Biomedical subjects

G A Truskey

Publications and source records attributed to G A Truskey.

At least 37 records · Page 2Linked to original sources

PubMed1998

Association between secondary flow in models of the aorto-celiac junction and subendothelial macrophages in the normal rabbit.

In order to examine the association between arterial fluid dynamics and the distribution of subendothelial macrophages in the normal rabbit aorta, steady and pulsatile particle flow visualization was performed in a geometrically realistic model of the rabbit aorto-celiac junction region. Over a range of aorto-celiac steady flow ratios, particle pathlines along the upstream lateral aortic walls curved to enter the celiac orifice, while two asymmetric regions of reversing spiral secondary flow originated along the downstream lateral portions of the orifice flow divider. These regions increased in size as either the Reynolds number or flow into the celiac artery increased. In pulsatile flow studies, particles along the lateral aortic walls near the celiac orifice began to spiral into the branch during peak systole. During systolic deceleration, the size of this spiral flow region increased as particles reversed direction to enter the celiac orifice. This contrasted with flow patterns directly upstream and downstream of the orifice, which remained unidirectional throughout this period even along the distal lip of the orifice. The highest frequency of subendothelial white blood cells in the normal rabbit aorta was associated with regions where secondary flow patterns occurred, and where the orientation of endothelial cell nuclei deviated from the major direction of aortic flow. Secondary flow patterns may aid the accumulation of monocytes and macrophages about the lateral regions of the celiac artery flow divider by transporting monocytes to the walls, allowing them time to attach to the endothelial cells, or by stimulating the endothelial cells to express leukocyte adhesion molecules. These same regions are associated with increased endothelial permeability to low density lipoprotein and, under hypercholesterolemic conditions, lesion origination.

Animals↗

PubMed1998

Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials.

Cell adhesion and function depend upon the formation of adhesive contacts between the cell and substrate. Determination of the cell substrate contact area is necessary in order to understand how biomaterial properties influence cell adhesion. In this review we describe the development and application of total internal reflection fluorescence microscopy (TIRFM) to quantify the separation distance of cells from a biomaterial surface. An approximate theory is presented for the straightforward calculation of separation distances when a fluor is placed in the cell membrane. The validity of this approach is discussed. TIRFM is compared to interference reflection microscopy and related techniques that measure cell/substrate separation distances. This approach is then applied to a number of important problems in cell substrate interactions, including changes in contact area and adhesion strength on biomaterial surfaces, analysis of bond strength, and real-time measurement of cell/substrate separation distances following exposure to flow.

Animals↗

PubMed1998

Effects of recirculating flow on U-937 cell adhesion to human umbilical vein endothelial cells.

We used a sudden-expansion flow chamber to examine U-937 cell adhesion to unactivated and tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC) in recirculating flow. For both unactivated and TNF-alpha-activated HUVEC, U-937 cells exhibited transient arrests within approximately 150 microm of flow reattachment. Few arrests occurred directly at the reattachment site. U-937 cell rolling was not observed. At all other locations within the recirculation zone, U-937 cells did not exhibit transient arrests or rolling. TNF-alpha activation increased the frequency of U-937 cell arrests near reattachment but did not change the median arrest duration. Numerically simulated cell trajectories failed to predict attachment near the reattachment point. Deviations between experiment and theory may result from the nonspherical shape and deformability of U-937 cells. These results demonstrate that U-937 cell transient arrests occur preferentially in the vicinity of the reattachment point in recirculating flow. Possible mechanisms for adhesion include low shear stress, curved streamlines, fluid velocity components normal to the endothelium, and formation of larger contact areas.

Cell Adhesion↗

PubMed1998

Improving endothelial cell adhesion to vascular graft surfaces: clinical need and strategies.

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p < 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.

Adsorption↗

PubMed1997

Engineering the tissue which encapsulates subcutaneous implants. I. Diffusion properties.

This report uses normal rat subcutis as a reference point to provide a quantitative analysis of small analyte transport through the tissue which encapsulates implants. Polyvinyl alcohol (PVA) with 60- and 350-micron mean pore size (PVA-60, PVA-350), nonporous PVA (PVA-skin), and stainless-steel cage (SS) specimens were implanted in the subcutis of Sprague-Dawley rats for 4 weeks to elicit a range of capsular wound-healing tissues. Histologic examination showed that the capsular tissue which formed around PVA-skin and SS specimens was densely fibrous and avascular. That forming around PVA-60 and PVA-350 was less densely fibrous and more vascular. The fibrous content of capsular tissue and subcutis was determined from eosin-stained histologic sections. Dual-chamber diffusion measurements of sodium fluorescein (Mw 376 g/mol) through capsular tissue and normal rat subcutis were used to quantitatively compare the effective diffusion coefficients of small analytes on the order of glucose. The two most fibrous capsular tissues exhibited diffusion coefficients that were statistically (p < 0.05) less than that determined for rat subcutis by 50 and 25% for PVA-skin and SS, respectively. The diffusion coefficients of the less dense capsular tissue which formed around the porous implants were not statistically different from subcutis. The experimentally measured diffusion coefficients of the two most fibrous capsular tissues were closely predicted by a simple two-component diffusion model consisting of an aqueous interstitium with an array of impenetrable bodies equal in volume fraction to the fibrous content of the tissue. This model overestimates the diffusion coefficients measured for the least fibrous tissues. Using the diffusion coefficient measured for the PVA-skin capsular tissue, a finite difference model predicts that a 200-microns-thick capsular layer would increase from 5 to 20 min the time required for subcutaneously implanted sensor to detect 95% of the blood analyte concentration. This study suggests that the fibrous capsule forming around a subcutaneously implanted smooth-surface sensor imposes a significant diffusion barrier to small analytes such as glucose, thus increasing the lag time of the sensor by as much as threefold. A corollary observation is that a sensor with a porous surface which allows tissue ingrowth may be more responsive to blood analyte fluctuations as a result of its a more vascular and less fibrous encapsulation tissue.

Animals↗

PubMed1996

Effect of fibronectin amount and conformation on the strength of endothelial cell adhesion to HEMA/EMA copolymers.

The effect of substrate surface hydrophobicity on fibronectin (Fn) adsorption and endothelial cell adhesion strength was studied. Bovine aortic endothelial cells (BAEC) were plated for 2 h with and without preadsorbed Fn on slides coated with homopolymers and copolymers of hydrophilic polyhydroxyethylmethacrylate (polyHEMA) and hydrophobic polyethylmethacrylate (polyEMA). The polarity of the substrate was determined by Wilhelmy plate contact angle. The amount of adsorbed Fn was determined using 125I-labeled Fn. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to detect gross conformational changes of adsorbed Fn on polyHEMA or polyEMA. BAEC were cultured in serum-free medium for 2 h and subjected to a brief exposure of laminar flow in a variable-height flow chamber that provided a range of shear stresses of 15-185 dynes/cm2. The critical shear stress to detach 50% of the cells increased with increasing EMA content to a maximum at 20% HEMA/80% EMA copolymer irrespective of the presence of preadsorbed Fn. However, the critical force increased even though there were similar amounts of Fn adsorbed on all substrates. ATR-FTIR spectroscopy showed only minor changes in beta-sheet structure of Fn adsorbed to polyHEMA and polyEMA. These results show that the force to detach cells did not increase solely with increasing amounts of adsorbed Fn; rather, these results indicate a more complex interplay involving both the amount and conformation of adsorbed Fn.

Animals↗

PubMed1996

A focal stress gradient-dependent mass transfer mechanism for atherogenesis in branching arteries.

A new arterial wall permeability function, based on the local wall shear stress gradient, has been developed and employed to simulate enhanced low density lipoprotein transfer across the endothelium. the atherosclerotic model used is that of the aorto-celiac junction of rabbits. The experimentally validated computer simulation model for convection mass transfer provides further evidence that the wall shear stress gradient is a reliable predictor of critical atherogenic sites in branching arteries. Some of the underlying biological aspects of atherogenesis due to locally significant and sustained wall shear stress gradient values are briefly discussed.

Animals↗

PubMed1996

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.

Adsorption↗

PubMed1995

The distribution of intimal white blood cells in the normal rabbit aorta.

Macrophages play an important role in atherogenesis and have been reported within the intima at lesion-prone sites in normocholesterolemic animals as well as infants and children. The objective of this study was to determine the spatial distribution of intimal white blood cells (WBC) in the normal rabbit aorta and the association of intimal WBC with replicating endothelial cells and sites of increased 125I-LDL permeability. Intimal WBC and macrophages were identified en face on whole aortic tissue and on Häutchen preparations based on their morphology, ingestion of exogenous horseradish peroxidase, non-specific esterase activity, and labeling with a monoclonal antibody for rabbit macrophages (RAM11). WBC were primarily located in the lesion-prone flow divider regions of the large abdominal branch arteries. Using [3H]thymidine autoradiography to determine cell proliferation, 4.4% of the WBC and 0.12% of the endothelial cells were labeled on the Häutchen preparations. The distribution of replicating endothelial cells was not localized to the arterial orifices and was not correlated with the distribution of intimal WBC. Intimal WBC were, however, spatially correlated with the distribution of 125I-LDL permeable sites about the celiac artery orifice and were directly associated with 31% of the LDL permeable spots. Moreover, mitotic endothelial cells accounted for only 8% of the total number of LDL permeable sites. The presence of intimal WBC at lesion-prone sites in the normocholesterolemic rabbit suggests that these cells may be important in the initiation of atherosclerotic lesions.

Animals↗

PubMed1995

Numerical investigation and prediction of atherogenic sites in branching arteries.

Atherosclerosis, a disease of large- and medium-size arteries, is the chief cause of death in the US and most of the western world. It is widely accepted that the focal nature of the disease in arterial bends, junctions, and bifurcations is directly related to locally abnormal hemodynamics, often labeled "disturbed flows." Employing the aorto-celiac junction of rabbits as a representative atherosclerotic model and considering other branching blood vessels with their distinctive input wave forms, it is suggested that the local wall shear stress gradient (WSSG) is the single best indicator of nonuniform flow fields leading to atherogenesis. Alternative predictors of susceptible sites are briefly evaluated. The results discussed include transient velocity vector fields, wall shear stress gradient distributions, and a new dimensionless parameter for the prediction of the probable sites of stenotic developments in branching blood vessels. Some of the possible underlying biological aspects of atherogenesis due to locally significant /WSSG/-magnitudes are briefly discussed.

Animals↗

PubMed1995

Characterization of a sudden expansion flow chamber to study the response of endothelium to flow recirculation.

In order to simulate regions of flow separation observed in vivo, a conventional parallel plate flow chamber was modified to produce an asymmetric sudden expansion. The flow field was visualized using light reflecting particles and the size of the recirculation zone was measured by image analysis of the particles. Finite element numerical solutions of the two and three-dimensional forms of the Navier-Stokes equation were used to determine the wall shear stress distribution and predict the location of reattachment. For two different size expansions, numerical estimates of the reattachment point along the centerline of the flow chamber agreed well with experimental values for Reynolds numbers below 473. Even at a Reynolds number of 473, the flow could be approximated as two-dimensional for 80 percent of the chamber width. Peak shear stresses in the recirculation zone as high as 80 dyne/cm2 and shear stress gradients of 2500 (dyne/cm2)/cm were produced. As an application of this flow chamber, subconfluent bovine aortic endothelial cell shape and orientation were examined in the zone of recirculation during a 24 h exposure to flow at a Reynolds number of 267. After 24 h, gradients in cell orientation and shape were observed within the recirculation zone. At the location of reattachment, where the wall shear stress was zero but the shear stress gradients were large, cells plated at low density were still aligned with the direction of flow. No preferred orientation was observed at the gasket edge where the wall shear stress and shear stress gradients were zero. At higher cell densities, no alignment was observed at the separation point.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor.

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+]i and NO release due to shear stress or agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 microL). The microsensor was positioned approximately 100 microns above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 +/- 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 +/- 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mumol/L) or Br-A23187 (10 mumol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mumol/L) chelation of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 +/- 0.07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64 +/- 0.32 nmol/L per second). These data argue that shear stress causes a Ca(2+)-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts.

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by back-ground noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 +/- 13 nm. Focal contact areas estimated by TIRFM (1.3 +/- 0.7 micron2) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 microns2).

Animals↗

PubMed1994

Characterization of sites with elevated LDL permeability at intercostal, celiac, and iliac branches of the normal rabbit aorta.

En face autoradiography of the endothelium was used to quantify the distribution, area, and permeability of sites with enhanced permeability to 125I-low-density lipoprotein (125I-LDL) around the intercostal and celiac arteries and at the iliac bifurcation of normal rabbit aortas. The density of such sites was highest in the upper thoracic aorta and around the celiac and superior mesenteric branches and was lowest in the lower abdominal aorta. Permeable sites occurred more frequently distal to the intercostal branch orifices and both lateral and distal to the orifice at the celiac branch. At the intercostal branch orifices, these sites were larger, with a lower permeability and higher frequency than those away from the branch. At the celiac flow divider, sites of elevated autoradiographic grain density were more permeable and larger than at other locations in the abdominal aorta. Mean regional permeabilities were obtained by weighted area averages of low- and high-permeability sites. Mean regional permeabilities around the intercostal branches were 1.5 times higher than values away from the intercostal branches. Within 0.25 and 1 mm away from the celiac flow divider, mean regional permeability was 3.1 and 1.3 times higher, respectively, than those away from the flow divider. Few sites of elevated permeability were found distal to the aortoiliac bifurcation, and the permeabilities at the medial and lateral walls of the iliac arteries were not different. Mitotic cells were associated with 13 +/- 8% of all sites with elevated permeability to 125I-LDL. The frequency of mitotic endothelial cells was not increased at branch sites, suggesting that mechanisms other than cell replication were responsible for increased LDL permeability in the rabbit. These results suggest that the permeability and frequency of occurrence of sites with elevated permeability around the celiac and intercostal branches may influence the distribution and severity of early lesions in rabbits fed a hypercholesterolemic diet.

Analysis of Variance↗

PubMed1993

Effect of the conformation and orientation of adsorbed fibronectin on endothelial cell spreading and the strength of adhesion.

The effect of surface hydrophobicity upon the conformation of the cell binding domain of fibronectin (Fn) and the influence of Fn conformation on bovine aortic endothelial cell (BAEC) adhesion were examined. The free sulfhydryl group of Fn located near the cell binding domain was selectively labeled with acrylodan, a polarity sensitive fluor. Fluorescence emission was monitored in solution and upon adsorption to hydrophilic glass and hydrophobic silanized glass. The acrylodan-labeled Fn emission maximum shifted to longer wavelengths upon adsorption and the shift was greater for acrylodan-labeled Fn adsorbed to hydrophilic glass than hydrophobic silane, suggesting that the acrylodan was in a more solvent accessible environment on glass than silane. BAEC, suspended in serum-free medium, attached for 15 or 120 min onto glass or silane surfaces containing preadsorbed Fn, after which cell spreading and the strength of adhesion in a parallel plate flow chamber were measured. Cell spreading was similar on both surfaces after 15 min attachment, but BAECs were more spread on glass than silane after 120 min. At low surface concentrations of Fn, BAECs were more adherent on glass than silane. At higher surfaces concentrations, adhesion was similar. After a 2-h incubation in serum-free medium, cells on glass showed more extensive development of focal contacts as determined by immunofluorescent staining for vinculin. Cell adhesion under flow was reduced on silane by inhibition of protein synthesis with cycloheximide, suggesting that cell attachment to silane was promoted by cellular synthesis of Fn. The results indicate that changes in the conformation of the Fn cell binding domain affect Fn affinity for its cell surface receptor.

Adsorption↗

PubMed1993

Relationship between 3T3 cell spreading and the strength of adhesion on glass and silane surfaces.

Cell detachment by laminar shear stresses was used to characterize cellular interactions with hydrophilic glass and hydrophobic silane. In this study, we examined whether smaller, rounder cells were preferentially detached by laminar flow, and whether cell detachment occurred by dissociation of adhesion proteins and their membrane receptors or rupture of the membrane. Shear-induced detachment from glass and silane were similar after 0.5 h static attachment to the surfaces, even though 3T3 cells had a greater projected area on silane. No particular cell size was preferentially detached by fluid shear stresses. After 2 h attachment and spreading, 3T3 cells were more easily detached from the silane surface even though the cells were more spread than on glass. On glass, smaller cells were preferentially detached below 30 dyne/cm2, increasing the mean projected area of the population. Above 30 dyne/cm2, larger cells also detached from the surface. Cell detachment from the silane surfaces did not show any size preference. The strength of adhesion and projected areas on both surfaces increased significantly when the surfaces were preincubated with fibronectin. Simple geometric models of spreading cells were used to estimate the forces exerted on cells. The hydrodynamic forces exerted on spreading cells were similar, but the bond density needed to resist detachment declined as the projected area increased. Analysis of Dil-C18(3) membrane fragments indicated that cell detachment by membrane rupture was a significant mechanism of cell detachment from glass for shear stresses above 40 dyne/cm2, but was unimportant for cell detachment from the silane surfaces. The results indicate that differences in the strength of 3T3 cell adhesion were probably due to differences in bond strength and the numbers of receptor-ligand bonds formed on the two surfaces and, on glass, cell detachment due to membrane failure at higher shear stresses.

3T3 Cells↗

PubMed1992

Measurement of endothelial permeability to 125I-low density lipoproteins in rabbit arteries by use of en face preparations.

A procedure of en face quantitative autoradiography of the endothelium (Hautchen preparations) was developed to examine regional variations in 125I-low density lipoprotein (125I-LDL) permeability in the arterial wall in vivo. Endothelial preparations from fixed arterial tissue and calibration standards consisting of known concentrations of 125I-albumin were dipped in nuclear emulsion, exposed for 1-3 months, developed, and stained with hematoxylin. Digital image analysis was used to analyze dark-field images of autoradiographs. Background grain densities on cold endothelial preparations were 30-100% higher than on glass, but the variability in grain densities on the two different surfaces was similar. Regression slopes of grain density versus concentration for calibration standards were the same for sections placed on cold tissue or glass. For 1-5-microns-thick calibration standards of the same concentration, the grain density was proportional to the total amount of radioactivity per unit area. The results indicated that errors arising from nonuniformities in preparation thickness were minimal, and permeabilities and intimal concentrations could be determined. Rabbits were killed 10 minutes after injection of 125I-LDL, and endothelial preparations were made. For regions of uniformly low grain density in the rabbit aorta, the 125I-LDL permeability was 1.9 +/- 0.8 x 10(-8) cm/sec, and the effective diffusion coefficient was 5.4 +/- 3.1 x 10(-10) cm2/sec. Errors in the estimated permeability arising from nonuniformities in tissue thickness were the same as the reported experimental variability. Analysis of elevated regions of permeability suggested that 125I-LDL was binding to the extracellular matrix. Approximately 25% of the sites of elevated grain density were associated with mitotic endothelial cells, and such regions had higher permeabilities than sites associated with nonmitotic cells. Around intercostal arteries, sites of highest permeability were distal and lateral to the vessels and occurred where lesions first develop in hypercholesterolemic rabbits.

Animals↗

PubMed1992

Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances.

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.

Animals↗