functions under mechanical loading may increase understanding of regulated functional adaptation by bone.
Effects of shear stress and cyclic strain on the expression of thrombin receptor gene in human vascular endothelial and smooth muscle cells
(2000) Nguyen, Kytai Truong; McIntire, Larry V.
Mechanical forces such as shear stress and cyclic strain have been shown to regulate expression of many genes that can alter vascular functions such as cell proliferation, leading to the development of vascular diseases including atherosclerosis. Thrombin receptor gene, protease-activated receptor-1 (PAR-1), mediates many important vascular functions such as thrombin-stimulated thrombosis, inflammation, and proliferation of vascular cells; however, the regulation of PAR-1 by mechanical forces has not previously been studied. This thesis investigates effects of shear stress and cyclic strain on gene regulation of PAR-1 in human vascular cells such as endothelial (ECs) and smooth muscle cells (VSMCs) and the molecular mechanisms involved in this regulation. This work finds that shear stress and cyclic strain differentially regulated PAR-1 expression in vascular cells, leading to alterations of cell functions in response to thrombin, and that these processes were mediated through various signaling pathways. Cultured cells were exposed to different levels of shear stress or cyclic strain using the parallel flow plate chamber or uni-axial cyclic strain system. After exposure, PAR-1 mRNA and protein were quantified by Northern blot and flow cytometry, respectively. In addition, inhibitors of various signal pathways such as protein kinases were used to investigate the molecular mechanisms. Arterial shear stresses decreased PAR-1 mRNA and protein both time- and dose-dependently in both macro- and microvascular ECs, leading to attenuation of thrombin-stimulated nitric oxide and endothelin-1 releases. Furthermore, protein kinase C partly mediated shear-reduced PAR-1 expression in both cell types. As in ECs, shear-downregulated PAR-1 expression in VSMCs caused decreases in thrombin-stimulated calcium mobilization and cell proliferation. The transcription mechanism, but not mRNA stability, regulated shear-reduced PAR-1 expression in VSMCs. In contrast to shear stress, high levels of cyclic strain increased PAR-1 expression in VSMCs time-dependently, leading to induction of cell proliferation in response to thrombin, and this process was mediated by reactive oxygen species, possibly through the NADPH pathway. These findings indicate important roles of mechanical forces in regulating vascular functions and thus provide a better understanding of how mechanical factors act to promote vascular diseases.
Effects of sickle erythrocytes on metabolism and gene regulation in cultured human endothelial cells under flow conditions
(1999) Shiu, Yan-Ting; McIntire, Larry V.
Sickle cell anemia is characterized by chronic hemolysis and episodic vasoocclusive crises. There are many factors contributing to vascular occlusion in sickle cell patients, with the enhanced, abnormal adhesion of sickle erythrocytes to endothelial cells being investigated most thoroughly. The vascular endothelium lining on blood vessel walls is in a unique position of direct exposure to these abnormal red blood cells. It is possible that interactions with sickle erythrocytes may modulate endothelial cells to create an environment sensitive to crisis-triggering factors and in favor of adhesion events. However, quantitative information on the metabolic and gene regulatory effects of these interactions on endothelial cells under flow is lacking. In this research, we developed an experimental system that simulates the physiological vascular flow environment to subject cultured human endothelial cell (EC) monolayers to sickle cell perfusion with a well-defined wall shear stress level of 1dyne/cm2 for times up to 24 hours. Effects of sickle erythrocytes on EC production of vasotone mediators (prostacyclin and endothelin-1) and on EC expression of cell adhesion molecules (ICAM-1 and VCAM-1) were examined at the transcription and/or protein levels. The production of prostacyclin and endothelin-1 both increased in ECs after exposure to sickle cell perfusion, in comparison to perfusion with normal red cells. The altered levels of prostacyclin and endothelin-1 may disturb the delicate balance of vasoactive materials, leading to vasotone instability in sickle cell patients. Gene expression and cell surface expression of ICAM-1 in ECs were profoundly elevated by perfusion with sickle erythrocytes. The elevation in membrane-bound ICAM-1 levels may increase the interactions between blood cells and ECs, especially leukocyte endothelium adhesion. This may lead to the entrapment of red cells in regions of low oxygen tension in the microcirculation and trigger the polymerization of sickle hemoglobin. VCAM-1 mRNA, but not membrane-bound VCAM-1, was also significantly increased by perfusion with sickle erythrocytes. The presence of VCAM-1 on ECs may enhance the adhesion of erythrocytes via binding to integrin VLA-4( a 4 b 1) expressed on sickle cell membranes, but not on normal red cell membranes. The release of soluble ICAM-1 and soluble VCAM-1 both increased in ECs after exposure to sickle cell perfusion, and may serve as an indicator of injury and/or activation of ECs. The presence of IL-1 b in the perfusion system, as a model of inflammatory cytokine effects, synergistically increased the production of prostacyclin, ICAM-1 and VCAM-1 in endothelial cells. This is consistent with the close association of inflammation and vasoocclusive crises observed in sickle cell anemia patients.

Browsing by Author "McIntire, Larry V."

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